- Email: [email protected]
Biochemical and immunological prospects for male contraception
Molec. Aspects Med. Vol. 3, pp. 225-293. © Pergamon Press Ltd., 1980. Printed in Great Britain.
0098-2997/80/0601-0225-$05.00/0
BIOCHEMICAL AND IMMUNOLOGICAL PROSPECTS FOR MALE CONTRACEPTION P. J. Quinn
Department of Biochemistry, Chelsea College, University of London, London SW3 6LX, UK
Contents I.
I ntrodu cti o n
226
2.
Control of the Hypothalamohypophyseal Axis 2.1. Mechanism of Action of Male Sex Hormones 2.1.1. Peptide hormones 2.1.2. Steroid hormones 2.2. Hormonal Regulation of Male Reproduction 2.2.1. Lutenising hormone - releasing hormone 2.2.2. P i t u i t a r y gonadotrophins 2.2.3. Inhibin
229 229 230 232 234 234 236 240
3.
Regulation of Testicular Function and Sperm Maturation 3.1. Interference of Spermatogenesis by Chemical Agents 3.2. Effects of Temperature and I r r a d i a t i o n 3.3. Biochemical and Immunological Aspects of Vasectomy
242 243 247 249
Survival of Spermatozoa in the Female Tract and F e r t i l i z a t i o n 4.1. Factors Affecting Sperm V i a b i l i t y and Transport 4.1.1. The action of prostaglandins on f e r t i l i t y 4.1.2. Steroid binding and sperm transport 4.2. Developmental A l t e r a t i o n s to Spermatozoa in the Female Tract 4.2.1. Capacitation and decapitation processes 4.2.2. The acrosome reaction 4.2.3. Control of capacitation and the acrosome reaction
259 259 260 262 263 263 264 268
Conclusions and Future Prospects
272
Acknowledgement
273
References
274
.
5.
225
Chapter 1
Introduction
Until comparatively recently contraceptive practices were reserved largely f o r breeding domestic animals.
Since B i b l i c a l
times the a t t i t u d e of man towards
contraception has been subject to theological constraints best exemplified by the teachings about the sin of Oman and his punishment.
The qradual separation of
pleasure from procreation ~ollowin~ the nrowth of Protestant ideology created a more positive a t t i t u d e to contraception as an unintended legacy to the secularized world of the eighteenth century.
The need for such practices, however, was not
dictated by demographic considerations but rather by the a t t i t u d e s of husbands towards the sufferings of t h e i r wives who were subjected to the anonies and dangers of repeated pregnancies and c h i l d b i r t h s .
There were also sound economic
reasons to practice contraception which emerged at this time.
~here the cost of
upbringing and launching a child into the world exceeded the value of his e x p l o i t a b l e labour and usefulness to his parents in t h e i r old ane there was c l e a r l y an incentive to l i m i t the size of the family. e l i t e of Europe were the f i r s t
The aristocracy and urban
group to experience these economic nressures
because they did not depend upon the labour of t h e i r children but nevertheless were obliged to bear the r i s i n g costs of t h e i r education and marriane. Furthermore, as respect f o r children developed within the society nreater care in t h e i r nurturing and upbringing was practiced and the level of care was dispensed to each individual
in d i r e c t proportion to the resources available.
Contraceptive methods and practices were at this time l a r q e l y the r e s p o n s i b i l i t y of the male partner.
They included oral or manual sex or c o i t u s i n t e r r u p t u s .
Practice of the l a s t method is c l e a r l y documented in the case of a young Massachusetts man cited by Perrenoud (Iq74) who, in 1771, was successfully
226
227
Biochemical and Immunological Prospects for Male Contraception
prosecuted in a paternity suit despite his protestation that ' I fucked her once, but I minded my pullbacks.'
Othermethods advocated in Elizabethan cookbooks,
herbals and medical treatises included the reduction of sexual desire by violent vomits, purges or bloodletting which rendered the victim too sick or weak to indulge in s e x .
Anotherapparently effective method was the application of
various ointments to the penis to prevent an erection.
The e a r l i e s t recorded
use of condoms was in the late seventeenth century but they did not come into common use until the eighteenth century.
The primary use of these devices,
however, was more than l i k e l y to avoid contracting venereal disease rather than simply to act as a contraceptive device.
Abortifacients, some with disastrous
after effects, were also employed in situations where contraceptive methods
f a i l e d to prevent unwanted pregnancies, Methods changed l i t t l e
until
comparatively recent times when i n s i g h t into the
hormonal regulation of human f e r t i l i t y female.
began to emerge, p a r t i c u l a r l y that of the
Over the past two decades three generations of hormonal female oral
contraceptives have evolved.
Sequential oral contraceptives that ostensibly
mimic the normal hormonal cycle in women and progestin-only compounds have been l a r g e l y withdrawn from use e i t h e r because of undesirable side e f f e c t s or unreliable contraceptive potency.
Of the remainder, the combination p i l l
has proved the
most successful but, judging from contemporary prospective and retrospective reviews of t h e i r usefulness combined with concerns of stroke, myocardial i n f a r c t i o n and hepatomes indicate that more s a t i s f a c t o r y methods of contraception are required.
Current methods of female contraception include the development of
improved combination p i l l s
based on antioestrogens and antiprogestins,
which have already proved suitable for selective use (David et a l . ,
some of
1979).
The development of r e l i a b l e and reversible male contraceptives has tended to lag somewhat behind those methods developed s p e c i f i c a l l y for women.
This may simply
r e f l e c t the fundamental differences in the processes of d i f f e r e n t i a t i o n
and
development of gametes in the male and female and the control mechanisms that regulate these processes.
This means that the basic approach to contraception,
the objectives of which are to r e v e r s i b l y prevent conception with the minimum inconvenience and undesirable side e f f e c t s , often requires a fundamentally d i f f e r e n t approach.
This can be i l l u s t r a t e d
by the simple f a c t that the germinal
c e l l s of the male gonad develop at puberty and p e r s i s t throughout reproductive life.
Consequently, chemical methods of contraception are often hazardous in
that they are l i a b l e to i n f l i c t
genetic damage during the production of mature
gametes that can introduce congenital defects into o f f s p r i n n .
This contrasts
with the female where germinal c e l l s have already entered into the f i r s t
meiotic
228
P.J. Quinn
d i v i s i o n by the time of p a r t u r i t i o n and therefore they are not as vulnerable to genetic a l t e r a t i o n s which could be transmitted to proqeny.
The other major
consideration with regard to male contraception is the need to preserve the psychic drive associated with sex i n s t i n c t or l i b i d o .
Suppression of spermato-
genesis can often lead to impotence which although s a t i s f y i n g the basic c r i t e r i a f o r contraception is obviously an untenable side e f f e c t . I t is against this background of the need to avoid genetic defects and the development of impotence that new and e f f e c t i v e methods of male contraception are being sought.
The following sections review the current status of research
in this area and evaluate the future prospects for male contraception.
Chapter 2
Control of Hypothalamohypophyseal Axis
The f i r s t stage of intervention in altering male reproductive capacity is at the level at which hormonal control of the process is exercised.
Hormonal control
of spermatogenesis and testicular function is achieved by a cascade mechanism subject to fee-back control by products produced in the testis.
The precise
checks and balances in the system are not yet completely understood but the role of the individual hormones involved is becoming clearer.
!,!hat is certain,
however, is that any disturbance in the hormonal balance invariably leads to dysfunction of the reproductive process.
Such disturbances can result from
abnormal release of one or several hormones from the endocrine system or some malfunction of the action of the hormone on i t s target cell.
Both these
processes are possible targets for contraception. 2.1. Mechanism of Action of Male Sex Hormones
Two types of hormone are concerned in male reproductive regulation; peptide hormones and steroid hormones.
The peptide or protein hormones include luteinis.
ing hormone-releasing hormone (LH-RH), produced by the hypothalamus and two gonadotrophin hormones, luteinising hormone (LH) and f o l l i c l e stimulating hormone (FSH) 6riginating from the anterior p i t u i t a r y .
LH-RH stimulates the synthesis
and release of gonadotrophins from the p i t u i t a r y (Yen et a t . , 1973; Schally, 1977; Guillemin, 1978).
Both gonadal functions, namely spermatogenesis and
androgen secretion, are s t r i c t l y regulated by the p i t u i t a r y gonadotrophins; FSH acts on Sertoli cells where i t is required to i n i t i a t e spermatogenesis and LH regulates testosterone production and secretion from the Leydig cells. I t is noteworthy that human chorionic gonadotrophin (hCG) has similar effects on androgen production by Leydig cells, suggesting the presence of receptor sites for hCG on Leydig cells.
229
230 2.l.l.
P.J. Quinn Peptide homones
The mode of action of LH-RH, LH and FSH on t h e i r target c e l l s is similar to that of most other peptide hormones (see Hsueh, 1978).
Hormone bindinq to specific
receptor sites located on the external surface of the plasma membrane of the target c e l l acts as a positive a l l o s t e r i c modulator of adenylcyclase, the c a t a l y t i c s i t e of which is accessible only from the cytoplasmic surface of the membrane.
These reactions are i l l u s t r a t e d
schematically in Fin. I.
peptidehormone hormone -eceptor
P~ ~ cyclic-AMP~ membrane effects phospho/ diesterase 5-AMP protein synthesis Fig. 1
)
cytoplasm
jr activation ~ inhibition
Schematic diagram of the mechanism of action of peptide hormones acting at s p e c i f i c receptor sites on the outer surface of the plasma membrane of the target c e l l .
This enzyme catalyses the formation of cyclic-AMP from ATP which in turn acts as an a l l o s t e r i c modulator of specific cytoplasmic enzymes.
FSH bindinq to the
plasma membrane of S e r t o l i c e l l s , for example, culminates in a v a r i e t y of c e l l u l a r changes including an elevation in the rate of protein biosynthesis mediated by c-AMP binding proteins and protein kinases.
Analysis of the protein products of
Sertoli c e l l s following FSH stimulation has so far f a i l e d to reveal any selective
Biochemical and Immunological Prospects for Male Contraception
231
synthesis of particular proteins and the effect on protein biosynthesis appears to be of a general nature. production induced by LH.
FHS also exerts a potentiating effect on androgen Binding of LH (or hCG) to the plasma membrane of
Leydig cells ultimately results in release of free cholesterol from an acyl ester located in i n t r a c e l l u l a r fat droplets.
This cholesterol then represents the
precursor of steroidonenesis (Gnodde et a t . , 1979), a pathway that is also believed to be i n d i r e c t l y modulated by synthesis of key regulator enzymes via an increased rate of mRNA transcription stimulated by the action of protein kinases (Rommerts e t a l . ,
1974).
The action of cAMP as a second messenger in these two examples leads to diverse responses in the two d i s t i n c t sets of target cells but i t s role in the p i t u i t a r y is less clear.
For example, both LH and FSH synthesis and release from the
p i t u i t a r y are stimulated by the action of LH-RH presumably by the second messenger cyclic-AHP but the plasma concentrations of each of the gonadotrophins is known to vary under d i f f e r e n t circumstances.
Certainly differences in
response of the p i t u i t a r y to LH-RH are clearly apparent between males and females. I t was o r i g i n a l l y thought that secretion of the nonadotrophins were independently controlled by two hynothalamic releasinn factors acting on two separate 9roups of pituitary cells, one secreting LH and the other FSH.
Studies of Schally et a l . ,
(1971) have tended to support the idea that LH-RH is the only hormone involved and that this stimulates release of LH and FSH from a sinmle pituitary cell type (Tongard et a l . , 1971).
To account for varying levels of the two gonadotrophins
in different stages of sexual maturity and in men with certain spermatonenic defects i t must be assumed that the rate of synthesis of the two hormones within the pituitary cell is independently controlled.
This would result in storane
of differing amounts of hormone in secretory vesicles or selective release of one
or other gonadotrophin upon stimulation with LH-RH.
The concept of a single
hormone responsible for triggering release of two separate products from a target cell other than in tandem i s , however, not completely satisfying. efforts continue to explore the original
Experimental
theory postulating the existence of
separate peptide hormones in the hypothalamus responsible for the release of FSH and LH respectively from the p i t u i t a r y but these have not yet proved successful. The p i t u i t a r i e s of males and females are known to d i f f e r in t h e i r response to LH-RH. This differenceisbelieved to be due to the particular hormonal environments which prevail during development of the brains of each sex (Gorski, 1971). Androgens and oestrogens, for example, are believed to play a direct role in the development of sexually dimorphic characteristics of the brain (Barraclough, 1968) and there is convincing evidence for the presence of both types of steroid
232
P.J. Ouinn
receptors in the hypothalamus of neonatal and prepubertal mice (Fox, 1975; Fox et a l . , 197R). More recently, the existence of androgen receptors has been established throughout the, so-called, c r i t i c a l period of brain sexual differentiation in mice using DNA-cellulose a f f i n i t y chromatrgraphy and velocity sedimentation and by analysis of the androgen-resistant mutant, testicular feminisation (Vito et at., 1979).
The role of steroids has been simply demon-
strated by exposure of the central nervous system of neonatal rats to testosterone. This results in a loss of the capacity of cyclic release of LH in the adult rat but does not affect tonic patterns of hormone secretion (Gorski, Iq68).
Using
sensitive radioimmunoassay methods, Tang (1978) observed the sex differences in synthesis and release of pituitary LH in an in v i t r o system.
I t was found
that LH-RH stimulated release of a siqnificantly greater proportion of total pituitary LH in female glands consistent with the essential mechanism required for the preovulatory LH surge at mid-cycle, whereas a lower response in the male system is geared to the tonic secretion of LH from male pituitary. The most convincing evidence that peptide hormones mediate their effects within the target cell exclusively by binding to a cell surface receptor has been derived by studies of prolactin.
Shiu and Friesen (1976) have succeeded in
purifying the membrane receptors for prolactin by a f f i n i t y chromatography and these were used as antigen to raise specific antibodies to the receptor site. Addition of these antibodies, which are obviously too large to pass across cell membranes, to mammary explants prevented binding of 1251-prolactin to membrane receptors and prolactin-mediated 2.1.2.
incorporation of radiolabelled leucine into casein
Steroid hormones
Unlike the peptide hormones, steroids penetrate cell membranes by a diffusion process and interact with specific receptor molecules located in the cell cytoplasm Despite the fact that some steroid hormone-induced responses can be mediated by effects on cell membranes, such as the well known oestrogen-induced permeability change in uterine cells, the primary mechanism of action is believed to be genome-mediated. The proposed mode of action of steroid hormones is shown diagrammatically in Fig. 2. The process appears to involve the migration of the steroid hormone-receptor protein complex into the cell nucleus where i t interacts with the non-histone acidic proteins associated with the chromosomal DNA.
The
interaction activates the transcriptional apparatus leading to the synthesis of new messenger RNA as well as ribosomal RNA and transfer RNA (O'Malley and Means, 1974).
The RNA then migrates to the cytoplasm where i t directs the translation
of proteins responsible for bringing about the altered function of the target cell.
Biochemical and Immunological Prospects for Male Contraception
testosteron~ e.~
OH
.~hesis / /
{j/
Fig. 2
~
j
/
"
Suggested mechanism of action of steroid hormones. The l i p o p h i l i c character of these hormones enables them to penetrate biological membranes and interact before or after enzymic modification with specific receptor proteins. The binding causes a conformational change in the protein which enters the nucleus and stimulates RNA synthesis. The RNA may be modified by interaction with the steroidcomplex when i t enters the cytoplasm and acts as a template for translation of specific proteins.
233
234
P.J. Quinn
Some steroid hormones l i k e oestradiol and progesterone bind to t h e i r respective receptor proteins in the target c e l l s in a chemically unmodified form.
Testosterone,
however, can be regarded as a prehormone since i t is the binding of a metabolite of testosterone, dihydrotestosterone, which represents the active receptor-protein binding complex.
The conversion of testosterone to dihydrotestosterone in the
target cell is mediated by a s p e c i f i c 5~-reductase enzyme.
Testosterone can also
be converted to oestrogens by both hypothalamus and n e r i f e r a l
f a t tissues where
the product binds to specific oestrogen receptors to modulate events in the cell nucleus.
2.2. Hormonal Regulation of Male Reproduction 2.2.1.
Lutenising Hormone - Releasing Hormone
The synthesis and release of gonadotrophins is u l t i m a t e l y regulated by the central nervous system and biogenic amines play an essential role in the a c t i v i t y of the hypothalamus-hypophysis axis.
A number of studies have shown that LH-RH is
located in neurons within the classical
hypophysiotrophic area of the medial basal
hypothalamus as well as in the medial preoptic area and septum (Barry e£ a t . , Barry and Dubois, 1976; and Silverman et a t . ,
Silverman and Zimmerman, 1978).
1973;
Krey and Silverman (1978)
(1979) have recently assessed the contribution of each of
these cell groups to LH-RH terminals in the median eminence of guinea pig brain usin~ localized lesion techniques.
They found that only destruction of the
arcuate nucleus resulted in diminution
of LH and testosterone levels in the plasma
of male guinea pigs, suggesting that this was the major but not necessarily the only source of LH-RH terminals in the median eminence.
These nerve terminals of
the hypothalamus which are under adrenergic control are believed to regulate the tonic secretion of gonadotrophin.
The role of LH-RH-containing neurons which
project into regions other than the median eminence is unknown but some function in the regulation of sexual behaviour cannot be excluded. As noted above the action of LH-RH on the p i t u i t a r y d i f f e r s in males and females probably as a consequence of the p a r t i c u l a r hormonal environments in each sex during development.
LH-RH, however, seems to influence t e s t i c u l a r function
d i r e c t l y and not e x c l u s i v e l y via stimulating release of gonadotrophin. A profusion of studies have been conducted to synthesize and examine the e f f e c t s on male reproductive capacity of agonists and antagonists of LH-RH following the original
observation of Vale et a t . ,
(1972) who showed that {des His 2) LH-RH is a
potent antagonist of LH-PH (Humphries et a t . , Moersch e t a t . ,
1979;
Schally et a t . ,
Ig76).
1978, 1979; Seprodi e t a t . ,
Ig78a;
The hormone is a decapeptide (see
Table I) featuring a type 116 bend hinged around a glycine residue at position 6
235
Biochemical and Immunological Prospects for Male Contraception TABLE 1
A n t a g o n i s t analogues of LH-~H
Amino 1
2
3
4
acid
5
sequence 6
7
8
9
I0
LH-RH (PyroGlu-
His
-
Trp
Ser -
Tyr -
Gly -
Leu
-
D-Phe-
Phe
D-Phe
-
-
D-Phe
-
D-Leu
-
-
D-Phe
-
D-AIa
-
-
D-Phe-
Pro
D-Trp
-
-
D-Phe-
Leu
D-Trp
-
-
D-Phe- DTrp
D-Phe
-
Arg -
Pro
Gly - HN2)
Antagonist
(I)
de la Cruz e t a l . ,
(2)
V i l c h e z - M a r t i n e z et a l . ,
(3)
Corbin and B e a t t i e (1975)
(4)
Humphries et a l . ,
(5)
Pedroza e t a l . ,
(I) (2) (3) (4) (4) (5)
(1976) (1976)
(1976) (1978)
which i s r e s p o n s i b l e f o r m a i n t a i n i n n the peptide i n an a c t i v e conformation.
The
bend can be c o n s i d e r a b l y s t a b i l i z e d by s u b s t i t u t i n ~ O-amino acids at 9 o s i t i o n 6, particularly
those w i t h bulky side chains.
Such analogues are found to possess
increased potency of gonadotrophin r e l e a s i n g c a g a c i t y . compared the b i o l o g i c a l
activity
Seprodi e t a l . ,
(Ig78b)
o f two l i n e a r LH-RH analogues w i t h t h e i r c y c l i c
c o u n t e r p a r t s and concluded t h a t the r e c e n t o r b i n d i n g conformation of the hormone consisted of a U shaped peptide i n which the C and N t e r m i n i were located in close p r o x i m i t y to each o t h e r . A selected nroup of s y n t h e t i c analogues o f LH-RH, a l l w i t h a n t a g o n i s t a c t i o n , i s presented i n Table I .
S u b s t i t u t i o n o f D-Phe at p o s i t i o n 2 appears to be an
important f a c t o r in antagonism o f LH-RH since replacement w i t h Phe rendered the analogue i n e f f e c t i v e .
The main s i t e o f a c t i o n of these 9eptides appears to be
at the l e v e l of the p i t u i t a r y the t e s t i s be e f f e c t i v e
(see below).
but there i s some evidence f o r a d i r e c t a c t i o n on
For example, some of these pegtides have been shown to
i n p r e v e n t i n g LH release i n men f o l l o w i n g s t i m u l a t i o n by LH-~H
236
P.J. Quinn
(Gonzalez-Barcena et a t . , 1977).
The contraceptive applications of these
peptides, however, may reside in their agonist rather than their antagonist action (Corbin et a t . , 1978).
The effects of long term treatment of male rats with
potent LH-RH agonists has been reported by Sandow et ai.,(1977) and Tcholakian et at.,
(1978).
Both groups reported that LH and FSH levels rise but serum testo-
sterone concentration
contrary to expectations, decrease markedly desnite the
absence of obvious changes in Leydig cell morphology.
The other most notable
feature of chronic LH-RH treatment is a marked decrease in size of the male accessory glands, particularly the seminal vesicles and ventral prostate, as well as the weight of the testes (Vale et a t . , 1977).
I t was suqnested that the
decrease in testosterone levels could be due to either a suppression of certain key steroidogenic enzymes responsible for testosterone biosynthesis or decreased numbers of LH/hCG receptors in the testis.
T h i s process has been considered as
a "desensitization" of the testis to LH following exposure to high levels of LH supported by pharmacological doses of LH-RH (Hsueh et a l . , Ig77).
The possibility
that large chronic doses of LH-RH render the pituitary refractory to further stimulation by LH-RH with a consequent decrease in serum gonadotrophins and atrophy of male reproductive organs has also been considered (Belchez et a t . , 1978; Sandow e t a t . , 1978).
Evidence has recently been presented (Hsueh and Erickson,
1979) to show that LH-RH acts directly on the Leydig cells of the testis to cause a decrease in testicular LH/hCG receptors and testicular steroidogenesis.
Irres-
pective of the precise mode of action of LH-RH analogues the compounds appear to offer, in pharmacological doses, an effective means of regulating male f e r t i l i t y . Another possible site of regulation of LH-RH action is at site of synthesis and storage of the hormone in the hypothalamus.
Active peptidases present in the
hypothalamus have been shown to rapidly degrade LH-RH (Griffiths et a l . , 1974, 1975; Swift and Crighton, 1978, 1979a).
The enzymes appear to be stimulated by
repeated injection of oestradiol and progesterone in the rat (Griffiths and Hooper, 1973).
The action of these steroids is apparently to bind to the
enzymes and thereby to exert a positive allosteric modulation of their activity or alternatively to remove some allosteric inhibitor (Swift and Crighton, 1979b). 2.2.2.
Pituitary Gonadotrophins
Hypophysectomy in men leads to testicular regression which can be restored to some extent by administration of hCG or human menopausal gonadotrophin (MacLeod, 1970). This observation emphasises the degree to which testicular function is controlled by the pituitary.
Russell and Clermont (1977) examined the degenerative changes
in germ cells of the rat during maturation of spermatozoa and compared these with changes associated with hypophysectomy.
~lormally a proportion of germ cells
237
Biochemical and Immunological Prospects for Male Contraception
of mature male rats degenerate at several stages during spermatogenesis includinq Type A spermatogonia, midpachytene spermatocytes, primary and secondary spermatocytes which are particularly vulnerable during their respective maturation divisions and mid and terminal stages of spermiogenesis.
Hypophysectomyappears
to cause a marked increase in degeneration of midpachytene spermatocytes and spermatids in the middle and terminal stages without notably affecting loss of cells at other stages of development.
Administration of FSH does not appear to
influence the course of events associated with hypophysectomy but LH, especially when augmented by FSH, reduces the number of degenerating cells to near normal values. Testicular function is also suppressed by active or passive immunization against gonadotrophins.
Antibodies against LH, for example, cause a reduction in testis
weight in the rat and a reduction and disturbance of spermatogenesis.
Changes in
Sertoli and Leydig cell functions are observed (Madhwa Raj and Dym, 1976) and spermatozoa develop abnormally (Talaat and Laurence, 1971).
In rabbits, maturation
of the gonads is prevented when immature animals are immunized against LH and spermatogonia
arrested at the spermatonia stage (Monastirsky et at., 1971).
Secretion of gonadotrophins is regulated by testosterone in a feedback control process. A diagrammatic representation of feedback control of pituitary gonadotrophins is illustrated in Fig. 3, where the effect of sex steroids on FSH and LH release from the anterior pituitary are summarised.
I t can be seen that while
oestrogens exert stimulatory effects on the release of both gonadotrophins at the pituitary level, androgens inhibit LH but stimulate FSH release.
Studies of
pituitary cells in culture (Drouin and Labrie, 1976) have suggested that androgens have not only specific but in certain circumstances an opposite effect at the pituitary level on the control of FSH and LH secretion.
Thus treatment of cells
with testosterone markedly inhibits LH response to LH-RH but stimulates FSH release.
The feedback control process is best exemplified by studies involvinq
active immunization of animals against testosterone.
Active immunization of
male rabbits, rats and monkeys against testosterone can produce UD to a lO0-fold increase in the total serum testosterone concentration.
The increase in testo-
sterone is accompanied by a parallel increase in serum antibody t i t r e un to about 12 weeks whereupon the steroid level reaches a constant value ( H i l l i e r et a t . , 1975). High t i t r e s of anti testosterone antibodies can also be provoked in castrated individuals but no elevation in total serum testosterone is observed confirming that the steroid is of testicular origin (Wickings, e t a t . , 1977). Although total serum testosterone increases most of this is complexed to antibody and, in fact, the amount of free testosterone decreases slightly in actively
238
P. J, Quinn
immunized animals (Hickings and K'ieschlag, 1978).
Brain
Hypothalamus
+1
I+ -
,
-
.
'
"
/
+
Oestrogens
Anterior pituitary
Gonads
Progestins Fig. 3
The effect of sex steroids on FSH and LH secretion from the anterior p i t u i t a r y .
239
Biochemical and Immunological Prospects for Male Contraception Hyperplasia of the Leydig cell population is a typical
histological
feature of
immunization and is similar to the e f f e c t s observed following hCG administration and blockage of androgen-sensitive receptors of the hypothalamus and p i t u i t a r y by drugs such as cyproterone (Heinert and Taubert, 197R).
As a d i r e c t consequence
of decreased unbound testosterone concentration in the serum, there is an increase in gonadotrophin l e v e l s ; following castration.
the increase, however, is not as marked as that observed Subsequent saturation of antibody-bindinq sites with
exogeneously-administered testosterone can restore LH levels to normal values but, contrary to expectation, FSH levels are unaffected by this procedure (Nieschlag et a t . ,
1975) suggesting that the feedback control of the two 9onadotrophins are
d i f f e r e n t in the way they respond to c i r c u l a t i n g free testosterone. As far as the contraceptive potential of immunization against testosterone is concerned there seems l i t t l e
scope.
Spermatogenesis, f o r example, does not
appear to be severely impaired by the reduction in free testosterone concentrations, c e r t a i n l y at the levels that can be achieved by active immunization against the steroid.
This might not be unexpected i f testosterone produced l o c a l l y in the
t e s t i s passes d i r e c t l y to the tubules and is not complexed to c i r c u l a t i n g a n t i testosterone antibodies of the serum which presumably do not penetrate the bloodtestis barrier.
Another serious manifestation of active immunization against
testosterone is the marked loss of l i b i d o which f u r t h e r detracts from this approach to contraception. Several drugs are known to suppress p i t u i t a r y function in men and some have been examined as potential contraceptive agents.
Medroxyprogesterone acetate, for
example, is a drug with potent long-acting gestogenic effects but l i k e other s i m i l a r drugs i t s administration is associated with some loss of l i b i d o Kretser, 1976).
(de
Loss of l i b i d o can be counteracted to some extent by combininq
gestogen administration with implants of long-acting testosterone analonues l i k e testosterone oenanthate.
Temporary suppression of spermatoqenesis without loss
of l i b i d o has been claimed from a number of c l i n i c a l combinations (Coutinho and Melo, 1973; et at.,
1978).
Moltz et a t . ,
gestogen, cyproterone acetate.
Frick, 1973;
trials
of androgen/gestogen
de Kretser, 1976; Neumann
(1978) have reported a detailed study of the The drug was found to cause a r e v e r s i b l e o l i q o -
spermia associated with an increase in the proportion of abnormal spermatozoa and a decrease in cell m o t i l i t y which e f f e c t i v e l y reduced the a b i l i t y of spermatozoa to penetrate ovulatory cervical mucus (Kremer t e s t ) .
No loss in l i b i d o was
observed despite a marked reduction in plasma testosterone concentrations which was restored, together with t e s t i c u l a r function, within I? weeks of withdrawal of the drug.
Large-scale c l i n i c a l
trials
of the drug under the auspices of the
P.J. Quinn
240
World Health Organization (7th Annual Report, 1978), however, led to the conclusion that cyproterone acetate has only limited contraceptive potential.
One
reason was that azoospermia is considered a necessary criterion for an effective contraceptive procedure. Drugs that suppress FSH and LH action on the gonads, thereby alterinn gonadal function, have also been investigated.
The contraceptive properties of one such
drug, danazol, an alkylated steroid (see Fin. 4) has been reviewed recently by r)mowski (1979).
Administration of danazol together with testosterone has been
found to cause severe oligospermia or complete azoospermia without reducing libido (Skoglund and Paulsen, 1973; Ulstein e t a t . ,
1975; Paulsen and Leonard, 1976). OH
~~.,i
Fig. 4
c-~CH
Structure of danazol.
A dose of 600 mg of danazol daily together with 200 mg of testosterone oenanthate given intramuscularly once a month was considered a useful contraceptive regimen and in normal men testicular function could be restored to pretreatment levels within 4 months of withdrawal from the drugs. 2.2.3.
Inhibin
Selective destruction of the germinal epithelium by radiation, drugs, testicular hyperthermy and ischemia and immunological aspermatogenesis has often led to elevation of FSH compared to serum LH, suggesting that production of another factor by cells of this tissue acts by a negative feedback process to regulate p i t u i t a r y FSH secretion (Setchell et a l . , 1977). Present evidence suggests that a protein of molecular weight about I0,000 produced by the Sertoli cells, referred to as inhibin, selectively suppresses FSH secretion by p i t u i t a r y cells in tissue culture (Steinberger and Steinberger, 1976) and offers contraceptive potential (Franchimont et a t . ,
1978).
Thus the development of synthetic analogues
of inhibin may suppress spermatogenesis without causing loss of libido (Davies et at.,
1978).
Our present state of knowledge of knowledge of the mechanism of hormonal regulation
Biochemical and Immunological Prospectsfor Male Contraception
241
of male reproduction does not yet provide a s a t i s f a c t o r y means of suppressing spermatogenesis while at the same time maintainin~ l i b i d o .
One of the main
reasons for this is the apparent close interdeDendence of LH and FSH release from the p i t u i t a r y under the influence of LH-RH on the one hand and feedback control by t e s t o s t e r o n e / i n h i b i n on the other.
Nevertheless, the differences
occasionally observed in LH and FSH release are s u f f i c i e n t l y encouraging to maintain current i n t e r e s t in i d e n t i f y i n g separate releasing hormones or factors f o r FSH as well as LH.
Independent regulation of these two gonadotrophins could
prove a useful approach to the development of male hormonal contraceptives,
Chapter 3
Regulation of Testicular Function and Sperm Maturation
Spermatogenesis involves a sequence of events including mitosis, meiosis and morphological changes which culminate in the d i f f e r e n t i a t i o n of highly specialized spermatozoa from the early spermatid progenitor.
Morpholooical features of the
process have been well documented by electron microscopic studies (de Kretser, 1969) and are i l l u s t r a t e d
in Fiq. 5.
Briefly,
the spermatogonia undergo m i t o t i c
d i v i s i o n s to provide groups of primary spermatogonia destined f o r f u r t h e r differentiation
and represent the immediate precursors of snermatocytes.
These
primary spermatocytes subsequently undergo a meiotic d i v i s i o n to form secondary spermatocytes followed by another d i v i s i o n , chromosome number by half.
thereby completing the reduction in
The haploid c e l l s represent the spermatids.
The
nuclei of the spermatids condense and occupy an eccentric position adjacent to the plasma membrane.
The acrosome is d i f f e r e n t i a t e d from elements of the Golgi
complex which become closely applied to the nucleus where i t approaches the plasma membrane.
The axial filament of the t a i l
and develops at the base of the nucleus.
arises from one of the c e n t r i o l e s
The cytoplasm condenses and is
e s s e n t i a l l y extruded from the base of the cell as the so-called residual body or cytoplasmic droplet.
During this process the mitochondria arrange around the
the proximal portion of the t a i l .
At this stage the now mature spermatozoa are
released from the Sertoli c e l l s into the lumen of the seminiferous tubules. Spermatogenesis is a continuous process in sexually mature males and i n t e r r u p t i o n at any stage reduces the rate of gamete production.
E f f e c t i v e contraceptive
intervention in the production of spermatozoa by the t e s t i s aims to eliminate spermatozoa from the ejaculate in such a way that the concentration of c e l l s can be r a p i d l y restored when treatment is discontinued.
By rapid, of course, one
must recognise that, in man, the production of mature spermatozoa requires period approaching 75 days from the nerminal cell stage.
242
Of p a r t i c u l a r concern
Biochemicaland ImmunologicalProspectsfor MaleContraception
243
in this approach, however, is the need to prevent the production of g e n e t i c a l l y abnormal spermatozoa.
spermatogonia I
proliferation
primary I spermatocyte
growth
secondary spermatocyte
maturation
/\
spermatid
1
I
1
transformation
spermatozoon
Fig. 5
Schematic representation of the process of spermatoqenesis
3.1. Inte~erenceof Spermatogenesisby ChemicalAgents A group of compounds that were o r i g i n a l l y developed as anti-cancer agents and bactericides, the n i t r o f u r a n s , were subsequently found to be e f f e c t i v e in r e v e r s i b l y blocking spermatogenesis at the level of the seminiferous tubules independently of the pituitary-gonadal
axis (~!elson et a t . ,
1954).
Hhen adminis-
tered in doses required to suppress sperm counts in men, however, there were marked g a s t r o i n t e s t i n a l
side e f f e c t s that eliminated these anents as e f f e c t i v e
contraceptives (Nelson and Bunge, I~57).
Likewise, diamine compounds are known
244
P.J. Quinn
to reversibly interfere with the function of seminiferous tubules and to lower sperm counts in a number of mammalian species, including primates, with no obvious effects on libido (Drobeck and Koulston, 1962) hut they also act as potent inhibitors of l i v e r enzymes responsible for drug metabolism (Heller et a t . , 1961). Selective damage to spermatogenesis by nitrofurans and manganese, which primarily injure spermatocytes and spermatids, at doses that have no obvious effects on other organs are l i k e l y to exert their effects by an interference with metabolic processes (Paul e t a l . ,
1953;
llematsu, 1966;
Husain e t a l . ,
1976).
Claims have
also been made that a l k y l a t i n g agents and related druQs used to t r e a t neoplasia (Fairley et a l . ,
IQ72;
Hinkes and P l o t k i n , I~73;
Sherins and ne Vita, 197~)
r e v e r s i b l y i n h i b i t spermatogenesis in men but t h e i r potential and usefulness as contraceptive agents have yet to be f u l l y evaluated.
In some experiments,
however, treatment of male mice with the a l k y l a t i n g agent triethylenemelamine (TEM) at a dose of N.Rmg/kg caused developmental retardation of embryos f e r t i l i z e d by defective spermatozoa ejaculated 20 days a f t e r i n t r a p e r i t o n e a l (Hitotsumachi and Kikuchi, 1977).
injection
Chromosomal aberrations such as breaks,
exchanges and premature chromosome condensations give rise to a high incidence of dominant l e t h a l i t y amongst affected embryos especially during the early, preimplantation stages of embryogenesis. damage may remain undetected u n t i l Sheridan, 1978).
Nevertheless TEM-induced chromosomal II
.
l a t e r stages of development (Burkl and
On this basis at least a l k y l a t i n g agents would seem to possess
limited contraceptive p o t e n t i a l . demonstrated a n t i f e r t i l i t y
The structures of some reaoents that have
e f f e c t s in shown in Fig. 6.
Certain metabolic i n h i b i t o r s appear to have selective effects on spermatogenesis. Fluoroacetate, f o r example, which i n h i b i t s the t r i c a r b o x y l i c acid cycle by conversion to f l u o r o c i t r a t e and blocking the enzyme cis-aconitase (Peters, 1952) has been examined in this regard.
The accumulation of c i t r a t e ,
reduction in ATP
concentration and effects of fluoroacetate on other substrate levels in the rat t e s t i s has been reported and found to be associated with tubular degeneration within the organ (Mazzanti et a l . ,
In65).
In a comparative study of the e f f e c t
of chronic exposure of rats to sublethal doses of fluoroacetate on the kidney, l i v e r and t e s t i s i t was concluded that the t e s t i s was the most vulnerable organ e x h i b i t i n g a s i g n i f i c a n t reduction in weight with a marked accumulation of c i t r a t e combined with decreased ATP levels.
~!o such changes were observed in the
other organs at comparable dosages of fluoroacetate.
Other studies have shown
that the p a r t i c u l a r lesion involves loss of t e s t i c u l a r c e l l u l a r elements and decrease of tubular volume resulting from t e s t i c u l a r atrophy with damage to the seminiferous epithelium but not Leydig cells (~azzanti et a l . , 1965).
High doses
of fluoroacetamide (Steinberger and Sud, 1970) and N-methyl-N(l-naphthyl)-fluoro
245
Biochemical and Immunological Prospects for Male Contraception acetamide (Hashimoto et a t . , 1968) cause similar histological
changes in rat
testes. In some instances, withdrawal of these drugs administered at r e l a t i v e l y low dose regimens results in some regeneration of the seminiferous tubules and the development of spermatids and spermatozoa in some tubules but not where residual damage to the tubules remains (Sullivan
a l . , 197g).
fluoroacetyl CoA
CoASH A.
et
FCH2--CO 2
FCH 2 - C - - SCoA
fluoroacetate
O
II
-FC H - - C - - SCoA ""
II
FCH--CO2H
O
I
HO--C-- C02H
I
÷
O=C--CO2H oxaloacetate I H2C--CO2H
citrate synthetase
4-
CH3--C--SCoA -
- -HCH--C--SCoA
II
""
O
I N O - - C -- C 02H
I CH2--CO2H
F C H 2 - C -- N H 2
II fluoroacetamide FCH2--C--N --OH 3
N - methyl - N ( I- naphthyl) fluoroacetamide
Fig. 6
CH2--CO 2
II
0
C,
fluorocitrate
O
acetyl CoA
B.
CH2-- C 0 2 H
The structure and site of action of metabolic i n h i b i t o r s which have a n t i f e r t i l i t y actions in males.
246
P . J . Quinn
Other metabolic i n h i b i t o r s such as thioqlucose (5-thio-D-qlucose), appear to i n h i b i t normal spermatogenesis without a f f e c t i n a Leydiq or S e r t o l i - c e l l of the t e s t i s or decreasina l i b i d o (Zysk e t a l . , High doses of the drug, ~-chlorohydrin, sterility
1975;
populations
Makamura and Hall, Ig77).
have been shown to cause permanent
(Kalla, IQ76) associated with occlusion of the epididymal duct and
subsequent degeneration of the germinal epithelium.
An e f f e c t of ~-chlorohydrin
on t e s t i c u l a r vascular has been suggested as the cause of these changes (Reijonen e t a l . ,
1975).
This is supported by the f a c t that vasoconstrictors
such as noradrenalin potentiate the lesion caused by a larae acute dose of the drug (30mg/kg) in rats whereas the vasodilator, sodium n i t r i t e ,
administered
before, but not a f t e r , a dose of 9hmg/kq ~-chlorohydrin prevented the t e s t i c u l a r epididymal lesion (Kalla and Sinah, 1979).
111trastructural chanaes of the t e s t i s
associated with administration of ~-chlorohydrin in amounts areater than 50mg/kg/ day to adult male rats have been systematically studied by James et a i . , ( 1 9 7 8 ) .
They
concluded that the gonads were the principal organ affected by the drug and that the main morphological changes were the atrophy and/or necrosis of the epididymus and seminiferous epithelium.
An increase in kidney and l i v e r weight did not
appear to be associated with any changes in biochemical functions in contrast to the reproductive system.
Thus, biochemical changes in the epidymus of the rat
especially in the biosynthesis and metabolism of l i p i d s metabolism of t e s t i c u l a r (Edwards et a l . , (!!ohri e t a l . ,
1975) and epididymal spermatozoa
1975) together with changes in the epididymal vasculature
(Reijonen et a l . , antifertility
(Voqlmayr, IO7~) and
1975) appear to underly many of the morphological changes.
action of this drug at the level of spermatozoan metabolism w i l l
The be
considered separately in Section 4.2.3. Other miscellaneous agents l i k e ethionine have also been shown to damage the seminiferous epithelium in a manner resembling gonadotrophin deprivation but the endocrine effects are uncertain (Dunn e t a l . ,
1962).
tadmium salts are known to
damage t e s t i c u l a r and epididymal vasculature but the a n t i f e r t i l i t y
effects
associated with cadmium may r e s u l t from a d i r e c t e f f e c t of the metal ion on isoenzymes of t e s t i c u l a r carbonic anhydrase (Hodgen et a l . , Another recent compound shown to possess a n t i f e r t i l i t y
1970). effects is gossypol, a
yellowish phenolic compound isolated from the seeds, stems and roots of the cotton plant.
Gossypol has been shown to i n h i b i t the f e r t i l i t y
of male rats (see
report of the National Coordinating Group on Male A n t i f e r t i l i t y Ultrastructural
Agents, 1978).
studies have shown that the spermatids are most sensitive to the
drug but with higher doses and prolonged exposure the spermatocytes are also damaged.
Examination of the epididymis revealed e x f o l i a t e d spermatids
247
Biochemical and Immunological Prospects for Male Contraception and spermocytes with numerous dead spermatozoa often with heads separated from tails.
Examination under the electron microscope showed structural
in the acrosomal membranes and mitochondria.
results in a gradual decrease in sperm count u n t i l trial
alterations
Continual exposure to gossypol azoospermia.
In a c l i n i c a l
involving 4~00 healthy male subjects receiving gossypol for a period of more
than six months, the a n t i f e r t i l i t y
e f f e c t evaluated by semen examination showed
the drug to be almost completely e f f e c t i v e .
A low incidence of mild side effects
were reported including the development of hypokalemia but whether this was related to gossypol treatment was uncertain.
Clearly gossypol shows promise as an
e f f e c t i v e male contraceptive and the results of f u r t h e r and more comnrehensive clinical
trials
Hale i n f e r t i l i t y
presently being undertaken are awaited with considerable i n t e r e s t . caused by exposure to certain chemicals in i n d u s t r i a l
have been described.
situations
Thus, azoospermia has been linked with chronic exposure
to reagents l i k e 1,2-dibromo-3-chloropentane which has long been used as an agricultural
nematocide.
The e f f e c t of the drug on suppressing spermatogenesis
has been examined by Potashnik et a l . ,
(1979) who found a selective atrophy of the
germinal epithelium in t e s t i c u l a r biopsy material characterised by i n t a c t Sertoli c e l l s and an increased number of morphologically-normal
Leydig c e l l s .
Hormone
studies showed elevated FSH levels with normal plasma LH and testosterone concentrations.
The use as a male a n t i f e r t i l i t y
agent, however, is precluded
because of the potent carcinogeneicity of the reagent.
3.2. Effects of Temperature and Irradiation I t has lonq been recognised that heating the t e s t i s to temperatures greater than 37°C causes atrophy of the tissues and function is lost.
Not all c e l l s are
affected to the same extent since primary spermatocytes during Dachytene and young spermatids appear to be the most sensitive c e l l s to elevated temperatures. The notion that c r i t i c a l
periods e x i s t in spermatogenesis such as between lepto-
tene and pachytene, another during the maturation d i v i s i o n and a third between the cap-phase and acrosome-phase spermatids, has been proposed by Collins and Lacy (1969) based on experiments involving the exposure of rat testes to a temperature of 43°C for various i n t e r v a l s .
Although the kinetics of spermato-
genesis are not affected by b r i e f periods of heating, populations sensitive to high temperature are depleted and are not replenished u n t i l development of new populations takes place followinQ temperature elevation. The mechanism of thermal s e n s i t i v i t y is unknown but i t has been suggested that increased metabolic a c t i v i t y induced at higher temperatures is not supported by a corresponding supply of oxygen and nutrients from the p e r i f e r a l blood (Setchell, 1978). role of glucose appears to be important because spermatids are remarkably
The
248
P.J. Quinn
dependent on g l y c o ] y t i c metabolism.
Whether g]yco]ysis is required to supply
the energy needed for protein synthesis is unknown but this process is markedly affected by temperature. Protein synthesis, for example, is known to be slower at body temperature (37°C) compared to lower temperatures of the scrotum.
This difference appears to be
largely due to the spermatids (Nakamura and Hail, ]978) since subce]]u]ar systems prepared from these ceils incorporate less amino acid into protein at 37°C compared with 34°C.
Other studies by these authors have shown that the most
temperature sensitive component of the protein synthesizing machine of spermatids are the ribosomes since incubation of a c e i l - f r e e system at temperatures greater than 34°C caused a dissociation of po]ysomes to monosomes.
The effects on polE-
ribosome dissociation could be due to, amongst other things, the reduced synthesis of mRNA strands suitable for translation, f a i l u r e of the i n i t i a t i o n
process or a
change in the s t a b i l i t y of rRNA. As with temperature, ionizing radiations such as X-rays cause selective damage to t e s t i c u l a r tissues.
Type A spermatogonia appear to be the most sensitive to
radiation damage and intermediate and type B spermatogonia are less sensitive. Higher doses are successively lethal to other germinal cells roughly in the order spermatids, spermatocytes and l a s t l y spermatozoa.
Some c e l l s , on the other hand,
are not as severely affected by X-radiation including the peritubular myoid c e l l s , while Sertoli cells are only affected at high doses p a r t i c u l a r l y in immature individuals (Erickson, 1976). Damage caused by ionizing radiations appears to be confined to chromosomal disruption and as a potential contraceptive method i t has severe l i m i t a t i o n s because of the p o s s i b i l i t y of congenital abnormalities.
The incidence of aneuploidy in
surviving human children, for example, is about 0.5% (Nielsen and Sillesen, 1975) although a frequency of 5-10% of aneuploid zygotes at conception has been estimated (Hansmann and Probeck, 1979). disjunction in mammalian spermatogenesis
X - i r r a d i a t i o n appears to induce nonbut an increase in the incidence of
aneuploidies among the F1 progeny is problematical The frequency of X-irradiation-induced
(Szemere and Chandley, 1975).
non-disjunction of sperm chromosomes by
examination of the progeny conceived by irradiated animals, however, tends to give a distorted picture of the total chromosomal aberrations because most irradiated spermatocytes do not mature into spermatozoa.
Many degenerate even after rela-
t i v e l y low doses of X-rays and those that do survive are a highly selected population. Furthermore, not many progeny survive to term because aneuploidy that results from loss or gain of an autosome generally results in early embryonic death.
249
Biochemical and Immunological Prospects for Male Contraception Some disturbance of endocrine function has been reported as a consequence of radiation damage to the t e s t i s .
Testosterone production, however, does not
appear to be affected by moderate doses of X-radiation but when spermatids are depleted from the germinal epithelium, serum LH and FSH become elevated, possibly due to a loss of inhibin production which helps to control p i t u i t a r y gonadotrophin secretion (Setchell e t a l . , Iq77).
Higher doses of X-radiation appear to a l t e r
steroidogenic pathways ( E l l i s , 1970).
3.3. Biochemical and Immunological Aspects of Vasectomy Vasectomy is a widely practiced method of male contraception but i t has the marked disadvantage of rendering the individual p o t e n t i a l l y i r r e v e r s i b l y s t e r i l e . Sterility, libido.
permanent or other wise, nevertheless is achieved with no loss of There are a number of interesting factors associated with b i l a t e r a l
l i g a t i o n of the vas deferencs that may provide a useful basis for the development of more satisfactory male contraceptives. The effect of vasectomy on spermatogenesis has been extensively reviewed (Neaves, 1975;
Heidger and Sawatzke, 1977) and the effect appears to d i f f e r from one
species to another. histological regard.
The effect of vasal obstruction on t e s t i s weight and
features of the seminiferous tubules has been investigated in this
In general, no marked changes in spermatogenesis are observed in the
b u l l , r a t or various primate species that can be attributed d i r e c t l y to vasectomy (Skinner and Rowson, 1968; Howards et a t . ,
1975).
P a l i f l e r and Foote, 1969;
Vare and Bansal, 1973;
Spermatogenesis in the guinea pig i s , however, severely
impaired and changes in the seminiferous epithelium of the rabbit are manifest after about 1 year.
In the dog, histological changes occur as an i n i t i a l
response to vasectomy but the tissue subsequently recovers to return to i t s normal appearance.
Certain other morphological and physiological alterations
in vasectomised individuals have also been observed (MacMillan e t a l . ,
1968;
Igboeli and Rakha, 1970) including changes in the blood-seminiferous tubule barrier that could have repercussions on spermatogenesis and/or sperm maturation (Turner e t a l . , 1979).
Biochemical studies of DNA synthesis in the t e s t i s of
guinea pigs, using t r i t i a t e d thymadine incorporation, has shown that spermatogenesis in vasectomised animals may be altered as a d i r e c t consequence of decreased t e s t i c u l a r DNA synthesis (Howards et a l . ,
1979).
The endocrine function of vasectomised men appears to be normal shortly after vasectomy (Naik e t a l . ,
1976;
Johnsonbaugh e t a l . ,
1977) as well as many years
following operation (Whitby e t a l . , 1979). However, some disturbance in the level of testosterone, dihydrotestosterone, LH, FSH and oestradiol at various intervals after vasectomy have been reported (Smith e t a l . ,
1976;
Purris e t a l . ,
250 1976;
P, J, Ouinn Kobrinsky e t a l . ,
as seriously abnormal.
1976;
Whitby e t a l . ,
1976) but none could be described
No morphological changes of Leydig cells were apparent
at the level of e i t h e r optical or electron microscopy (Kubota, 1969) and no functional
impairment could be detected (Whitby et a l . ,
I t has frequently been reported that low f e r t i l i t y vasectomy (Kar and Phadke, 1975;
Ig79).
is associated with vaso
Schmidt, 1975) and there is some evidence that
the presence of antisperm antibodies in the serum is related to this i n f e r t i l i t y . Antisperm antibodies have been detected in the sera of both sexually mature males and females.
Indeed, a high incidence of such antibodies in the sera of
subjects with otherwise unexplained i n f e r t i l i t y 1954;
Franklin and Dukes, 1964;
antibodies in causing i n f e r t i l i t y
has often been reported (Wilson,
Shulman, 1971).
The precise role of these
is, however, not known (Jones, I~77).
Tests
for detecting antisperm antibodies are f a r from completely r e l i a b l e (see WHO reference bank f o r reproductive immunology, 1977). these antibodies are i l l u s t r a t e d
in Fig. 7.
classes can be i d e n t i f i e d by t h e i r a b i l i t y
Two methods of detecting
Antibodies of the IgM and some IgG to react with antigens located on the
plasma membrane of the c e l l , to f i x complement and lyse the cell - these a n t i bodies are referred to as immobilizing antibodies.
Another method is to observe
spermagglutination which occurs when s p e c i f i c bivalent antibodies cross-link neighbouring c e l l s in a manner similar to haemagqlutination.
The d i f f i c u l t y
about the agglutination tests is that spermatozoa are prone to agglutinate under a v a r i e t y of conditions in the absence of antisperm antibody.
T h u s certain
microorganisms including viruses, some drugs and defined factors present in fresh autologous serum can agglutinate sperm (Boettcher, et a l . ,
1970;
Boettcher, 1974).
Anti-sperm antibodies, nevertheless, can be detected by macroscopic reactions but other factors present in semen are also auto antigenic. and enzymes such as hyaluronidase (Metz, e t a l . ,
Lactoferrin-type proteins
1976) as well as soluble DNA
polymers (Witkin and Higgins, 1978) are included in this category. Antigens of the ABO blood group have been i d e n t i f i e d on sperm (Edwards
et al.,
1964) as have the blood group antigens M, ~! and Tja which, unlike the ABO antigens, are absent from the seminal plasma (Edwards, 1964). Attempts to detect Rh antigens have so f a r proved unsuccessful; i f this were possible then cells from an Rh positive Dd heterozygote genotype could be separated into two populations with obvious advantages.
Histocompatibility antigens (H-2) of mouse sperm have
been observed by fluorescent antibody techniques (Barth and Russell, 1964; Goldberg e t a l . ,
1970) and some of these appear to be products of the H-2 d region
of the major h i s t o c o m p a t i b i l i t y
complex (Vojtiskova e t a l . ,
s p e c i f i c HL-A tissue typing sera and a m i c r o c y t o t o x i c i t y
196~).
The use of
test has indicated that
251
Biochemical and Immunological Prospects for Male Contraception at least some antigens determined by the HI-A locus are present in high conconcentration on human sperm (Fel]ons and Dausset, 1970).
When spermatozoa from
donors heterozygous for certain HL-A antigens were tested with monospecific a n t i serum lysis of about half of the sperm population was observed, suggesting haploid expression of the HL-A antigens.
In other experiments, antigenical]y
d i f f e r e n t types of sperm were detected in semen from a single fonor.
These
observations have yet to be s a t i s f a c t o r i l y explained.
A. acrosome antibody surface
nucleus
sperm head
| 'ane
s-linking pody
midpiece
Fig.7
Tests for serum antisperm antibodies. A. Crosslinking and binding of IgG or IgM antibodies directed against antigens present on the cell surface i n i t i a t e s the classical pathway of complement f i x a t i o n leading ultimately to lysis by terminal complement factor and loss of sperm m o t i l i t y . B. Sperm agglutinating antibodies crosslink antigens on the surface of adjacent cells.
252
P.J. Quinn
Another category of sperm antigens are those associated with subcellular constituents of the spermatozoa.
These include the nucleoprotamines which are
confined to the nuclei of spermatids and spermatozoa c . f . the nucleohistones which are widely distributed in somatic cell nuclei of animals.
They are low-
molecular-weight proteins which are synthesized during the early and middle stages of spermatid d i f f e r e n t i a t i o n when they replace the histones (Louie and Dixon, 1972) Although o r i g i n a l l y believed to be non-immunogenic (they are widely used as heparin antagonists, for example), antibodies to human protamine have recently been observed in the sera of a number of i n f e r t i l e al.,
1974; Kolk and Samuel, 1975;
and vasectomised men (Kolk et
Samuel et a l . , 1975, 1976, 1977; Samuel, 1977)
In other more recent studies, Kay and Alexander (1978) report a change in the antigenic s p e c i f i c i t y of spermatozoa that have been damaged.
T h e y subjected
spermatozoa to mild centrifugation and examined the damaged cells by immunofluorescent methods.
This indicated that new antigens were expressed when the
plasma membrane was stripped off to reveal the underlying subcellular organelles. The t a i l and principal piece region of the cells appeared to be p a r t i c u l a r l y vulnerable to damage and showed a high concentration of the newly-expressed antigens. Numerous studies performed on the serum of normal f e r t i l e men have conclusively shown that the mammalian reproductive system is remarkably unresponsive to autoantigens.
Thus spermatozoa and other products of the male genital t r a c t are
recognised as being f u l l y antigenic in animals (Katsh and Bishop, 1958) and in humans (Mancini, 1974) yet under normal conditions autoimmune responses are rarely detectable.
Similarly many subcellular organelle membranes and soluble
constituents including mitochondrial constituents are antigenic but autoimmunity to such antigens arises only in certain pathological conditions. Antibodies specific to sperm antigens in serum detected by agglutination and immobilization tests, have been associated with vasectomy of various species including men (Ansbacher, 1971, 1973, 1974;
Ansbacher et
al.,
1976;
Gupta et
at.,
1975; Tung, 1975; Linnet and Hjort, 1977; Hellema and R~mke, 1978) guinea-pigs (Alexander, 1973) rabbits (Bigazzi e t a l . , 1976 a and b) rats (R~mke and Titus, 1970; 1977).
Brannen and Coffey, 1974) and monkeys (Alexander
et
a l . , 1974;
Alexander,
I t should be noted that in men, where most of these studies have been
performed, vasectomy is not invariably associated with the presence of antisperm antibodies in the serum.
In general, only 60-70% of vasectomised men e x h i b i t
any appreciable t i t r e s of agglutinating antibodies and somewhat fewer men have sperm immobilizing antibodies.
Typical data is i l l u s t r a t e d in Table 2.
Biochemical and TABLE 2
Immunological Prospects for Male Contraception
253
Number of sera with t i t r e s of antisperm antibodies amongst f e r t i l e and b i l a t e r a l l y vasectomised males. Antisperm antibodies
Group F e r t i l e males (16)
Aqglutinating
ImmobilisinQ
none
none
9
5
Vasectomised males (12)
Numbers in parenthesis r e f e r to the t o t a l number per group In general, sperm immobilizing antibodies are only found in sera which contain sperm agglutins.
Thus in sera from 160 vasectomised men studied by Hallima and
Rumke (1978) only 5 showed sperm immobilizina antibodies, at t i t r e s below that used to detect sperm agglutinating antibodies.
Table 3 presents representative t i t r e s
of d i f f e r e n t antibodies in the serum of vasectomised men. TABLE 3
T i t r e s of sperm aaglutinins and sperm immobilizins in the sera of b i l a t e r a l l y vasectomised men.
Sample Immobilizing
Titre Aqglutinating
Tyne of , agglutination
1
2
32
T
2
0
16
T
3
4
32
T
4
8
256
T
5
0
8
T
6
0
16
7
0
8
8
0
32
T
9
4
16
H
I0
0
16
T
II
32
256
H
0
8
12 * T =
tail-to-tail;
H = head-to-head.
Many studies have established a hiah c o r r e l a t i o n between t i t r e s of sDermagglutinatinq and immobilizing antibodies. detected by an i n d i r e c t immunofluorescence
Antibodies against human protamine, t e s t on swollen sperm heads (Kolk et
254
P.J. Quinn
al.,
1974) are also associated with antibodies specific for antiaens displayed
on the sperm plasma membrane (Samuel et a l . , Ig75) and antibodies directed towards the acrosome, the principal piece and other regions have a high incidence in sera of vasectomised men (Tung, 1975).
The latter study was of particular interest
because the antigen to the principal piece, designated ~P, was reactive only after the cells had been disrupted with detergent, subjected to proteolysis and fixed briefly with methanol suggesting that the antigen was of subcellular origin. Because the antigen was confined to the principal piece of the cell i t was thought to be associated with mitochondrial membranes.
The anti-Mp antibody appeared to
differ from other antibodies directed primarily to the principal piece of the cell since these were accessible to the antigen in live, unfixed cells (Feltkamp et al.,
1965).
Four auto antigens, designated S, P, T and Z have been described in guinea-pig sperm (Voisin and Toullet, 1968).
The f i r s t three have been shown to have
different immunological, physicochemical and pathological al.,
1973; Toullet and Voisin, 1974).
properties (Toullet et
Furthermore, female ouinea-pigs react
against all of these three autoantigens producing antibodies that are
indistino-
uishable from male autoantibodies directed against the same autoantigens. Although the autoantigens of human spermatozoa have not been examined in as great a detail as the guinea-pig, cross-reacting and autoantigens and autoantibodies between these two species have recently been described (D'Almeida and Voisin, 1977) as well as between human, bull and rabbit spermatozoa (Hansen, In72). Attempts to demonstrate cross-reactivity between human and guinea-pio testis have been performed but the results are ambiguous.
Otani and Behrman (1963), for
example, could find no evidence for cross-reactivity although common antigenicity could be observed with tissue extracts (Katsh, 1962). The relationship between f e r t i l i t y and the presence of antisperm antibodies in the serum has been the subject of many investigations.
I t has been noted, for
example, that vasectomised men who have undergone successful reversal of the operation, exhibit poor f e r t i l i t y which appears to be correlated with the t i t r e of sperm agglutinating, but not sperm immobilizing, antibodies in the serum (Sullivan and Howe, 1977). In about half the cases studied by these workers, in which pregnancy was achieved after reanastamosis of the vas deferens, agglutinating antibodies were present suggesting that these antibodies were not primarily responsible for poor f e r t i l i t y of the spermatozoa.
This appears to contrast with
studies by Menge and Protzman (1967) and later by Menge (1971) who observed low conception rates in female rabbits inseminated with semen pretreated with antisperm antibodies.
Normal conception rates were achieved, however, i f antiseminal
255
Biochemical and Immunological Prospects for Male Contraception
plasma antibodies were used to p r e t r e a t the sperm despite the f a c t that about the same extent of agglutination occurred in both qroups. corroborated this finding by performing a r t i f i c i a l
Hetz and Anika (1970)
insemination in the r a b b i t with
sperm pre-treated with univalent antisperm antibody fragments (Fab) agglutinate or immobilize sperm.
which did not
Pates of conception were again low even though
no agglutination or immobilization of the sperm was observed.
I t is apparent
that antibodies directed against other antigens often contribute to poor f e r t i l i t y in post-vasectomised men.
This appears to contrast with e a r l i e r findings that
antisperm antibodies that agglutinate spermatozoa i n t e r f e r e d with the migratory potential of the c e l l s and thus t h e i r a b i l i t y In this regard the a b i l i t y
to reach the ovum (Wilson, 1956).
to migrate through the mucus present in the cervix of
the female reproductive t r a c t would present a formidable b a r r i e r especially i f immobilizing cytotoxic antibodies are also involved (see F j a l l b r a n t ,
1968).
9estruction of the nerve supply to the vas deferens during vasectomy has also been considered as a factor contributing to the low f e r t i l i t y vasectomy (Pabst et a t . ,
observed a f t e r vaso-
1979).
Despite the f a c t that a high c o r r e l a t i o n between spermagglutinin t i t r e s in blood serum and the degree of sperm agglutination in the semen has been claimed (Fjallbrant,
1968;
necessarily
imply that these antibodies are capable of reaching the seminal
fluid,
Ansbacher et a t . ,
1976) high t i t r e s in the serum do not
where they would come into contact with the sperm.
Some types of c i r c u -
l a t i n g sperm autoantibodies, p a r t i c u l a r l y those of the IgH class have not u n t i l recently been detected in semen (Azim e t a t . ,
1978).
The Igr! class of antibodies
is believed to be p r i n c i p a l l y involved in head-to-head agglutination of spermatozoa (Friberg, 1974).
Other classes of serum immunoglobulins such as IgG have been
reported in semen and these presumably arise from transudation from the c i r c u l a t i o n into the secretions of the prostate gland. to cause head-to-tail
agglutination of spermatozoa.
These antibodies are believed IgA has been detected in
semen but the precise source of the immunoglobulin is not known.
Diffusion from
II
blood serum has been suggested (Helleme and Rumke, 1976) while a local secretion into the semen is a mechanism favoured by other workers (Uehling, 1971).
The
l a t t e r mechanism most l i k e l y explains the occasional incidence of t i t r e s higher in the semen than the serum and in which the seminal plasma spermagglutinins belong to the IgA class compared to IgG or IgM spermagglutinins in the blood serum. The presence of sperm agglutins in serum of women has been considered as a cause of human i n f e r t i l i t y .
Remarkable v a r i a b i l i t y
in the presence of these antibodies
and t h e i r t i t r e s have been reported which probably r e f l e c t s
differences in assay
250
P.J. Quinn
sensitivity from one investigator to another.
However, the general trend that
can be discerned from such studies is that relatively hinh frequencies of spermagglutinating sera are associated with certain arouns of i n f e r t i l e women, esoecially those with unexplained i n f e r t i l i t y as compared with non-preanant women with presumably normal f e r t i l i t y or proven f e r t i l i t y (Isojima et a t . , 1974; Shulman et at.,
1975).
A recent detailed study has confirmed that agglutinatinq
antibodies occur in a significantly hiqher frequency and in hiaher t i t r e s in i n f e r t i l e women and the presence of B-spermagglutinins was confined exclusively to this group (Ingersler and Hjort, 197g). The escape of antiaens from the reproductive tract into the host environment and the entry of antibodies and immune cells into the tract is prevented by a system of zona occludens, tight junctions that seal the epithelia lining the male reproductive tubules and ducts. barrier.
The system is referred to as the blood testis
The barrier has been investinated in detail by electron-opaque inter-
cellular tracer studies and high maqnification electron microscopy.
This has
shown a system of tight junctions between adjacent Sertoli cells which subdivide the seminiferous epithelium into two compartments, basal and adluminal (Dym and Fawcett, 1970; Dym, 1973).
Theseunique Sertoli cell junctional complexes are
characterised by bundles of fine filaments adjacent to the plasma membranes and profiles of endoplasmic reticulum next to the filaments (see Nicander, 1967; Flickinger and Fawcett, 1967).
It is the tight junctions unitinq the Sertoli
cells that are believed to represent the structural basis of the blood-testis barrier (Dym and Cavicchia, 1977).
The barrier prevents intercellular diffusion
and serves to protect the individual from sensitisation to his own spermatozoan antigens. The barrier may be breached by disease, trauma or obstruction of the tubules or ducts in which case the incidence of sensitisation to autoantigens may be s i g n i f i cant (Schoenfelt
et
ai.,1976;
Tunn, 1975).
I t should be noted that escape of
the entire or intact spermatozoan from the tract may not be required for the formation of autoantibodies;
presentation of soluble or particulate antigens through
leaky epithelial junctions may be sufficient to sensitise the individual. The detailed mechanism whereby obstruction of the reproductive tract e l i c i t s antisperm autoantibody formation is unknown.
I t seems likely that antigen escapes
from distended ducts into the blood stream where they interact with the host immune system so that the humoral and/or cellular manifestations of sensitisation to spermatozoa are observed.
In the case of vasectomy, exposure of the immune
system to sperm is likely to occur, in the f i r s t instance, at operation particularly i f this is performed by resection rather than by ligation alone. Post-
257
Biochemical and Immunological Prospects for Male Contraception operatively, exposure through sperm granulomas is orobably the principal route.
The granulomas,which form in a high proportion of vasectomised men (Alexander and Schmidt, 1977) consist of a central mass of packed sperm and cell d6bris bordered by epithelioid histiocytes with an i n f i l t r a t i o n of polymorphonuclear leucocytes. Granulomas are thought to develop as an inflammatory response to extravasated spermatozoa which are surrounded by a wall of leucocytes.
Early granuloma
formation, however, does not appear to be the result of an inflammation associated with an autoimmune reaction (Brannen et a t . , 197~).
No particular type
of antisperm antibody has been shown to be associated exclusively with the presence of a granuloma;
however, more men with granulomas appear to have immobil-
ising antibodies than men in which such antibodies cannot be detected.
The
sequence of exposure to antigen may also be a factor in the antibody response. Exposure of the immune system to antigen at operation could have the effect of e l i c i t i n g a primary antibody immune response.
I t is well known that the uptake
of soluble antigen both through the gastrointestinal (Andr~ et al., 1974) and respiratory (Stokes et a l . , 1975; Thrall et a l . ,
1978) tracts is considerably
reduced in immunised animals and a similar situation may exist with regard to the uptake of soluble antigens derived from spermatozoa or other seminal constituents. Many questions regarding the way sperm antigens interact with the host immune system, however, remain unanswered.
I t is not known, for example, how the
quantity of antigen and the rate of its exnosure influence the nature of the immune response.
The type of exposure may also be an imnortant factor in deter-
mining the expression of immunity, e.g. escape of intact spermatozoa compared with the leakage of soluble antigens.
The immune system may also react to certain
spermatozoan antigens in a tolerant manner but be sensitised by others. A further consideration that must be taken into account in regard to the isolation of the reproductive tissues from the immune system is the possible involvement of phagocytic cells from the host.
The directed migration of polymorphonuclear
leucocytes from the vascular compartment into the tissues is known to be mediated by a variety of humoral agents derived from microorganisms, tissues and plasma proteins including the complement, kinin, f i b r i n o l y t i c and coagulation systems (see review by Hilkinson, 1974).
These chemotactic factors have been identified
i n s i t u in areas of inflammation and in normal biological fluids.
The importance
of the leucocytic influx in the host response to infection is well known. Factors that are released by polymorphonuclear leucocytes that have a toxic effect on adjacent host tissues have also been identified and these are believed to act on spermatozoa in the female reproductive tract.
Muir et a l . (1977) have carried
out a detailed study of the possibility that cell mediated immune reactions may be involved in the abnormalities resulting in the semen of men who had undergone a
258
P, J. Quinn
reversal vasectomy.
Oligospermia with ooor m o t i l i t y and pleomorphism of the
spermatozoa had been suggested e a r l i e r to be due to autoimmune reactions (Shulman, 1971).
An assay for cell mediated immunity to human sperm extracts
was developed using the a b i l i t y
of the antigen to transform peripheral lymphocytes
in c u l t u r e , but they were unable to find any evidence f o r s p e c i f i c stimulation of leucocytes even in patients that had been vasectomised f o r periods of up to one year.
Similarly,
skin tests performed on guinea-pigs using heat-treated homo-
logous epididymal sperm extracts f a i l e d to e l i c i t
a delayed-type h y p e r s e n s i t i v i t y .
I t was concluded that cell-mediated immunity to sperm does not develop in e i t h e r man or guinea-pig following vasectomy despite the obvious humoral response and the c l e a r l y demonstrated cell-mediated immunity to sperm in immunised guinea-pigs (Harcus et a l . , 1973, 197£).
Thesestudies appear to be in complete contrast
to those of Nagarkatti and Pao (1976) who used an inhibition of leucocyte migration test in the presence of sperm antigens as an index of cell mediated immunity.
They reported clear positive results in many males following vasectomy
and an increased incidence with the length of time following operation.
Further-
more, i t has been shown recently that T-cells from long-term vasectomised rhesus monkeys have reduced mitogen reactivity in mixed lymphocyte cultures (Wilson et a t . , 1977) and this appears to be associated with histocompatibility type (Wilson et a l . ,
197q).
Clearly more data is required to determine whether or not cell
mediated immune reactions are a feature of vasectomy in humans.
Chapter 4
Survival of Spermatozoa in the Female Tract and Fertilization
In order that successful f e r t i l i z a t i o n
of the egg may be achieved the inseminated
spermatozoa must be transoorted through the female genital t r a c t to the site of fertilization changes.
and they must have undergone certain certain crucial developmental
Interruption of either processes can provide a potential method of
preventing f e r t i l i z a t i o n .
4.1. Factors Affecting Sperm Viability and Transpo~ Assessment of semen quality a b i l i t y of the spermatozoa.
in
vitro
has been used as a guide to the f e r t i l i z i n g
Spermatozoa that are defective morphologically or
show poor m o t i l i t y are at an obvious disadvantage when compared with normal cells. Furthermore, sperm concentrations appear to be related to f e r t i l i t y .
Other
factors present in the seminal plasma and female t r a c t secretions also assist in sperm transport and maintain v i a b i l i t y of the cells.
Such factors, for example,
have been shown to protect spermatozoa from damage associated with l i p i d autooxidation.
Human spermatozoa have been shown to be p a r t i c u l a r l y susceptible to
damage by oxidative processes. Peroxidation of the membrane phospholipids, p a r t i c u l a r l y docosahexanoic acid associated with plasmalogens, causes a progressive and i r r e v e r s i b l e loss of structural
i n t e g r i t y and v i a b i l i t y manifested by loss
of m o t i l i t y and metabolic a c t i v i t y (Jones and Mann, 1976).
Not only are endogen-
ous l i p i d peroxidation products detrimental to spermatozoa but exogenous f a t t y acid peroxides are potent spermicidal agents (Jones and Mann, 1977).
The
harmful effects of autoxidation processes can be reduced by serum proteins (Stocks e t a l . , 1974) human caeruloplasmin (AI-Timini and Dormandy, 1977) and seminal plasma (Jones et
al.,
1979) presumably by binding the degraded l i p i d s .
The amelioration of these effects by agents present in the female t r a c t is also l i k e l y to be one factor concerned in the v i a b i l i t y of spermatozoa.
259
260
P.J. Quinn
Those factors which are normally present in human semen that may affect f e r t i l i t y w i l l be examined since therapeutic intervention in the male could influence the level of such factors in the eiaculate and hence represent notential male contraceptives. in vitro
I t should be emphasized, however, that postulations based on
studies of spermatozoa should not be regarded as conclusive since
deficiencies recognized in the ejaculate may be compensated by adequate supplies of the substance in secretions from the female t r a c t .
As we shall see, these
secretions appear to provide key substrates for the modification of spermatozoa that are not supplied in the ejaculate. 4.1.1.
The action of prostaglandins on f e r t i l i t y
One factor considered important for transport and v i a b i l i t y of spermatozoa is the presence of prostaglandins in the seminal plasma.
Ejaculates of normally f e r t i l e
men contain about 1 mg of prostaglandin, mainly of the F-tynes includina PGEI , PGE2, 19-OHPGE1 and 19-OHPGE2 (Templeton e t a l . ,
I~7~).
The function of these
prostaglandins is uncertain but a correlation between f e r t i l i t y concentration in semen has been suaaested (Bygdeman et a l . ,
and prostaglandin
I°70).
This should
be considered in conjunction with the fact that no apparent correlation exists between prostaglandin concentrations and other commonly used parameters of semen quality such as sperm count, m o t i l i t y , volume (Asplund, 1947; C o l l i e r et a l . ,
1975).
Bygdeman e t a l . ,
longevity of the spermatozoa or seminal 1970~ Brummer and Gillespie, In72;
This is somewhat surprising considering the fact that
pharmacological doses of prostaglandins affect seminal characteristics of domestic animals.
Thus PGF2~ has been shown to cause an increase in ejaculated
cell numbers in the s t a l l i o n al.,
(Cornwell e t a l . ,
197a), rabbit and bull (Hafs e t
1974) presumably by nromoting smooth muscle contraction of the epididymus
serving to deliver more spermatozoa to the deferent duct.
~nother rather
anomalous situation is the recent report that the prostaglandin content of ejaculates from polyzoospermic men (sperm concentrations qreater than 3xlO 8 c e l l s / ml) are s i g n i f i c a n t l y less than in ejaculates of lower sperm counts (Kelly e t a l . , 1979). Aspirin, in therapeutic doses, has been shown to depress the prostaglandin concentration in semen (Collier and Plower, 1971) but the drua does not appear to reduce fertility,
suggesting that lowered concentrations of prostaglandins are not
primarily responsible for i n f e r t i l i t y .
Prostafllandins, however, are known to
affect sperm metabolism, for example, 19-~H PG~ (but not PGE or PGF) reduce respiration of washed spermatozoa in v i t r o but do not affect alycolysis (Kelly, 1977).
Furthermore, endogenous synthesis of prostaalandins
vesicle is related to the androgen status of the individual.
by the human seminal Testosterone has
261
Biochemical and Immunological Prospects for Male Contraception been shown, for example, to increase the synthesis of 19-NH PGE in hypo~onadal males (Skakkeback e t a t . ,
1976).
Two conceivable mechanisms could be considered
with regard to the action of prostaglandins as male contraceptive aqents.
Firstly,
there is some evidence to suggest that prostanlandins affect sperm transport through the female genital t r a c t by altering tubal or uterine a c t i v i t y .
In this
respect certain prostaglandins have been shown to increase the rate of transport of spermatozoa (Hafs, 1974; Hargrove and E l l i s , 1976) so that removal of prostaglandins from the ejaculate or i n h i b i t i n g t h e i r action on the smooth muscles of the female t r a c t could interfere with sperm transport.
Secondly, by altering the
metabolic a c t i v i t y of spermatozoa the a c t i v i t y of the cells could be impaired to an extent where they may exhibit reduced a b i l i t y to penetrate the cervical mucus and the final barrier to f e r t i l i z a t i o n ,
the investments surrounding the eqg.
This aspect w i l l be dealt with in greater detail in a l a t e r section. Apart from affecting sperm transport and v i a b i l i t y
prostaglandins appear to be
involved in the contraceptive efficiency of intrauterine devices (Thompson, I°73). For example, an increased content of prostaglandins in uteri of rats, hamsters (Saksena e t a l . ,
1974) mice (Lau e t a l . ,
1974) and rabbits (Saksena and Harper,
1974) in which intrauterine devices were inserted has been reported and that administration of indomethacin, a potent i n h i b i t o r of prostaglandin synthesis, suppresses the contraceptive action of the device (Saksena and Harper, 197~).
In
addition, the decidual cell reaction appears to be suppressed by the presence of an intrauterine device and although prostaglandins have been shown to be l e u t o l y t i c (Chatterjee, 1972) i t is not certain whether or not i n h i b i t i o n of decidualization is mediated through alterations in ovarian steroidogenesis alone.
Bronson and
Hamada (1979) have reported experiments on ovariectomised, hormone-replaced pregnant mice treated with PGE2 and PGF2~ to distinguish the extraovarian actions of prostaglandins upon the decidual cell reaction from those mediated by alterations in ovarian steroidogenesis.
They found that the effect of PGF2~ on decidualization
was mediated via an alteration in ovarian steroidogenesis, but that PGE2 acted independently of the ovary.
T h e y suqgested on the basis of these experiments
that the r a t i o of E to F prostaglandins e l i c i t e d within the endometrium by the presence of an intrauterine device i n h i b i t s decidualization at the uterine level possibly mediated by the impairment of histamine release from endometrial mast cells. The prominent i n f l u x of leucocytes into the t r a c t in response to the presence of spermatozoa or other seminal constituents has been repeatedly observed (Mattner, 1969;
Soupart, 1970) and suggests that semen contains leucocyte chemotactic
factors, or agents, that can interact with other proteins to generate chemotactic
262
P.J. Quinn
factors.
Indeed, the
studies of ~laroni and colleagues (~!aroni
in vitro
et al.,
1971, 1972) have demonstrated selective chemotaxis of macrophages towards human and guinea-pig spermatozoa and seminal f l u i d . macrophages there was l i t t l e
Although the spermatozoa attracted
a c t i v i t y for polymorphonuclear leucocytes whereas
seminal f l u i d a c t i v a t i o n of serum or plasma generated chemotactic factors r e l a t i v e l y s p e c i f i c for polymorphs.
The chemotactic factor was not i d e n t i f i e d
but this was l a t e r shown to be due to the i n t e r a c t i o n of both spermatozoa and seminal plasma with the serum complement system to generate a ootent polymorphonuclear leucocyte chemotactic f a c t o r from the f i f t h (Clark and Klebanoff, 1976).
component of complement
The role of prostaglandins in these chemotactic
processes has yet to be assessed. Prostaglandins and p a r t i c u l a r l y the balance between E and F-type prostaglandins are also believed to be responsible for the physiological control of ovum transport in the female t r a c t .
Recent studies of the influence of hCG and steroid treatment
on prostaglandin metabolism by rabbit uterus and oviduct has suggested that changes in metabolism of PGE2 and PGF2m by the oviduct is the primary mechanism regulating ovum transport (Bodkhe and Harper, lq7q).
The use of Drostaglandins
as contraceptive agents has been suggested because they i n t e r f e r e with sperm and ovum transport in the female t r a c t and they could be involved in leucocytic processes.
One of the major d i f f i c u l t i e s
in the use of naturally-occurring
prostaglandins as contraceptives is the high incidence of side effects diarrhea and vomiting and the f a c t that they are not completely r e l i a b l e .
The
quest for synthetic analogues of the prostaglandins which are more e f f e c t i v e and clinically 4.1.2.
acceptable continues. Steroid binding and sperm transport
Specific steroid-binding sites on the surface of human spermatozoa have been identified
(Hyne and Boettcher, 1977).
These include a l o w - a f f i n i t y ,
specific
oestradiol-binding s i t e for which progesterone and a number of s t r u c t u r a l l y related steroids are e f f i c i e n t competitors compared with testosterone. series of binding sites appear to have a high a f f i n i t y
for testosterone.
of sperm migration through cervical mucus or buffered albumin solutions
Another Studies in vltro
have shown that m o b i l i t y is i n h i b i t e d by progesterone or synthetic progestogens (Kesseru e t a l . ,
1975;
Beck e t a l . ,
1976) suggesting that binding of steroids
to spermatozoa may be an e f f e c t i v e method of regulating f e r t i l i t y antifertility
and explain the
e f f e c t s of steroid-releasing i n t r a u t e r i n e (Hagenfeldt, 1976) and
intravaginal devices (Mishell, 1975).
These devices release pharmacological
doses of steroids into the lumen of the female genital t r a c t where they compete for oestradiol-binding sites on the surface of the spermatozoa.
In a detailed
263
Biochemical and Immunological Prospects for Male Contraception study of the binding of steroids to human spermatozoa Hyne and Boettcher (1978) showed that some steroids such as progesterone, lynestrenol and norethynodrel markedly disrupt sperm migration and m o t i l i t y ,
whereas other steroids such as
oestrone have no detectable e f f e c t on these processes.
Some of the steroids
appeared to i n h i b i t oxidative metabolism and t h e i r e f f e c t s may be ascribed to interference with energy requiring steps in f e r t i l i z a t i o n , seemed to i n h i b i t migration and m o t i l i t y d i r e c t l y .
while other steroids
I t was concluded that
synthetic progestogens released from devices within the female t r a c t compete strongly f o r the oestrodiol-binding receptors on the plasma membrane of the spermatozoa where they a l t e r the permeability c h a r a c t e r i s t i c s of the membrane or, in some way, d i s r u p t a membrane function associated with sperm m o t i l i t y .
I t is
noteworthy that the s i t e of action of these steroid hormones is d i f f e r e n t from the i n t r a c e l l u l a r protein-binding sites c h a r a c t e r i s t i c of the mode of action of these hormones as described in Section 2.1.2.
4.2. Developmental AIterations to Spermatozoa in the Female Tract When semen is deposited in the female t r a c t the spermatozoa undergo a series of complex biochemical and morphological changes which are required to render them capable of successfully f e r t i l i z i n g
the egg.
These events are time-dependent
and they appear to rely upon substances provided in secretions of the female t r a c t . The nature of these processes w i l l
be examined in turn and l i k e l y avenues for
contraceptive i n t e r v e n t i o n explored. 4.2.1.
Capacitation and decapacitation processes
Freshly ejaculated spermatozoa from mammalian species are incapable of immediately fertilizing
eggs;
they require a period of incubation e i t h e r i n v l v o or i n v i t r o
during which they acquire the canacity to f e r t i l i z e (see Austin, 196~).
or are said to be capacitated
The process can be reversed at any stage by exposure of the
spermatozoa to fresh seminal plasma or epididymal f l u i d which contains the putative decapacitation f a c t o r whereupon capacitation must recommence from i t s initial
state (Chang, 1957).
The period required to capacitate spermatozoa
varies from one species to another and, for example, a 6-I0 hour incubation is needed to f u l l y capacitate r a b b i t spermatozoa whereas in the mouse only 2 hours is s u f f i c i e n t .
These times r e f e r to the minimum period of incubation under
optimal conditions that fresh epididymal or ejaculated spermatozoa require to complete the changes involved in capacitation.
Since the discovery that the
decapacitation factor could be removed from seminal plasma by u l t r a c e n t r i f u g a t i o n (Bedford and Chang, 1962) a number of studies have been undertaken in an attempt to characterize the f a c t o r or factors.
There is s t i l l
uncertainty about the nature
264
P.J. Quinn
of the decapacitation f a c t o r with some claims that i t is a large protein or elycoprotein constituent of the seminal plasma (mavis, l q 7 l ) , or a ] i n i d - ] i k e (Dukelow e t a l . ,
component
1967) while other workers suggest that i t is a small lysine-
containing peptide (McRorie and Williams,
1974).
There i s , however, general
agreement that the decapacitation factor originates from the male accessory glands as well as the testes and the male genital t r a c t .
Furthermore, the notion that
capacitation of spermatozoa involves the removal or i n a c t i v a t i o n of decapacitation f a c t o r t i g h t l y bound to the plasma membrane of the cell receives popular support (Weinman and Williams,
1964).
A plausible mechanism of action of bound decapacit-
ation f a c t o r has been suggested by Bedford (1977) in which sites of importance in the induction of the next stage of the developmental process, the acrosome reaction, are e f f e c t i v e l y blocked by the factor and u n t i l ment is prevented.
they are unmasked f u r t h e r develop-
In a recent study of the decapacitation f a c t o r in human
seminal plasma (Kanwar e t a l . ,
1979) a macromolecular substance or factors
associated with macromolecules were found to e f f e c t i v e l y i n h i b i t penetration of human spermatozoa into zona-free hamster eggs. The f a c t that seminal plasma contains substances that i n h i b i t capacitation suqgests a number of ways in which contraception might be achieved.
Seminal plasma is
e f f e c t i v e l y separated from spermatozoa in species where semen is deposited in the vagina (e.g. humans and rabbits) by the b a r r i e r provided by the cervix and i t s associated mucus.
I f this b a r r i e r were removed then presumably seminal plasma
would be able to accompany the spermatozoa to the s i t e of f e r t i l i z a t i o n penetration into the egg.
and block
An a l t e r n a t i v e approach could be to attempt to
s t a b i l i z e the i n t e r a c t i o n between the decapacitation factor and i t s receptor site on the surface of the spermatozoa. considerably more information w i l l
Clearly, in order to pursue the second route, be required concerning the chemical nature of
the decapacitation f a c t o r ( s ) and the precise function that the f a c t o r ( s ) 4.2.2.
perform.
The acrosome reaction
When spermatozoa are f u l l y
capacitated they are able to enter the next phase of
development referred to as the acrosome reaction. the acrosome reaction is not r e v e r s i b l e .
Hnlike the capacitation process
Once spermatozoa have undergone the
acrosome reaction i t is u n l i k e l y that they survive any considerable length of time so that f o r successful f e r t i l i z a t i o n the egg.
the reaction must occur in the proximity of
Indeed the question of whether or not spermatozoa that have undergone
the acrosome reaction in situations remote from the egg are at all capable of subsequently f e r t i l i z i n g
an egg is debatable.
The acrosome reaction involves extensive morphological changes in the head region of the sperm including vesiculation and blebbing of the plasma membrane and the
265
Biochemical and Immunological Prospectsfor Male Contraception outer acrosomal membrane.
This process serves to release factors required to
facilitate
the penetration of the spermatozoa through the i nvesi~ents surrounding
the egg.
Another feature of sperm a c t i v i t y associated with i n i t i a t i o n
of the
acrosome reaction is the development of a pronounced whiplash type of m o t i l i t y (Yanagimachi, 1970, 1972; motility.
Fraser, 1977) often observed as a general a c t i v a t i o n of
I t is possible that such patterns of m o t i l i t y are complementary to the
enzymic attack on the egg investments mounted by enzymes released from the sperm acrosome.
The various impediments to sperm nenetration include the outer cumulus
oophorus and corona radiata layers which envelop the zona pellucida which the egg i t s e l f .
bounds
The structure of these investments are shown schematically in
Fig. 8.
..
'...
.
" d}°" ° o • ', • .u..... " . . . . ' . :........,X..:.~;..O!i~6..:~?,~...: ",. .~J.'.;;~..~7-,~r'~ ~.~.,~r,.,~Jri :.;. •
, •
.-".
: ;'.~t"
'".;:
cm,
°®~'~
",.~'t~,k_).
:-.,~..'/.. - . :..,,.0 : : " ,,..ft.:,.
; ,'...~X'~...~<.
{ :: '.:: ::i .,kI.t z=,. ,,,,u°=
~5 "':
•"
o
~
.' "
Fig. 8
~
~ (.'.
":: , : ~ i i t e l l i l ' ~ -
.,,..
membrane
-v,,.,= :.'..- .,~.: ~ c~";7%'."
Diagram of the investments surrounding the typical mammalian egg.
The factors released from the acrosome are mainly enzymes capable of digesting these layers and allowing spermatozoa to fuse with the egg plasma membrane at the centre. I d e n t i f i c a t i o n of acrosomal enzymes and characterisation of t h e i r putative roles in f e r t i l i z a t i o n
have been undertaken by detergent extraction or sonication pro-
cedures which release the enzymes from the i n t a c t acrosome.
The extracted
enzymes have been p u r i f i e d and t h e i r e f f e c t s on i n t a c t eggs observed,
llnfortun-
a t e l y , these methods do not provide any information concerning the concerted or sequential release of acrosomal enzymes or t h e i r temporal role in the f e r t i l i z a t i o n process.
Moreover, the treatments required to remove "acrosomal components" are
rather drastic and c e r t a i n l y lack s u f f i c i e n t s e l e c t i v i t y to enable precise
266
P.J. Quinn
locations of p a r t i c u l a r enzymes within the structure to be determined.
For
example, contamination of detergent or sonicated extracts with typical mitochondrial enzymes is frequently observed.
~levertheless, a number of enzymes
believed to be released when spermatozoa undergo the acrosome reaction have been i d e n t i f i e d and include 6-glucuronidase, hyaluronidase and other proteases, acid phosphatase, phospholipases and arylsulphatases. Hyaluronidase is one of the more important enzymes involved in the acrosome reaction.
I t is released in some quantity simply by freezing spermatozoa suggest-
ing that i t is r e l a t i v e l y loosely compartmentalized within the c e l l . is responsible f o r penetration of the cumulus oophorus.
The enzyme
I t was o r i g i n a l l y thought
that the entire cumulus layer had to be digested but the evidence was derived mainly from experiments where large quantities of sperm-derived hyaluronidase were employed.
Motile, i n t a c t ejaculated spermatozoa from a v a r i e t y of species have
been shown to approach and adhere to the cumulus but f a i l
to penetrate the layer
unless hyaluronidase is released, a process that is associated with disruption of the plasma and acrosomal membranes (Austin, 1960).
Additional information on
the role of hyaluronidase has been obtained from immunological studies of the r a b b i t system.
I t has been shown, for example, that anti-hyaluronidase antibodies
which bind s p e c i f i c a l l y to the enzyme and block a c t i v i t y prevents f e r t i l i z a t i o n . Another enzyme believed to be released in the acrosome reaction of certain spermatozoa is the corona penetrating enzyme.
I t appears to act in a similar
manner to hyaluronidase but instead causes dispersion of cells present in the corona radiata.
There is no evidence indicating protease a c t i v i t y by the f a c t o r
which may also be present in uterine secretions since the corona is completely removed following f e r t i l i z a t i o n .
Acrosomal enzymes are also present which allow
spermatozoa to penetrate the zona pellucida.
This layer consists mainly of
neutral and acidic glycoproteins linked by disulphide bridges.
Hyaluronic acid
and other glycosidic components have also been detected in this layer.
Since a
v a r i e t y of d i f f e r e n t components are present in the zona pellucida, i t is thought that a combination of enzymes may be required for penetration of the spermatozoa. One enzyme believed to be active in this region has been described in r a b b i t , boar and human spermatozoa and is referred to as acrosin. be present as an inactive zymogen, proacrosin.
This protease is thought to
I t is a calcium-requiring serine
protease which hydrolyses peptide bonds adjacent to h i s t i d i n e residues. Human spermatozoa possess an i n h i b i t o r of acrosin referred to as acrostatin.
It
has been sugqested that acrostatin may be required to prevent l y t i c a c t i v i t y of acrosin when the enzyme is released from spermatozoa during the acrosome reaction since the enzyme would otherwise damage uterine and tubal tissues.
Acrostatin is
267
Biochemical and Immunological Prospects for Male Contraception presumed to be removed from the spermatozoa or i t s e l f
inactivated while present in
the female genital t r a c t , possibly in association with the capacitation process. What r o l e , i f any, secretions from the female t r a c t play in removal of acrostatin is unknown.
The precise locations of acrosin and acrostatin in the sperm head
are also unknown.
Some studies have suggested that acrosin is associated with
the inner acrosomal membrane (Srivastava, 1973;
Brown and Hartree, 1974) while
others provide evidence for a binding s i t e on the acrosome surface (Yanagimachi and Teichman, 1972;
Stambaugh and Buckley, 1972;
Garner et a t . ,
1975).
Any
acrosin that is associated with the outer acrosomal membrane is u n l i k e l y to be involved with penetration of the sperm through the zona pellucida because this membrane is shed before the sperm reaches this layer.
Nevertheless, the p o s s i b i l -
i t y that acrosin may be responsible f o r diaestion of the plasma and outer acrosomal membranes during the acrosome reaction cannot be e n t i r e l y discounted, especially since protease i n h i b i t o r s are known to i n h i b i t the acrosome reaction (Meizel and Lui, 1976).
In a careful detergent-extraction study Bhattacharyya and Zaneveld
(1978) concluded that both acrosin and acrostatin e x i s t at two locations on the spermatozoa; the f i r s t
representina about 10% of the total enzyme and i n h i b i t o r
associated with the outer acrosomal membrane and the remainder at a second s i t e closely adherent to the inner acrosomal membrane.
The acrosin at the l a t t e r s i t e
appears to be predominantly in the proacrosin form. The development of low molecular weight synthetic compounds capable of binding to acrosin and preventing p r o t e o l y t i c action has been suggested as a method which has considerable contraceptive potential
(Zaneveld, 1976).
The compound p - n i t r o p h e n y l
p'-guanidinobenzoate appears to have the most potent i n h i b i t o r y action on human acrosin and to i n h i b i t f e r t i l i z a t i o n at.,
1976).
fertilization
in several snecies tested (Bhattacharyya et
Further agents that i n t e r f e r e with acrosomal factors and block are being a c t i v e l y sought.
Penetration of the zona pellucida must be considered as a more complicated process than simply the enzymic digestion of the zona material.
The existence
of s p e c i f i c sperm receptors, for example, has been suggested on the basis of studies in the hamster.
Addition of plant l e c t i n s such as wheat germ agglutinin
to hamster eggs results in the binding of the l e c t i n to the n-acetyl neuraminic acid residues located in the zona which prevents f e r t i l i z a t i o n .
A neuraminidase
had been characterized in rabbit spermatozoa which is t i g h t l y membrane-bound and removes n-acetyl neuraminic acid residues from the zona. prevents f e r t i l i z a t i o n
Action of the enzyme
and i t s role in penetration of spermatozoa is unknown.
The role of calcium in these processes has been recognised. be required for capacitation (Yanagimachi
and Usui, 1974;
Calcium is known to Rogers and Yanagimachi,
268
P.J. Quinn
1976) and the calcium ionophore, A23187, has been reported to accelerate the capacitation of rabbit spermatozoa i n v i t r o
(Reyes et a l . ,
1978).
Related to
the effect of calcium on capacitation is the concentration of this ion in tubular secretions.
I t has been shown, for example, that calcium concentration increases
in the tubal secretions of the rabbit (Holmdahl and Mastroianni, 1965) and rhesus monkey (Mastroianni et a l . , 4.2.3.
1973) following ovulation.
Control of capacitation and the acrosome reaction
Many studies have been undertaken to investigate the process of capacitation and the acrosome reaction and the development of successful in v l t r o techniques has greatly assisted these studies.
fertilization
One such system has been devised
in the mouse which enables the temporal events in the process to be clearly distinguished (Fraser, 1979).
B r i e f l y the system consists of incubating epidi-
dymal spermatozoa under experimental conditions for a designated time, mixing the spermatozoa with eggs and then f i x i n g the eggs 75 min l a t e r for microscopic examination.
I f spermatozoa are f u l l y capacitated at the time they are added to
the eggs, by the time of f i x a t i o n the f e r t i l i z e d eggs w i l l all have completed meiosis I I , telophase can easily be recognised and the second polar body w i l l observed.
be
Meanwhile, the penetrating spermatozoa shows evidence of usually
complete decondensation with the egg cytoplasm.
I f spermatozoa are not f u l l y
capacitated at the time of addition to the eggs but undergo the acrosome reaction at some later stage, this w i l l be reflected in a correspondignly e a r l i e r stage of development of the f e r t i l i z e d egg.
This technique essentially avoids the need
to assess capacitation on the basis of examination of eggs reaching the two-cell stage 24 hours after adding spermatozoa. Of particular interest in these studies has been the substrate requirements for capacitation and the acrosome reaction.
Despite the fact that fructose is by far
the most abundant hexose substrate in the semen of a wide variety of manTnalian species (Mann, 1964) this sugar does not support capacitation and f e r t i l i z a t i o n those species so far examined.
Thus no f e r t i l i z a t i o n
in
was obtained, for example,
in in vitro systems of mouse (Hoppe, 1976) and rat (Niwa and I r i t a n i , 1978) gametes when the sole exogenous substrate was fructose, although when glucose was present high levels of f e r t i l i z a t i o n were achieved. for
in v i t r o
Most culture media employed
f e r t i l i z a t i o n and preimplantation embryonic development contain
pyruvate, lactate and glucose, and i t was originally suggested that all three substrates were required to support f e r t i l i z a t i o n (Mihamoto and Chang, 1973). Subsequently i t was found that lactate is not an essential ingredient (Hoppe, 1976) A comprehensive study of substrate requirements in the murine system has been reported (Fraser and Quinn, 1980). I t was shown that spermatozoa preincubated
Biochemical and Immunological Prospects for Male Contraception
269
in the absence of exogenous substrates for sufficient time to permit f u l l capacitation in complete medium failed to f e r t i l i z e eggs until supplied with glucose;
f e r t i l i z a t i o n then proceeded immediately and nuclear development advanced
to stages that were indistinguishable from those obtained with glucose present throughout.
Experiments performed under completely anaerobic conditions and in
the presence of inhibitors of oxidative phosphorylation showed that some metabolite of glycolysis was obligatory for i n i t i a t i n g the acrosome reaction and inducing the characteristic whiplash m o t i l i t y .
I t was also confirmed that fructose could
not substitute for glucose in these processes, thereby demonstrating that ejaculated spermatozoa cannot proceed beyond the terminal stages of capacitation without a supply of glucose from female tract secretions. These observations suggest methods for regulating the development of spermatozoa for contraceptive purposes and provide a plausible explanation for the antifertility
action of ~-chlorohydrin.
Metabolic, morphological
and other studies of the effect of various dose levels of
~-chlorohydrin on males of different species have suggested that one possible antifertility
effect of this compound is via an effect on the metabolism of spermatozoa.
Thus when ~-chlorohydrin is administered intravenously i t enters sperm cells and acts as a competitive inhibitor of glycerol phosphorylation (Edwards et a l . , mediated by glycerol kinase (Brooks e t a t . ,
1974).
1976)
The metabolic disposal of
[36CI] and [l~c]-~-chlorohydrin has been investigated in some detail in the rat. Compounds containinq labile halogens are normally detoxified in the l i v e r by conjugation with glutathione and eventually excreted as S-substituted cysteine conjugates in the urine.
Fig. 9 shows the various metabolic pathways delineated for
~-chlorohydrin metabolism.
The phosphorylated product of ~-chlorohydrin is a
potent i n h i b i t o r of glyceraldehyde-3-phosphate al.,
1978).
dehydrogenase (Brown-Woodman et
Irreversible inhibition of this glycolytic enzyme has been shown
to cause a reduction in ATP concentration in the cell (Chulavatnatol et a l . ,
1977).
resulting in a decrease of sperm m o t i l i t y (Brown and White, 1973; Brown-Woodman et al.,
1974).
The effect on general sperm m o t i l i t y , in the l i q h t of studies on
the murine system (Fraser and nuinn, 19nO), may however be secondary to the specific effects that Qlycolytic products exert on the acrosome reaction and whiplash m o t i l i t y . The contraceptive usefulness of ~-chlorohydrin has been shown to be limited by the manifestation of undesirable side effects.
Nevertheless, in the knowledge that
the action of ~-chlorohydrin is to interfere with glycolysis, other strategies seem possible.
The use of less toxic analogues of ~-chlorohydrin or other
reagents that i n h i b i t glyceraldehyde-3-phosphate
dehydrogenase could be developed.
270
P. J. Quinn 3- chloropropan1,2-diol-l-phosphate
CH,CI I CHOH
0
AH 2--0
bl
OH CH,CI I CHOH F
l
7H,CI
co,
I
f
CI
YHOH CO,H
cl-:0 P-chbro lactaldehyde
C02H I Co2H oxalate
p-~ZtYe
or-chlorohydrin CO,H
J CH,- S-glutathibne glycidol
glycerol
-
I CH,OH
CH,OH
I YHPH
glycerolipids
CHOH
I
CH,OH Fig. ?
CH,-S -CH2+H CHOH CH,OH
NH:!-++ excretory pathway as cysteine metabolites
r” ‘t
CO,
Pathways of metabol ism of a-chl orohydrin in mammals
The antibiotic, pentalenolactone (Fig. 10 ) which can be isolated from the fermentation broth of various species of Streptomyces (Aizawa et a~., 197g), for
Fig. 10
Structure of pentalenolactone E.
example, is also known to inhibit glycolysis by forming an irreversible complex with qlyceraldehyde-3-phosphate dehydrogenase (Hartmann et al., 1978).
The anti-
271
Biochemical and Immunological Prospects for Male Contraception b i o t i c i r r e v e r s i b l y i n h i b i t s the enzyme ~rom a variety of sources and when
examined in plant systems, which have three classes of enzyme, there appears to be a high s p e c i f i c i t y for the enzyme concerned with glycolysis (Mann and Mecke, 1977). Another possible contraceptive device may be to supply glucose in the ejaculate, or in the epididymus, with the aim of inducing premature i n i t i a t i o n of the acrosome reaction and whiplash m o t i l i t y , can successfully f e r t i l i z e
the egg.
thereby reducing chances that the spermatozoa Removal of glucose from female t r a c t
secretions would also serve to prevent f e r t i l i z a t i o n
by blocking f u l l
development
of the spermatozoa. Apart from the supply of essential glucose, female t r a c t secretions are believed to contain other factors required to maintain sperm v i a b i l i t y genital t r a c t and assist in the f e r t i l i z a t i o n
process.
in the female
Such factors appear to
assist in the binding and penetration of spermatozoa through the zona nellucida, aid in the dispersal of corona cells and provide immunoloqical protection for the zygote.
Oviduct secretions may also supply enzymes, possibly amylases, respon-
sible for removing decapacitation factor(s) from the seminal plasma.
Other
substances present in these secretions also act to prevent sperm penetration into the egg.
The action of acrosin, for example, can be affected by protease
i n h i b i t o r s which have been shown to be present in tubal secretions of women (~1oghissi, 1970; Hirschhauser et a l . , 1971) and rhesus monkeys (Stambaugh e£ a l . , 1974).
Chapter 5
Conclusions and Future Prospects
In the preceeding sections the hormonal regulation of male reproduction has been outlined and possible sites for developing suitable male contraceptives i d e n t i f i e d . Hany of these offer potential for avoiding the two major hazards associated with contraception in the male, namely, the danger of introducing genetic damage and secondly the loss of l i b i d o . Possibly the most promising area for contraceptive development is that where cooperation between the gametes and t h e i r environment in the female genital t r a c t is required to achieve successful f e r t i l i z a t i o n
and development of the embryo.
Clearly, more research is necessary to i d e n t i f y the molecular bases of these cooperations especially in respect of sperm capacitation and penetration of the egg. Survival of the f e r t i l i z e d
egg is another area receiving special attention with
respect to the development of effective contraceptives.
Amongst the most
promising lines of investigation in this connection is the i d e n t i f i c a t i o n of specific tubal and uterine secretions upon which f e r t i l i z a t i o n
and/or maintenance
of the embryo depend, with the intention of provoking autoimmune responses aimed at blocking t h e i r action. difficulties immunity.
This work, however, is confronted by serious technical
as well as the hazards associated with the development of autoAn alternative approach advocated by Aitken (197ga) is to concentrate
an immune attack on antigens of embryonic origin.
This has the d i s t i n c t
advantage that the respective antigens are exposed only b r i e f l y to the maternal immune system.
No factors known to be released from the developing embryo and to
affect oviduct or uterine secretions have, as yet, been f u l l y characterised.
Other
immunological methods that show promise include the active immunization of women against chorionic gonadotrophin (Talwar et a t . , 1976a & b;
272
Hearn, 1979).
The
273
Biochemical and Immunological Prospects for Male Contraception strategy here has been to design an immunogen that is capable of provoking an
autoantibody reaction against the hormone so that the developino embryo f a i l s to rescue the corpus luteum during early pregnancy.
The use of human chorionic
gonadotrophin and i t s fragments coupled to heterologous proteins as immunogens to produce active immunization in women is currently being investigated, but this approach in all events appears to be nonreversible.
Passive immunization with
gonadotrophin antisera would therefore seem to offer greater prospects.
In this
connection, antibodies against the B-subunit of ovine lutenizing hormone have been shown to cross react strongly with monkey chorionic gonadotrophin and block fertility et at.,
(Thau et a l . ,
1979), i n h i b i t ovulation and terminate pregnancy (Hondgal
1978) in immunized monkeys.
Despite these results major d i f f i c u l t i e s
remain in generating and maintaining antibody t i t r e s of high s p e c i f i c i t y for human chorionic gonadotrophin.
F i n a l l y , the presence of autoantibodies directed against
zona pellucida antigens has been detected in some i n f e r t i l e women (Shivers and Dunbar, 1977) and active immunization of mice a~ainst these antiqens leads to a temporary state of i n f e r t i l i t y et at.,
1977).
during which f e r t i l i z a t i o n
is prevented (Gwatkin
Other immunological, hormonal and chemical methods for f e r t i l i t y
control in the female have been reviewed recently by Talwar (1978) and Aitken (1979b). I t may be concluded that despite considerable research experience, acceptable and r e l i a b l e methods og f e r t i l i t y
control in men have not vet been realized.
Nevertheless, a number of approaches appear promisinn and with continued e f f o r t should yield an ef#ective male contracentive in the not too distant future.
Acknowledgement Christine Quinn is thanked for her helpful discussions and for her patience during preparation of the manuscript.
References
Aitken, R.J. (1979a). Tubal and uterine secretions; the possibilities for contraceptive attack. J.Reprod.Fertil., 55, 247-254. Aitken, R.J. (1979b). 35, 199-204.
Contraceptive research and development.
Brit.Med.Bull.,
Aizawa, S., H. Akutsu, T. Satomi, S. Kawatata, and K. Sasaki (1978). AA-57, a new ~ntibiotic related to pentalenolactone. J.Antibiotics, 31, 729-731. Alexander N.J. (1973). Autoimmune hypospermato~enesis in vasectomised ~uinea pi~s. Contraception, 8, 147-164. Alexander N.J. (1977). Fertility in rhesus monkeys after vasovasectomy. Scand.J. Immunol., 6, 675. Alexander N.J., and S.S. Schmidt (1977). Incidence of antisperm antibody levels and granulomas in men. Fertil. Steril., 28, 655-657. Alexander N.J., B.J. Wilson, and G.D. Patterson (1974). Vasectomy: immunologic effects in rhesus monkeys and men. Fertil. Steril., 25, 149-156. AI-Timimi D.J., and T.L. Dormandy (1977). The inhibition of lipid autoxidation by human caeruloplasmin. Biochem. J., 168, 283-288. Andre, C. R. Lambert, H. Bazin and J.F. Heremans (1974). Interference of oral immunization with the intestinal absorption of heterologous albumin. Immunol., 4, 701-704. Ansbacher, R. (1971). Sperm-agglutinating and sperm-immobilizing antibodies in vasectomized men. Fertil. Steril., 22, 629-632. Ansbacher, R. (1973). 792.
Vasectomy:
sperm antibodies.
Ansbacher, R. (1974). 9, 227-237.
Bilateral vas ligation:
274
Fertil. Steril., 24, 788-
sperm antibodies.
Contraception,
Biochemical and Immunological Prospects for Male Contraception
275
Ansbacher, R., P. Hodge, A. Willams and D.M. Mumford (1976). Vas ligation: humoral sperm antibodies. Int. J. Fertil., 21, 258-260. Asplund, J. (1947). A quantitative determination of the content of contraceptive substances in human sperm and their significance for the motility and vitality of the spermatozoa. Acta Physiol. Scand., 13, 103-108. Austin, C.R. (1960). Capacitation and the release of hyaluronidase from spermatozoa. J.Reprod. Fertil., I, 310-311. Austin, C.R. (1969). Sperm capacitation - biological significance in various species. In C.G. Hartman (Ed.), Mechanisms Concerned with Conception, Pergamon, Oxford. pp.285-320. Azim, A.A., S. Fayad, A.A. Fattah, and M. Habeib (1978). male infertility. Fert. Steril., 30, 426-429.
Immunologic studies of
Barraclough, C.A. (1968). In A. McLaren (Ed.), Advances in Reproductive Physiology, Logos, London. pp. Barry, J., and M.P. Dubois (1976). Immunoreactive LFR neurosecretory pathways in mammals. Acta anat., (Basel) 94, 497-503. Barry, J., M.P. Dubois, and P. Poulain (1973). LRF-producing cells of the mammalian hypothalamus. Cell Tiss. Res., 146, 351-366. Barth, R.F., and P.S. Russell (1964). The antigenic specificity of spermatozoa. I. An immunofluorescent study of the histocompatibility antigens of mouse sperm. J. Immunol., 93, 13-19. Beck, K.J., S. Herschel, R. Hungershofer, and E. Schwinger (1976). The effect of steroid hormones on motility and selective migration of X- and Y-bearing human spermatozoa. Fertil. Steril., 27, 407-412. Bedford, J.M. (1977). Sperm/egg interaction: spermatozoa. Anat. Rec., 188, 477-488. Bedford, J.M., and M.C. Chang (1962). Nature, 193, 898-899.
the specificity of human
Fertilization of rabbit ova in vitro.
Belchetz, P.E., T.M. Plant, Y. Nakai, E.J. Keogh, and E. Knobil (1978). Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasin~ hormone. Science, 202, 631-633. Bhattacharyya, A.K., and L.J.D. Zaneveld (1978). Release of acrosin and acrosin inhibitor from human spermatozoa. Fertil. Steril., 30, 70-78. Bhattacharyya, A.K., L.J.D. Zaenveld, B.M. Dragoje, G.F.B. Schumacher, and J. Travin (1976). Inhibition of human sperm acrosin by synthetic agents. J. Reprod. Fertil., 47, 97-1OO. Bi~azzi, P.E., L.L. Kosuda, L.L. Harnick, R.C. Brown, and N.R. Rose (1976a). Antibodies to testicular antigens in vasectomised rabbits. Clin. Immunol. Immunopath., 5, 182-194. Bigazzi, P.E., L.L. Kosuda, K.C. Hsu, and G.A. Andres (1976b). Immune complex orchitis in vasectomised rabbits. J. exp. Med., 143, 382-404.
276
P.J. euinn
Bodkhe, R.R., and M.J.K. Harper (1979). Influence of hCG injection and steroid treatment on prostaglandin metabolism by rabbit uterus and oviduct. J. Reprod. Fert., 57, 101-112. Boettcher, B. (1974). The molecular nature of spermagglutinins and sperm antibodies in human sera. J. Reprod. Fert.,(suppl.) 21, 151-168. Boettcher, B., J. Hay, D.J. Kay, B.A. Baldo, and T.K. Roberts (1970). Spermagglutinating activity in some human sera. Int. J. Fertil., 143-158.
15,
Brannen, G.E., and D.S. Coffey (1974). Immunologic implications of vasectomy: II. Serum-mediated in~nunity. Fertil. Steril., 25, 515-520. Brannen, G.E., J.C. Eggleston, J.S. Adams, and D.S. Coffey (1974). The effects of immuno suppression on sperm granulomas in vasectomised rats. J. Urol., 112, 793-797. Bronson, R., and. Y. Hamada (1978). The .effect of prostaglandins F~Z ~ and E~ on . . . . . pregnancy in mlee during implantation. F e r t l l . S t e r l l . , 30, 354-36~. Brooks, D.E., D.W. Hamilton and A.H. Mallek (1974). Carnitine and glycerylphosphorylcholine in the reproductive tract of the male rat. J. Reprod. Fert., 36, 141-160. Brown, C.R., and E.F. Hartree (1974). Distribution of trypsin-like proteinase in ram spermatozoa. J. Reprod. Fertil., 36, 195-198. Brown, P.D.C., and I.G. Ifhite (1973). Studies on the male anti-fertility druE 3-chloro-l,2-propanediol. J. Reprod. Fert., 32, 337-338. Brown-Woodman, P.D.C., H. Mohri, T. Mohri, D. Suter, and I.G. White (1978). Mode of action of ~-chlorohydrin as a male antifertility agent. Biochem. J., IZ~, 23-37. Brown-Woodman, P.D.C., S. Salamon, and I.G. White (1974). Effect of ~-chlorohydrin on the fertility of rams. Acta. Europ. Fert., 5, 193-197. Brurmner, H.C., and Gillespie, A. (1972). Clin. Endocrin., I, 363-368.
Seminal prostaglandins and fertility.
Burki, K., and W. Sheridan (1978). Expression of TEM-induced damage to postmeiotic stages of spermatogenesis of the mouse during early embryogenesis II cytological investigations. Mut. Res., 52, 107-115. Bygdeman, M., B. Fredricsson, K. Svanborg, and B. Samuelsson (1970). The relation between fertility and prostaglandin content of seminal fluid in man. Fertil. Steril., 21, 622-629. Chatterjee, A. (1972). The possible mode of action of prostaglandins: I. Interruption of decidual reaction by PGF~ and its prevention by using prolactin or progesterone. Acta Endocrlnol. (Kbh), 70, 781-785. Chulavatnatol, M., I. Hasibuan, S. Yindepit, and T. Eksittikul (1977). Lack of effect of ~-chlorohydrin on the ATP content of rat, mouse and human spermatozoa. J. Reprod. Fert., 50, 137-139. Clark, R.A., and S.J. Klebanoff (1976). Generation of a neutrophil chemotactic agent by spermatozoa: role of complement and regulation by seminal plasma factors. J. Immunol., 117, 1378-1386.
Biochemical and Immunological Prospects for Male Contraception
277
Collier, J.G., and R.J. Flower (1971). Effect of aspirin on human seminal prostaglandins. Lancet, 2, 852-853. Collier, J.G., R.J. Flower, and S.L. Stanton (1975). Seminal prostaglandins in infertile men. Fertil. Steril., 26, 868-871. Collins, P., and D. Lacy (1969). Studies on the structure and function of the mammalian testis. II. Cytological and histochemical observations on the testis of the rat after a single exposure to heat applied for different lengths of time. Proc. Roy. Soc., B., 172, 17-38. Corbin, A., C.W. Beattie, J. Tracy, R. Jones, T.J. Foell, J. Yardley, and R.W.A. Rees (1978). The anti-reproductive pharmacology of LH-RH and agonistic analogues. Int. J. Fertil., 23, 81-92. Cornwell, J.C., K.L. Koonce and J.L. Kreider (1974). F2~ on seminal characteristics of the stallion.
Effect of prostaglandin J. Anim. Sci., 38, 226.
Coutinho, E.M., and J.F. Melo (1973). Successful inhibition of spermatogenesis in man without loss of libido: a potential new approach to male contraception. Contraception, 8, 207-217. D'Almeida, M., and G.A. Voisin (1977). Human and guinea-pig cross-reacting spermatozoa autoantigens and autoantibodies. Clin. exp. Immunol., 28, 163-172. Davis, B.K. (1971). Macromolecular inhibitor of fertilization in rabbit seminal plasma. Proc. Natl. Acad. Sci., U.S.A., 68, 951-955. David, S.S., G.R. Huggins, C-R Garcia, and C. Busacca (1979). A synthetic , steroid (R2323) as a once-a-week oral contraceptive. Fert. Steril., 31 278-281. de Kretser, D.M. (1969). Ultrastructural features of human spermJo~enesis. Z. Zellforsch. Mikrosk. Anat., 98, 477-485. de Kretser, D.M. (1976). Towards a pill for men. In R.V. Short and D.T. Baird (Eds.), Contraceptives of the Future, The Royal Society, London. pp.161-174. de la Cruz, A., D.H. Coy, J.A. Vilchez-Martinez, A. Arimura, and A.V. Schally (1976). Blockade of ovulation in rats by inhibitory analogues of luteinizing hormone-releasing hormone. Science, 191, 195-197. Dmowski, ~q.P. (1979). Fertil. Steril.,
Endocrine properties and clinical application of danazol. 31, 237-251.
Drobeck, H.P., and F. Coulston (1962). Inhibition and recovery of spermatogenesis in rats, monkeys and dogs medicated with bis(dichloroacetyl) diamines. Exp. Mol. Pathol., I, 251-258. Drouin, J., and F. Labrie (1976). Selective effect of androgens on LH and FSH release in anterior pituitary cells in culture. Endocrinology, 98, 1528-1534. Dukelow, W.R., H.H. Chernoff, and W.L. Williams (1967). Properties of decapacitation factor and presence in various species. J. Reprod. Fertil.,
1_4, ~93-399. Dunn, M.S., K. Sakamoto, and E.A. Murphy (1962). Effect of ethionine in castrated male rats, with and without testosterone. Proc. Soc. Exp. Biol. Med., IIO, 50~-513.
278
P.J. Quinn
Dym, M. (1973). The fine structure of the monkey (Macaca) Sertoli cell and its role in maintaining the blood-testis barrier. Anat. Rec., 175, 639-656. Dym, M., and J.C. Cavicchia (1977). Further observations on the blood-testis barrier in monkeys. Biol. Reprod., 17, 390-403. Dym, M., and D.W. Fawcett (1970). The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol. Reprod., ~, 308-326. Edwards, E.M., J-L Dacheux, and G.M.H. Waites (1976). Effects of ~-chlorohydrin on the metabolism of testicular and epididymal spermatozoa of rams. J. Reprod. Fertil., 48, 265-270. Edwards, E.M., A.R. Jones and G.M.H. Waites (1975). The entry of ~-chlorohydrin into body fluids of male rats and its effect upon the incorporation of glycerol into lipids. J. Reprod. Fertil., 43, 225-232. Edwards, R.G. (1964. Immunological control of fertility in female mice. Nature, 203, 50-53. Edwards, T.G., L.C. Ferguson, and R.R.A. Coombs (1964). Blood group antigens on human spermatozoa. J. Reprod. Fertil., ~, 153-161. Ellis, L.C. (1970). In A.D. Johnson, W.R. Gomes, and N.L. Van Demark (Eds.), Radiation Effects, Vol. III. Academic Press, New York. pp.333-376. Erickson, B.H. (1976). Effect of 60Co y radiation on the stem and differentiating spermatogonia of the post puberal rat. Radiat. Res., 433-448. Fairley, K.F., J.U. Barrie, and W. Johnson (1972). atrophy related to cyclophosphamide therapy.
68,
Sterility and testicular Lancet, I, 568-569.
Feltkamp, T.E.W., K. Kruyff, N.C.J.J. Ladiges, and P. R~mke (1965). Autospermagglutinins: immunofluorescent studies. Annal. N.Y. Acad. Sci., 124, Fellons, M., and J. Dausset (1970). antigens on human spermatozoa.
Probable haploid expression of HL-A Nature, 225, 191-193.
Fjallbrant, B. (1968). Interrelation between high levels of sperm antibodies, and sterility in men. Acta obstet, gynec. Scand., 47, 102-118. Flickinger, C., and D.W. Fawcett (1967). The junctional specializations of Sertoli cells in the seminiferous epithelium. Anat. Rec., 158, 207-221. Fox, T.O. (1975). 441-444.
Oestradiol receptor of neonatal mouse brain.
Nature,
258,
Estrogen and androgen receptor Fox, T.O., C.C. Vito, and S.J. Wieland (1978). proteins in embryonic and neonatal brain: hypotheses for roles in sexual 18, 525-537. differentiation and behaviour. Am. Zool., Franchimont, P., A. Demoulin, J. Verstraelen-Proyard, M.T. Hazee-Hagelstein, J.S. Walton and G.M.H. Waites (1978). Nature and mechanisms of action of inhibin: perspective in regulation of male fertility. Int. J. Androl., suppl, no.2, pp.69-80.
Biochemical and Immunological Prospects for Male Contraception
279
Franklin, R.R., and C.D. Dukes (1964). Further studies on sperm agglutinating antibody and unexplained infertility. J. Am. Med. Assoc., 190, 682-683. Fraser, L.R. (1977). Motility patterns in mouse spermatozoa before and after capacitation. J. exp. Zool., 202, 439-444. Fraser, L.R. (1979). Rate of fertilization in vitro and subsequent nuclear development as a function of the post-ovulatory age of the mouse egg. J. Reprod. Fertil., 55, 153-160. Fraser, L.R., and P.J. Quinn (1980). A glycolytic product is obligatory for initiation of the acrosome reaction and whiplash motility required for the fertilization of mouse gametes. J. Reprod. Fertil., (in press). Friberg, J. (1974). A simple and sensitive micro-method for demonstration of sperm-agglutinating activity in serum from infertile men and women. Acta obstet, gynec. Scand., (Suppl.) 3-6, 21-29. Frick, J. (1973). Control of spermatogenesis in men by combined administration of progestin and androgen. Contraception, 8, 191-206. Garner, D.L., M.P. Easton, M.E. Munson, and M.A. Doane (1975). Immunofluorescent localization of bovine acrosin. J. Exp. Zool., 191, 127-134. Gorski, R.A. (1968). Influence of age on responses to paranatal administration of a low dose of androgen. Endocrinology, 82, 1001-1004. Gorski, R.A. (1971). Gonadal hormones and the perinatal development of neuroendocrine function. In L. Martini and W.F. Ganong (Eds.), Frontiers of Neuroendocrinology, Oxford Univ. Press, New York. pp. 237-290. Gnodde, H.P., J.A.M.J. Van Dieten, G.P. Van Rees, and I. Rothchild (1979). Conversion of progesterone to testosterone in the rat Leydig cell. Proc. Soc. Exp. Biol. Med., 160, 130-133. Goldberg, E.H., T. Aoki, E.A. Boyse, and D. Bennett (1970). Detection of H-2 antigens on mouse spermatozoa by the cytotoxicity test. Nature, 228, 570-572. Gonzalez-Barcena, D., A.J. Kastin, D.H. Coy, K. Nikoliss, and A.B. Schally (1977). Suppression of gonadotropin release in man by an inhibitory analogue of LH-RH. Lancet, ~, 997-998. Griffiths, E.C., and K.C. Hooper (1973). The stimulatory influence of gonadal steroids and methallibure (ICI 33, 828) on peptidase activity in the female rat hypothalamus. Fertil. Steril., 24, 269-274. Griffiths, E.C., K.C. Hooper, D. Hutson, S.L. Jeffcoate, and D.T. Holland (1975). The presence of peptidases in the rabbit hypothalamus capable of inactivating luteinizing hormone-releasin~ hormone. Fertil. Steril., 26, 802-806. ' ' Griffiths, E.C., K.C. Hooper, S.L. Jeffcoate, and D.T. Holland (1974). The presence of peptidases in the rat hypothalamus inactivating luteinizing hormone-releasing hormone (LH-RH). Acta Endocrinol. , 77, 435-442. Guillemin, R. (1977). Peptides of cerebral origin: their importance in growth physiology and medicine. Neuro-Horm. Hypothal., Masson: Paris. pp.3-16.
280
P. J, Ouinn
Gupta, J., S. Dhawan, G.D. Goel, and K. Saha (1975). Low fertility rate in vasovasostomized males and its possible immunologic mechanism. Int. J. Fert., 20, 183-191. Gwatkin, R.B.L., D.T. Williams and D.J. Carlo (1977). Immunization of mice with heat-solubilized hamster zonae: production of antizona antibody and inhibition of fertility.. Fertil. Steril., 28, 871-887. Hafs, D.H., T.M. Louis, and J.N. Stellflug (1974). Increased sperm numbers in the deferent duct after prostaglandin F2~ in rabbits. Proc. Soc. Exptl. Biol. Med., 145, 1120-1124. Hagenfeldt, D. (1976). The modes of action of medicated intrauterine devices. J. Reprod. Eertil., (Suppl.), 25, 117-132. Hansen, K.B. (1972). Immunofluorescent studies on mammalian spermatozoa: antigenic relationship between human, bull and rabbit spermatozoa. J. Reprod. Fertil., 29, 19-28. Hansmann, I., and H-D. Probeck (1979). The induction of non-disjunction by irradiation in marmnalian oogenesis and spermatogenesis. Mut. Res., 61, 69-76. Hargrove, J.L., and L.C. Ellis (1976). Autonomic nerves versus prostaglandins in the control of rat and rabbit testicular capsular contractions in vivo and in vitro. Biol. Reprod., 14, 651-657. Hartmann, S., J. Neeff, U. Heer, and D. Mecke (1978). Arenaemycin (pentalenolactone): a specific inhibitor of glycolysis. FEBS Lett., 93, 339-342. Hashimoto, Y., T. Makita, H. Miyata, T. Noguchi, and G. Ohta (1968). Acute and subchronic toxicity of a new fluorine pesticide, N-Methyl-N-(l-Naphthyl) fluoroacetamide. Toxicol. appl. Pharmacol., 12, 536-547. Hearn, J.P. (1979). Material Recognition of Pregnancy. (Ciba Foundation Symposium, new series no.64). Excerpta Medica, Amsterdam. pp.353-365. Heidger, P~.M., and C.L. Sawatzke (1977). Fine structural effects of vasectomy. In R.D. Yates and M. Gordon (Eds.), The Male Reproductive System, Masson Inc., New York. pp.131-153. Heinert, G., and H.D. Taubert (1973). Effect of cyproterone and cyproterone acetate on testicular function in the rat: a karyometric study. Endokrinologie, 61, 168-178. Hellema, H.W.J., and P.L. R~mke (1976). Comparison of the tray agglutination technique with the gelatin agglutination technique for the detection of spermagglutinating activity in human sera. Fertil., Steril., 27, 284-292. Hellema, H.W.J., and P. Rumke (1978). Sperm autoantibodies as a consequence of vasectomy. Clin. exp. I~m~unol., 31, 18-29. Heller, C.G., D.J. Moore, and C.A. Paulsen (1961). Suppression of spermatogenesis and chronic toxicity in men by a new series of bis (dichloroacetyl) diamines. Toxicol. Appl. Pharmacol., 3, I-II.
281
Biochemical and Immunological Prospects for Male Contraception Hillier, S.G., G.V. Broom, A.R. Boyns, and E.H.D. Cameron (1975). The active immunisation of intact adult rats against steroid-protein conjugates: effects on circulating hormone levels and related physiological processes. In E.H.D. Cameron, S.G. Hillier and K. Griffiths (Eds.), Steroid Immunoassays, Alpha Omega Publ., Cardiff Wales. pp.97-110. Hinkes, E., and D. Plotkin (1973). patient with acute leukemia.
Reversible drug-induced sterility in a J. Amer. Med. Assoc., 223, 1490-1491.
Hirschhauser, C., V. Kionke, and E. Daume (1971). Trypsin inhibitors in the female genital tract. Acta endocr. Copenh., 68, 413-416. Hitotsumachi, S., and Y. Kikuchi (1977). Chromosome aberrations and dominant lethality of mouse embryos after paternal treatment with triethylenemelamine. Mut. Res., 42, 117-124. Hodgen, G.D., W.R. Gomes, and N.L. Vandemark (1970). In vitro and in vivo effects of cadmium chloride on isoenzymes of carbonic anhydrase in rat testes and erythrocytes. Biol. Reprod., 2, 197-201 Holmdahl, T., and L. Mastroianni (1965). Continuous collection of rabbit oviduct secretions at low temperature. Fertil. Steril., 16, 587-595. Hoppe, P.C. (1976) Glucose requirement for mouse sperm capacitation in vitro. Biol. Reprod., 15, 39-45. Howards, S.S., S.J. Jessee and A.L. Johnson (1975). Micropuncture studies of the blood seminiferous tubule barrier. Biol. Reprod., 14, 264-296. Howards, S.S., C.J. Flickinger, D.W. Holler, and W.B. Panko (1979). The effects of vasectomy on testicular metabolism in the guinea-pig. J. Reprod. Fertil., 55, 401-405. Hsueh, A.J.W. (1978). Current developments in the mechanism of action of reproductive hormones. Clin. Obstet. Gynecol., 21, 53-66. Hsueh, A.J.W., M.L. Dufau, and K.J. Catt (1977). Gonadotropin-induced regulation of luteinizing hormone receptors and desensitization of testicular 3':5'-cyclic AMP and testosterone responses. Proc. Natl. Acad. Sci., U.S.A., 74, 592-595. Hsueh, A.J.W., and G.F. Erickson (1979). Extra-pituitary inhibition of testicular function by luteinising hormone releasin~ hormone. Nature, 66-67.
281,
Humphries, J., J.P. Wan, K. Flokers, and C.Y. Bowers (1976). Presence of proline in position 3 for potent inhibition of the activity of the luteinizing hormone-releasing hormone and of ovulation. Biochem. Biophys. Res. Commun., 72, 939-944. Humphries, J., Y.P. Wan, K. Folkers, and C.Y. Bowers (1978). Inhibitory analogues of the luteinizing hormone-releasing hormone havinB D-aromatic residues in positions 2 and 6 and variation in position 3. J. Med. Chem., 21, 120-126. Humphries, J., Y.P. Wan, T. Wasiak, K. Folkers, and C.Y. Bowers (1979). Structural requirements in positions I, 2, 3 and 6 of the luteinizing hormone-releasin~ hormone (LH-RH) for antiovulatory activity. J. Med. Chem., 22, 774-777.
282
P.J. Quinn
Husain, R., P.K. Seth, and S.V. Chandra (1976). dehydrogenase by manganese in rat gonads. 16, 118-121.
Early inhibition of succinate Bull. Env. Contam. Toxicol.,
Hyne, R.V., and B. Boettcher (1977). The selective binding of steroids by human spermatozoa. Contraception, 15, 163-174. Hyne, R.V., and B. Boettcher (1978). Binding of steroids to human spermatozoa and its possible role in contraception. Fertil. Steril., 30, 322-328. Igboeli, G., and A.M. Rakha (1970). Bull testicular and epididyrnal functions after long-term vasectomy. J. Anim. Sci., 31, 72-75. Ingerslev, H.J., and T. Hjort (1979). Spermagglutinating antibodies and B-spermagglutinins in sera from infertile and fertile women. Fertil. Steril., 31, 496-502. Isojima, S., K. Koyama, and K. Tsuchinya (1974). The effect of infertility in women of circulating antibodies against spermatozoa. J. Reprod. Fertil. (Suppl.), 21, 125-132. James, R.W., R. Heywood, J. Colley, and B. Hunter (1978). The oral toxicity of l-Amino-3-chloro-propanol hydrochloride (cl 88236) in rats. Toxicology, ii, 235-243. Jones, W.R. (1977). Irmnunological aspects of infertility. In J.S. Scott and W.R. Jones (Eds.), Immunology of Human Reproduction, Grune and Stratton, New York. pp. 375-413. Jones, R., and T. Mann (1976). Lipid peroxides in spermatozoa, role of plasmalogen and physiological significance. Proc. Roy. Soc. Lond. B., 193, 317-333. Jones, R., and T. Mann (1977). endogenous phospholipids.
Damage to spermatozoa by peroxidation of J. Reprod. Fertil., 50, 261-268.
Jones, R., T. Mann, and R. Sherins (1979). Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides and protective action of seminal plasma. Fertil. Steril., 31, 531-537. Johnsonbaugh, R.E., C.L. Czerwinski, and M. Edson (1977). Serum hormone levels before and two years after vasectomy. Contraception, 16, 563-568. Kalla, N.R. (1976). Chlorohydrins in male fertility regulation. Reprod., 28, 261-267.
Bibliogr.
Kalla, N.R., and B. Singh (1979). Effect of sodium nitrite on the ~-chlorohydrin-induced lesion of the testis-epididymis complex in the rat. J. Reprod. Fertil., 56, 149-151. Kanwar, K.C., R. Yanagimachi, and A. Lopata (1979). Effects of human seminal plasma on fertilizing capacity of human spermatozoa. Fertil. Steril., 31, 321-327. Kar~ J.K., and A.M. Phadke (1975). 26, 743-756.
Vaso-eDididymal anastomosis.
Fertil. Steril.,
283
Biochemical and Immunological Prospects for Male Contraception Katsh, S. (1962).
Antigenicity of human testis.
J. Urol.,
87,
896-902.
Katsh, S., and D.W. Bishop (1958). The effects of homologous testicular and brain and heterologous testicular homogenates combined with adjuvant upon the testis of guinea pigs. J. Embryol. exp. Morph., 6, 94-104. Kay, R., and N.J. Alexander (1978). Immunofluorescence titres of sperm centrifuged at different speeds. Clin. exp. Immunol., 32, 299-301 Kelly, R.W. (1977). Effect of seminal prostaglandins on the metabolism of human spermatozoa. J. Reprod. Fertil., 50, 217-222. Kelly, R.W., I. Cooper and A.A. Templeton (1979) Reduced prostaglandin levels in the semen of men with very high sperm concentrations. J. Reprod. Fertil., 56, 195-199.
Kesseru, E., P. C~acho-Ortega, G. Landahn, and G. S c h o p f l l n (1975). In v i t r o a c t i o n of progestogens on sperm m i g r a t i o n i n human c e r v i c a l mucus. Fertil. Steril., 26, 57-61. Kobrinsky, N.L., J.S. Winter, F.I. Reyes, and C. Faiman (1976). Endocrine effects of vasectomy in man. Fertil. Steril., 27, 152-156. Kolk, A.H.J., and T. Samuel (1975). Isolation, chemical and immunological characterization of two strongly basic nuclear proteins from human spermatozoa. Biochim. Biophys. Acta, 393, 307-319. Kolk, A.H.J., T. Samuel and P. Rumke (1974). Auto-antigens of human spermatozoa. I. Sohbilization of a new auto-antigen detected on swollen sperm heads. Clin. exp. Immunol., 16, 63-76. Krey, L.C., and A.J. Silverman (1978). The luteinizing hormone-releasing hormone (LH-RH) neuronal networks of the guinea-pig brain: II. The regulation on gonadotropin secretion and the origin of terminals in the median eminence. Brain Res., 157, 247-256. Kubota, R. (1969). Electron microscopic studies of the testis after vasectomy in rats and men. Jap. J. Urol., 60, 373-388. Lau, I.F., S. K. Saksena, and M.C. Chang (1974). Prostaglandin F in the uterine horns of mice with intrauterine devices. J. Reprod. Fert., 429-432.
37,
Linnet, L., and T. Hjort (1977). Sperm agglutinins in seminal plasma and serum after vasectomy; correlation between immunological and clinical findings. Clin. exp. Immunol., 30, 413-420. Louie, A.J., and G.H. Dixon (1972). Trout testis cells. II. Synthesis and phosphorylation of histones and protamines in different cell types. J. biol. Chem., 247, 5498-5505. MacLeod, J. (1970). The effects of urinary gonadotrophins following hypophysectomy and in hypogonadotrophic eunchoidism. Adv. Explt. Med. Biol., IO, 577-586. MacMillan, K.L., C. Desjardins, K.T. Kirton, and H.D. Hafs (1968). Gonadal and extragonadal sperm reserves after unilateral vasoligation in rabbits. Fertil. Steril., 19, 982-990.
284
P.J. Quinn
Madhwa Raj, H.G., and M. Dym (1976). The effects of selective withdrawal of FSH or LH on spermatogenesis in the immature rat. Biol. Reprod., 14, 489-494. Mancini, R.E. (1974). Immunologic and testicular response to a damage induced in the contralateral gland. In R.E. Mancini and L. Martin (Eds.), Male Fertility and Sterility. Academic Press, London. pp.271-3OO. Main, S.J., R.V. Davies, and B.P. Setchell (1978). Feedback control by the testis of gonadotropin secretion: an examination of the inhibin hypothesis. J. Endocrin., 79, 255-270. Mann, K., and D. Mecke (1979). Inhibition of spinach glyceraldehyde-3-phosphate dehydrogenases by pentalenolactone. Nature, 282, 535-536. Mann, T. (1964). The Biochemistry of Semen and the Male Reproductive Tract. Methuen, London. Marcus, Z.H., J.H. Freisheim, J.L. Houk, J.H. Herman, and E.V. Hess (1978). In vitro studies in reproductive immunology. I. Suppression of cellmedicated immune response by human spermatozoa and fractions isolated from human seminal plasma. Clin. Immunol. Immunopath., 9, 318-326. Marcus, Z.H., Y. Soffer, A. Ben-David, S. Peleg, and L. Nebel (1973). Studies on sperm antigenicity. I. Delayed hypersensitivity to spermatozoa. Europ. J. Immunol., 3, 75-78. Maroni, E.S., D.N.K. Symon, and P.C. Wilkinson (1972). Chemotaxis of neutrophil leucocytes towards spermatozoa and seminal fluid. J. Reprod. Fertil., 28, 359-368. Maroni, E.S., and P.C. Wilkinson (1971). Selective chemotaxis of macrophages towards human and guinea-pi~ spermatozoa. J. Reprod. Fertil., 27, 149-152. Mastroianni, L., M. Urzua, and R. Stambaugh (1973). The internal environmental fluids of the oviduct. In S.J. Segal, R. Crozier, P.A. Corfman, and P.G. Condliff (Eds.), The Regulation of Mammalian Reproduction, Charles C. Thomas, Springfield, Ill. pp.376-384. Mattner, P.E. (1969). Differential leucocytic responses to spermatozoa in the cervic and the uterus in ewes. J. Reprod. Fertil., 18, 297-303. Mazzanti, L., M. Lopez, and M.G. Verti (1965). Atrofia del testicolo prodotta dal monofluoroacetato sodico nel ratto albino. Experentia, 21, 446-447. McRorie, R.A., and W.L. Williams (1974). Biochemistry of mammalian fertilization. Ann. Rev. Biochem., 43, 777-833. Meizel, S., and C.W. Lui (1976). Evidence for the role of a trypsin-like enzyme in the sperm acrosome reaction. J. Exp. Zool., 195, 137-140. Menge, A.C. (1971). Antiserum inhibition of rabbit spermatozoal adherence to ova. Proc. Soc. exp. Biol. Med., 138, 98-102. Menge, A.C., and W.P. Protzman (1967). Origin of the antigens in rabbit semen which induce antifertility antibodies. J. Reprod. Fertil., 13, 31-40.
Biochemical and Immunological Prospects for Male Contraception
285
Menge, A.C., and W.P. Protzman (1967). Origin of the antigens in rabbit semen which induce antifertility antibodies. J. Reprod. Fertil., 13, 31-40. Metz, C.B., and J. Anika (1970). Failure of conception in rabbits inseminated with nonagglutinating, univalent antibody-treated semen. Biol. Reprod., 2, 284-290. Metz, C.B., M.G. O'Rand, and C.T. Cordle (1976). Agglutination and immobiliza6on of human spermatozoa. In E.S.E. Hafez (Ed.), Fertility Regulation in Men, C.V. Mosby Co., St. Louis, pp.276-286. Mishell, D.R. (1975). Intravaginal rings for contraceptive use: comment. Contraception, 12, 249-252.
an editorial
Miyamoto, H., and M.C. Chang (1973). The importance of serum albumin and metabolic intermediates for capacitation o~ spermatozoa and fertilization of mouse e~gs in vitro. J. Reprod. Fertil., 32, 193-205. Moersch, G.W., M.C. Rebstock, E.Lo Wittle, F.J. Tinney, E.D. Nicolaides, M.P. Hutt, T.E. Mich, J.M. Vandenbelt, R.E. Edgren, J.R. Reel, W.C. Dermody, and R.R. Humphrey (1979). Synthesis and biological activity of peptide antagonists of Luliberin (Luteinizing hormone-releasing hormone). J. Med. Chem., 22, 935-943. Moghissi, K.S. (1970). Human Fallopian tube fluid. Fert. Steril., 21, 821-829.
I. Protein composition.
Mohri, H., D.A.I. Suter, P.D.C. Brown-Woodman, I.G. White, and D.D. Ridley (1975). Identification of the biochemical lesion produced by ~-chlorohydrin in spermatozoa. Nature, 255, 75-77. Moltz, L., A. Roemmler, U. Schwartz, and J. Hammerstein (1978). Effects of cyproterone acetate (CPA) on pituitary ~onadotropin release and on androgen secretion before and after LH-RH double stimulation in men. Int. J. Androl. (suppl) 2, 713-719. Monastirsky, R., K.A. Laurence, and E. Tovar (1971). The effects of gonadotrophin immunization of prepubertal rabbits on gonadal development. Fertil. Steril., 22, 381-325. Moudgal, R.N., V.R. Mukku, S. Prahalada, G.S.R.C. Murty, and C.H. Li (197B). Passive immunization with an antibody to the 6-subunit of ovine luteinizin~ hormone as a method of early abortion - a feasibility study in monkeys (Macaca radiata). Fertil. Steril., 30, 223-229. Muir, V.V., J.L. Turk, and H.G. Hanley (1977). Comparison of allergic aspermatogenesis with that induced by vasectomy. II in vitro studies of cell-mediated immunity to sperm after vasectomy in man and guinea-pi~. Clin. exp. Immunol., 28, 461-466. Nagarkatti, S., and S.S. Rao (1976). Cell mediated ir~nunity to homologous spermatozoa followin~ vasectomy in the human male. Clin. exp. Immunol., 26, 239-242. Naik, V.K., A.N. Thaleur, A.R. Sheth, U.M. Joshi, S.S. Rao, D.S. Pardanani, J.K. Kulsreshtla, and R.K. Handa (1976). The effect of vasectomy on pituitary-gonadal function in men. J. Reprod. Fertil., 48, 441-448.
286
P.J. Quinn
Nakamura, M., and P.F. Hall (1977). Effect of 5-thio-D-glucose on protein synthesis in vitro by various types of cell from rat testes. J. Reprod. Fertil., 49, 395-397. Nakamura, M., and P.F. Hall (1978). The influence of temperature upon polysomes of spermatids of rat testes. Biochem. Biophys. Research Commun., 85, 756-761. National Coordinating Group on Male Antifertility Agents (1978). Gossypol: new antifertility agent for males. Chin. Med. J. (Engl.Ed.), i, 417-428. Neaves, W.B. (1975). Biological aspects of vasectory. ~, 7 pp.382-404.
In Handbook of Physiology
Nelson, W.O., and R.G. Bunge (1957). The effect of therapeutic doses of nitrofurantoin (Furadantin) upon spermatogenesis in man. J. Urol., 275-282.
7--7,
Nelson, W.O., E. Stemberger, and A. Boccabella (1954). Recovery of spermatogenesis in rats treated with nitrofurans. Anat. Rec., 118, 333-338. Neumann, F., R.C. Nicholson, B. Schenck, and H. Steinbeck (1978). The physiological action of progesterone and the pharmacological effects of progestogens - a short review. Int. J. Androl., suppl, no.2 , pp.147-154. Nicander, L. (1967). An electron microscopical study of cell contacts in the seminiferous tubules of some mammals. Z.Zellforsch.Mikroskop.Anat., 83, 375-397. Nielsen, J., and J. Sillesen (1975). among 11,148 newborn children.
Incidence of chromosome aberrations Hum. Genet., 30, 1-12.
Nieschlag, E., K-H. Usadel, E.J. Wickings, H.K. Kley, and W. Wuttke (1975). Effects of immunization with steroids on endocrine functions in male animals. In E. Nieschlag (Ed.), Immunization with Hormones in Reproductive Research, North Holland, Amsterdam. pp.155-170. Niwa, K., and A. Iritani (1978). Effect of various hexoses on sperm capacitation and penetration of rat eggs in vitro. J. Reprod. Fertil., 53, 267-271. O'Malley, B.W., and A.R. Means (1974). Female steroid hormones and target cell nuclei. Science, 183, 610-620. Otani, Y., and J. Behrman (1963). homologous testis and sperm.
Immunization of the guinea pig with Fertil. Steril., 14, 456-467.
Pabst, R., O. Martin, and H. Lippert (1979). Is the low fertility rate after vasectomy caused by nerve resection during vasectomy? Fertil. Steril., 13, 316-320. Paufler, S.K., and R.H. Foote (1969). Spermatogenesis in the rabbit following ligation of the epididymidls at different levels. Anat. Rec., 164, 339-348. Paul, H.E., M.F. Paul, F. Kopko, R.C. Bender, and G. Everett (1953). Carbohydrate metabolism studies on the testis of rats fed certain nitrofurans. Endocrinology, 53, 585-592.
Biochemical and Immunological Prospects for Male Contraception
287
Paulsen, C.A., and J.M. Leonard (1976). Clinical trials in reversible male contraception. I. Combination of danazol plus testosterone. In C.H. Spilman, T.J. Lobl and K.T. Kirton (Eds.), Regulatory Mechanisms of Male Reproductive Physiology, Excerpta Medica, Amsterdam. pp.197-210. Pedroza, E., J.A. Vilchez-Martinez, D.H. Coy, A. Arimura, and A.V. Schally (1978). Correlation between in vivo inhibition of gonadotropin release induced by LH-RH and the blockade of ovulation by synthetic analogues of LH-RH. Int. J. Fertil., 23, 294-299. Perrenoud, A. (1974). Malthusianisme et Protestantisme; Weberien. Annal. E.C.S., 29, 983-985. Peters, R.A. (1952).
Lethal synthesis.
un modNle d~mographique
Proc.Roy. Soc., B,
139, 143-170.
Potashnik, G., N. Ben-Aderet, R. Israeli, I. Yanai-Inbar, and I. Sober (1979). Suppressive effect of 1,2-dibromo-3-chloropropane on human spermatogenesis. Fertil. Steril., 30, 444-447. Purvis, K., S.K. Saksena, Z. Cekan, E. Diczfalusy, and J. Giner (1976). Endocrine effects of vasectomy. Clin. Endocrinol., 5, 263-269. Reijonen, K., M. Kormano, and R.J. Ericsson (1975). Studies on the rat epididymal blood vessels following alpha-chlorohydrin administration. Biol. Reprod., 12, 483-490. Reyes, A., B. Goicoechea, and A. Rogado (1978). Calcium ion requirement for rabbit spermatozoal capacitation and enhancement of fertilizing ability by ionophore A23187 and cyclic 3':5'-monophosphate. Fert Steril., 29, 451-455. Rogers, B.J., and R. Yanagimachi (1976). Competitive effect of magnesium on the calcium-dependent acrosome reaction in guinea-pig spermatozoa. Biol. Reprod., 15, 614-619. Rommerts, F.F.G., B.A. Cooke, and H.J. van der Molen (1974). The role of cyclic AMP in the regulation of steroid biosynthesis in testis tissue. J. Steroid Biochem., 5, 279-285. It
Rumke, P., and M. Titus (1970). Spermagglutinin formation in male rats by subcutaneously injected syngeneic epididymal spermatozoa and by vasoligation or vasectomy. J. Reprod. Fertil., 21, 69-79. Russell, L.D, and Y. Clermont (1977). Degeneration of germ cells in normal hypophysectomised and hormone treated hypophysectomised rats. Anat. Rec., 187, 347-366. Saksena, S.K., and M.J.K. Harper (1974). Prostaglandin-mediated action of the intrauterine devices: F-prostaglandins in the uterine horns of pregnant rabbits with unilateral intrauterine devices. Fertil. Steril., 25, 121-126. Saksena, S.K., I.F. Lau, and V.D. Castrachane (1974). Prostaglandin mediated action of IUD's. II. F-prostaglandins (PGF) in the uterine horn of pregnant rats and hamsters with intrauterine devices. Prostaglandins, 97-106. Samuel, T. (1977). Antibodies reacting with salmon and human protamines in sera from infertile men and from vasectomized men and monkeys. Clin. exp. Immunol., 30, 181-187.
,
288
P.J. Quinn II
Samuel, T., N.J. Alexander, and P. Rumke (1977). Reproducibility of the indirect immunofluorescence test on swollen human spermheads. Clin. exp. Immunol., 28, 223-225. Samuel, T., A.H.J. Kolk, P. Rumke, L.A. Aarden, and M. Bustin (1976). and DNA detection in swollen spermatozoa and somatic cells, by immunofluorescence. Clin. exp. Immunol., 24, 63-71.
Histone
Samuel, T., A.H.J. Kolk, P. Rumke, and J.M.J. Van Lis (1975). Autoimmunity to sperm antigens in vasectomized men. Clin. exp. Immunol., 21, 65-74. Sandow, J., W. Von Rechenber~, and G. Jerzabek (1977). Endocrine effects o£ chronic treatment with the LH-RH analogue D-SER (TBU)6 ethylamide. Acta Endocrinol., 84, 33-41. Sandow, J., W. Von Rechenberg, G. Jerzabek, and W. Stoll (1978). Pituitary gonadotropin inhibition by a highly active analog of luteinizing hormonereleasing hormone. Fert. Steril., 30, 205-209. Schally, A.V. (1977). Aspects of hypothalamic regulation of the pituitary gland with major emphasis on its implications for the control of the reproductive processes. In: Prix Nobel pp.201-234. Schally, A.V., A. Arimura, A.J. Kastin, H. Matsuo, Y. Baba, R.W. Redding, R.M.G. Nair, and L. Debeljuk (1971). Gonadotropin-releasing hormone: one polypeptide regulates secretion of luteinizing and folliclestimulating hormones. Science, 173, 1036-1037. Schally, A.V., A.J. Kastin and D.H. Coy (1976). LH-releasing hormone and its analogues: recent basic and clinical investigations. Int. J. Fertil., 21, 1-30. Schmidt, S.S. (1975). 47, 309-314.
Vas anastomosis:
a return to simplicity.
Brit. J. Urol.,
Schoenfeld, C., R.D. Amelar, and L. Dubin (1976). Clinical experience with sperm antibody testing. Fertil. Steril., 27, 1199-1203. Seprodi, J., D.H. Coy, J.A. Vilchez-Martinez, E. Pedroza, and A.V. Schally (1978a) Branched-chain analogues of luteinizing hormone-releasing hormone. J. Med. Chem., 21, 276-280. Seprodi, J., D.H. Coy, J.A. Vilchez-Martinez, E. Pedroza, W-Y. Huang, and A.V. Schally (1978b). Cyclic analogues of luteinizing hormone-releasing hormone with significant biological activities. J. Med. Chem., 21, 993-995. Setchell, B.P. (1978).
The Mammalian Testis, Paul Elek, London.
Setchell, B.P., R.V. Davies, and S.J. Main (1977). Inhibin. In A.D. Johnson and W.R. Comes (Eds.), The Testis, Vol. IV. Academic Press, New York. pp. 189-238. Sherins, R.J., and V.T. De Vita (1973). Effect of drug treatment for lymphoma on male reproductive capacity. Studies of men in remission after therapy. Ann. Intern. Med., 79, 216-219.
289
Biochemical and Immunological Prospects for Male Contraception Shiu, R.P.C., and H.G. Friesen (1976). Blockade of prolactin action by an antiserum to its receptors. Science, 192, 259-261. Shivers, C.A.,and B.S. Dunbar (1977). Autoantibodies to zona pellucida: possible cause of infertility in women. Science, 197, 1082-1084.
a
Shulman, S. (1971). Antigenicity and autoimmunity in sexual reproduction: a review. Clin. exp. Immunol., 9, 267-288. Shulman, S,, H. Jackson, and M.L. Stone (1975). Comparative studies of sperm agglutinating activity in groups of infertile and fertile women. Am. J. Obstet. Gynecol., 125, 139-145. Silverman, A.J., L.C. Krey, and E.A. Zimmerman (1979). A comparative study of the luteinizing hormone releasing hormone (LH-RH) neuronal networks in mammals. Biol. Reprod., 20, 98-110. Silverman, A.J., and E.A. Zimmerman (1978). Pathways containing luteinizing hormone-releasing hormone (LH-RH) in the mammalian brain. In D.E. Scott (Ed.), Neural Hormones and Reproduction, Third Brain-Endocrine Interaction Symposlum. Karger, Basel. pp. 83-96. Skakkebaek, N.E., R.W. Kelly, and C.S. Corker (1976). Prostaglandin concentrations in the semen of hypogonadal men during treatment with testosterone. J. Reprod. Fertil., 47, 119-121. Skinner, J.D., and L.E.A. Rowson (1968). Some effects of unilateral cryptorchism and vasectomy on sexual development of the pubescent ram and bull. J. Endocrin., 42, 311-321. Skoglund, R.D., and C.A. Paulsen (1973). Danazol-testosterone combination: a potentially effective means of reversible male contraception, a preliminary report. Contraception, 2, 357-361. Smith, K.D., R.K. Tcholakian, M. Chowdhury, and E. Steinberger (1976). An investigation of plasma hormone levels before and after vasectomy. Fertil. Steril., 27, 144-151. Soupart, P. (1970). Leukocytes and sperm capacitation in the rabbit uterus. Fertil. Steril., 21, 724-756. Srivastava, P.N. (1973).
Location of zona lysin.
Biol. Reprod.
9,
84.
Stambaugh, R., and J. Buckley (1972). Histochemical subcellular localization of the acrosome proteinase effecting dissolution of the zona pellucida using fluorescein-labelled inhibitors. Fertil. Steril., 23, 348-352. Stambaugh, R., H.M. Seitz, and L. Mastroianni (1974). Acrosomal proteinase inhibitor in rhesus monkey oviduct fluid. Pert. Steril., 25, 352-357. Steinberger A., and E. Steinberger (1976). Secretion of an FSH-inhibiting factor by cultured Sertoli cells. Endocrinology, 99, 918-921. Steinberger, E., and B.N. Sud (1970). Specific effect of fluoroacetamide on spermatogenesis. Biol. Reprod., 2, 369-375. Stocks, J., J.M.C. Gutteridge, R.J. Sharp and T.L. Dormandy (1974). The inhibition of lipid autoxidation by human serum proteins and ~-tocopherol. Clin. Sci. Mol. Med., 47, 223-230
290
P.J. Quinn
Stokes, C.R., J.F. Soothill, and M.W. Turner (1975). function of I g A . Nature, 255, 745-746.
Irmnune exclusion is a
Sullivan, M.J.,and G.E. Howe (1977). Correlation of circulatin~ antisperm antibodies to functional success in vasovasectomy. J. Urol., 117, 189-191. Sullivan, J.L., F.A. Smith, and R.H. Garman (1979). Effects of fluoroacetate on the testis of the rat. J. Reprod. Fertil., 56, 201-207. Swift, A.D., and D.B. Crighton (1978). Relative activity, plasma elimination and hypothalamic and pituitary degradation of synthetic luteinizing hormone releasing hormone and certain of its analogues. J. Endocrinol., 77, 35P. Swift, A.D., and D.B. Crighton (1979a). Relative activity, plasma elimination and tissue degradation of synthetic luteinizing hormone releasin~ hormone and certain of its analogues. J. Endocrinol., 80, 141-152. Swift, A.D., and D.B. Crighton (1979b). The effects of certain steroid hormones on the activity of ovine hypothalamic luteinizin~ hormone-releasing hormone (LH-RH)-degrading enzymes. FEBS Letts., IO0, 110-112. Szemere, G., and A.C. Chandley (1975). Trisomy and Triploidy induced by X-irradiation of mouse spermatocytes. Mut. Res., 3_33, 229-238. Talaat, M., and K.A. Laurence (1971). Impairment of spermatogenesis and libido through antibodies to luteinizing hormone. Fertil. Steril., 113-118. Talwar, G.P. (1978). contraception.
2--2,
The present and future of immunologic approaches to Int. J. Gynaecol. Obstet., 15, 410-414.
Talwar, G.P., N.C. Sharma, S.K. Dubey, M. Salahuddin, C. Das, S. Ramakrishnan, S. Kumar, and V. Hingorani (1976a). Isoin~nunization against human chorionic gonadotropin with conjugates of processed 6-subunit of the hormone and tetanus toxoid. Proc. Natl. Acad. Sci. U.S.A., 73, 218-222. Talwar, G.P., N.C. Sharma, S.K. Dubey, M. Salahuddin, N. Shastri, and S. Ramakrishnan (1976b). Processing of the preparations of 6-subunit of human chorionic gonadotropin for minimization of cross-reactivity with human luteinizing hormone. Contraception, 13, 131-139. Tang, L.K.L. (1978). Sex difference in LH response to LHRH and DBcAMp and effect of testosterone. Amer. J. Physiol., 235, E291-E294. Tcholakian, R.D., A. de la Cruz, A.K. Chowdhury, M. Chowdhury, A.V. Schally, and A. Steinberger (1978). Effects of an LH-RH analogue in male rats pretreated with oestradiol benzoate. J. Reprod. Fert., 54, 441-445. Templeton, A.A., I. Cooper, and R.W. Kelly (1978). Prostaglandin concentrations in the semen of fertile men. J. Reprod. Fert., 52, 147-150. Thau, R.B., K. Sundaram, Y.S. Thornton, and L.S. Seidman (1979). Effects of immunization with the 6-subunit of ovine luteinizing hormone on corpus luteum function in the rhesus monkey. Fert. Steril., 31, 200-204.
291
Biochemical and Immunological Prospects for Male Contraception Thompson, I.E. (1973). The IUD and prostaglandins: Obstet. Gynaecol., 42, 617-620.
a review of the evidence.
Thrall, R.S., L.B. Peterson, J.H. Lineham, P. Abramoff, and V.L. Moore (1978). The effect of immunization on the uptake of intratracheally administered antigen. Clin. Immunol. Ir~nunopath., IO, 136-147. Tougard, C., B. Kerdelhue, A. Tixier-Vidal, and M. Jutisz (1971). Localisation par cyto-immunoenzymologie de la LH, de ses sous-unit~s ~ et B e t de la F SH dans l'ad~nohypophyse de la ratte castr~e. Comp. Rend. Acad. Sci. Paris, S~rie D. 273, 897-900. Toullet, F., and G.A. Voisin (1974). Spermatotoxic, spermagglutinating and cytotoxicactivities of guinea-pig autoantibodies to sperm autoantigen T. J. Reprod. Fertil., 37, 299-313. Toullet, F., G.A. Voisin, and M. Nemirovsky (1973). Histoimmunochemical localization of three guinea-pig spermatozoal autoantigens. Immunology, 24, 635-653. Tung, K.S.K. (1975). Human sperm antigens and antisperm antibodies. on vasectomy patients. Clin. exp. Immunol., 20, 93-104.
I. Studies
Turner, T.T., D.A. D'Addario, and S.S. Howards (1979). Effects of vasectomy on the blood-testis barrier of the hamster. J. Reprod. Fertil., 55, 323-328. Uehling, D.T. 769-773.
(1971).
Secretory IgA in seminal fluid.
Fertil. Steril.,
22,
Uematsu, K. (1966). Testicular changes of rats induced by nitrofurazone; a light and electron microscopic study. Med. J. Osaka Univ., 16, 287-306. Ulstein, M., N. Netto, J. Leonard, and C.A. Paulsen (1975). Changes in the sperm morphology in normal men treated with danazol and testosterone. Contraception, 12, 437-444. Vale, W., G. Grant, M. Amoss, R. Blackwell, and R. Guillemin (1972). of enzymatically dispersed anterior pituitary cells. Functional validation of a method. Endocrinology, 91, 562-572.
Culture
Vale, W., C. Rivier, M. Brown and J. Rivier (1977). Pharmacology of thyrotropin releasing factor (TRF), luteinizing hormone releasing factor (LRF) and somatostatin. Adv. exp. med. Biol., 87, 123-156. Vare, A.M., and P.C. Bansal (1973). Changes in the canine testes after bilateral vasectomy - an experimental study. Fertil. Steril., 24, 793-797. Vilchez-Martinez, J.A., D.H. Coy, E.J. Coy, A. Arimura, and A.V. Schally (1976). Comparison of the anti-LH/FSH-RH and anti-ovulatory activities of [D-Phe2,D-Leu 6] - LH-RH and [D-Phe2,D,AIa 6] - LH-RH. Endocr. Res. Commun., ~, 231-238. Vito, C.C., S.J. Wieland, and T.O. Fox (1979). Androgen receptors exist throughout the 'critical period' of brain sexual differentiation. Nature, 282, 308-310.
292
P.J. Ouinn
Volgmayr, J.K. (1974). Alpha-chlorohydrin-induced changes in the distribution of free myo-inositol and prostaglandin F2~ , and synthesis of phosphatidylinositol in the rat epididymis. Biol. Reprod., II, 593-600. Voisin, G.A., and F. Toullet (1968). Etude sur l'orchide aspermatogenetique autoimmune et les autoantigenes de spermatozoides chez le cobaye. Ann. Inst. Pasteur, Paris, 114, 727-755. Vojtigkov~, M., M. Pol~kova and Z. Pokornfi (1969). Histocompatibility on mouse spermatozoa. Folia Biol.(Prague) 15, 322-332.
antigens
Weinman, D.E., and W.L. Williams (1964). Mechanism of capacitation of rabbit spermatozoa. Nature, 203, 423-424. Whitby, R.M., R.D. Gordon and B.R. Blair (1979). vasectomy: a prospective five-year study. ~0
The endocrine effects of Fertil. Steril., 21, 518-520.
Reference Bank for Reproductive Immunology (1977). Auto- and iso-antibodies to antigens of the human reproductive system. Clin. exp. Immunol., 30, 173-180.
Wickings, E.J., and E. Nieschlag (1978). The effects of active immunization with testosterone on pituitary-gonadal feedback in the male rhesus monkey (Macaca mulatta). Biol. Reprod., 18, 602-607. Wickings, E.J., C.A. Tafurt, H. Hoogen, and E. Nieschlag (1977). of titre and specificity of antibodies to testosterone. J. Steroid Biochem., 8, 147-150.
The development
Wilson, L. (1954). Sperm agglutinins in human semen and blood. Biol. Med., 85, 652-655.
Proc. Soc. exp.
Wilson, L. (1956). ~, 262-267.
Sperm agglutination due to autoantibodies.
Fertil. Steril.,
Wilson, B.J., N.J. Alexander, G. Porter, and D.L. Fulgham (1977). Cell-mediated immunity in vasectomised rhesus monkeys. Fertil. Steril., 28, 1349-1355. Wilson, B.J., G. Porter, and A. Goldstein (1979). Reduced T cell reactivity in vasectomised rhesus monkeys: association with histocompatibility type. Pertil. Steril., 31, 434-440. Wilkinson, P.C. (1974). Edinburgh,
Chemotaxis and Inflammation.
Churchill Livingstone,
Witkin, S.S., and P.J. Higgins (1978). Effects of vasectomy and antisperm antibodies on human seminal fluid deoxyribonucleic acid polymerase activity. Fertil. Steril., 29, 314-319. Yanagimachi, R. (1970). The movement of golden hamster spermatozoa before and after capacitation. J. Reprod. Fert., 23, 193-196. Yanagimachi, R. (1972). Penetration of guinea-pig spermatozoa into hamster eggs in vitro. J. Reprod. Fertil., 28, 477-480. Yanagimachi, R., and R.J. Teichman (1972). Cytochemical demonstration of aerosomal proteinase in mammalian and avian spermatozoa by a silver proteinate method. Biol. Reprod., 6, 87-97.
Biochemical and Immunological Prospects for Male Contraception Yanagimachi, R., and N. Usui (1974). Calcium dependence of the acrosome reaction and activation of guinea-pig spermatozoa. Exptl. Cell Res., 161-174.
293 89,
Yen, S.S.C., R. Rebar, G. Vandenberg, F. Naftolin, H. Judd, Y. Ehara, K.J. Ryan, J. Rivier, M. Amoss, and R. Guillemin (1973). Clinical studies with synthetic LRF (luteinizing hormone-releasing factor). In C° Gual and E. Rosemberg (Eds.), Hypothalamus Hypophysiotropic Hormones. Excerpta Medica, Amsterdam. pp.217-229. Zaneveld, L.J.D. (1976). Sperm enzyme inhibitors as antifertility agents. In E.S.E. Hafez (Ed.), Human Semen and Fertility Regulation in Men. C.V. Mosby Co., St. Lou'is. pp.570-582. Zysk, J.R., A.A. Bushway, R.L. Whistler, and W.W. Carlton (1975). Temporary sterility produced in male mice by 5-thio-D-glucose. J. Reprod. Fertil., 45, 69-72.
0098-2997/80/0601-0225-$05.00/0
BIOCHEMICAL AND IMMUNOLOGICAL PROSPECTS FOR MALE CONTRACEPTION P. J. Quinn
Department of Biochemistry, Chelsea College, University of London, London SW3 6LX, UK
Contents I.
I ntrodu cti o n
226
2.
Control of the Hypothalamohypophyseal Axis 2.1. Mechanism of Action of Male Sex Hormones 2.1.1. Peptide hormones 2.1.2. Steroid hormones 2.2. Hormonal Regulation of Male Reproduction 2.2.1. Lutenising hormone - releasing hormone 2.2.2. P i t u i t a r y gonadotrophins 2.2.3. Inhibin
229 229 230 232 234 234 236 240
3.
Regulation of Testicular Function and Sperm Maturation 3.1. Interference of Spermatogenesis by Chemical Agents 3.2. Effects of Temperature and I r r a d i a t i o n 3.3. Biochemical and Immunological Aspects of Vasectomy
242 243 247 249
Survival of Spermatozoa in the Female Tract and F e r t i l i z a t i o n 4.1. Factors Affecting Sperm V i a b i l i t y and Transport 4.1.1. The action of prostaglandins on f e r t i l i t y 4.1.2. Steroid binding and sperm transport 4.2. Developmental A l t e r a t i o n s to Spermatozoa in the Female Tract 4.2.1. Capacitation and decapitation processes 4.2.2. The acrosome reaction 4.2.3. Control of capacitation and the acrosome reaction
259 259 260 262 263 263 264 268
Conclusions and Future Prospects
272
Acknowledgement
273
References
274
.
5.
225
Chapter 1
Introduction
Until comparatively recently contraceptive practices were reserved largely f o r breeding domestic animals.
Since B i b l i c a l
times the a t t i t u d e of man towards
contraception has been subject to theological constraints best exemplified by the teachings about the sin of Oman and his punishment.
The qradual separation of
pleasure from procreation ~ollowin~ the nrowth of Protestant ideology created a more positive a t t i t u d e to contraception as an unintended legacy to the secularized world of the eighteenth century.
The need for such practices, however, was not
dictated by demographic considerations but rather by the a t t i t u d e s of husbands towards the sufferings of t h e i r wives who were subjected to the anonies and dangers of repeated pregnancies and c h i l d b i r t h s .
There were also sound economic
reasons to practice contraception which emerged at this time.
~here the cost of
upbringing and launching a child into the world exceeded the value of his e x p l o i t a b l e labour and usefulness to his parents in t h e i r old ane there was c l e a r l y an incentive to l i m i t the size of the family. e l i t e of Europe were the f i r s t
The aristocracy and urban
group to experience these economic nressures
because they did not depend upon the labour of t h e i r children but nevertheless were obliged to bear the r i s i n g costs of t h e i r education and marriane. Furthermore, as respect f o r children developed within the society nreater care in t h e i r nurturing and upbringing was practiced and the level of care was dispensed to each individual
in d i r e c t proportion to the resources available.
Contraceptive methods and practices were at this time l a r q e l y the r e s p o n s i b i l i t y of the male partner.
They included oral or manual sex or c o i t u s i n t e r r u p t u s .
Practice of the l a s t method is c l e a r l y documented in the case of a young Massachusetts man cited by Perrenoud (Iq74) who, in 1771, was successfully
226
227
Biochemical and Immunological Prospects for Male Contraception
prosecuted in a paternity suit despite his protestation that ' I fucked her once, but I minded my pullbacks.'
Othermethods advocated in Elizabethan cookbooks,
herbals and medical treatises included the reduction of sexual desire by violent vomits, purges or bloodletting which rendered the victim too sick or weak to indulge in s e x .
Anotherapparently effective method was the application of
various ointments to the penis to prevent an erection.
The e a r l i e s t recorded
use of condoms was in the late seventeenth century but they did not come into common use until the eighteenth century.
The primary use of these devices,
however, was more than l i k e l y to avoid contracting venereal disease rather than simply to act as a contraceptive device.
Abortifacients, some with disastrous
after effects, were also employed in situations where contraceptive methods
f a i l e d to prevent unwanted pregnancies, Methods changed l i t t l e
until
comparatively recent times when i n s i g h t into the
hormonal regulation of human f e r t i l i t y female.
began to emerge, p a r t i c u l a r l y that of the
Over the past two decades three generations of hormonal female oral
contraceptives have evolved.
Sequential oral contraceptives that ostensibly
mimic the normal hormonal cycle in women and progestin-only compounds have been l a r g e l y withdrawn from use e i t h e r because of undesirable side e f f e c t s or unreliable contraceptive potency.
Of the remainder, the combination p i l l
has proved the
most successful but, judging from contemporary prospective and retrospective reviews of t h e i r usefulness combined with concerns of stroke, myocardial i n f a r c t i o n and hepatomes indicate that more s a t i s f a c t o r y methods of contraception are required.
Current methods of female contraception include the development of
improved combination p i l l s
based on antioestrogens and antiprogestins,
which have already proved suitable for selective use (David et a l . ,
some of
1979).
The development of r e l i a b l e and reversible male contraceptives has tended to lag somewhat behind those methods developed s p e c i f i c a l l y for women.
This may simply
r e f l e c t the fundamental differences in the processes of d i f f e r e n t i a t i o n
and
development of gametes in the male and female and the control mechanisms that regulate these processes.
This means that the basic approach to contraception,
the objectives of which are to r e v e r s i b l y prevent conception with the minimum inconvenience and undesirable side e f f e c t s , often requires a fundamentally d i f f e r e n t approach.
This can be i l l u s t r a t e d
by the simple f a c t that the germinal
c e l l s of the male gonad develop at puberty and p e r s i s t throughout reproductive life.
Consequently, chemical methods of contraception are often hazardous in
that they are l i a b l e to i n f l i c t
genetic damage during the production of mature
gametes that can introduce congenital defects into o f f s p r i n n .
This contrasts
with the female where germinal c e l l s have already entered into the f i r s t
meiotic
228
P.J. Quinn
d i v i s i o n by the time of p a r t u r i t i o n and therefore they are not as vulnerable to genetic a l t e r a t i o n s which could be transmitted to proqeny.
The other major
consideration with regard to male contraception is the need to preserve the psychic drive associated with sex i n s t i n c t or l i b i d o .
Suppression of spermato-
genesis can often lead to impotence which although s a t i s f y i n g the basic c r i t e r i a f o r contraception is obviously an untenable side e f f e c t . I t is against this background of the need to avoid genetic defects and the development of impotence that new and e f f e c t i v e methods of male contraception are being sought.
The following sections review the current status of research
in this area and evaluate the future prospects for male contraception.
Chapter 2
Control of Hypothalamohypophyseal Axis
The f i r s t stage of intervention in altering male reproductive capacity is at the level at which hormonal control of the process is exercised.
Hormonal control
of spermatogenesis and testicular function is achieved by a cascade mechanism subject to fee-back control by products produced in the testis.
The precise
checks and balances in the system are not yet completely understood but the role of the individual hormones involved is becoming clearer.
!,!hat is certain,
however, is that any disturbance in the hormonal balance invariably leads to dysfunction of the reproductive process.
Such disturbances can result from
abnormal release of one or several hormones from the endocrine system or some malfunction of the action of the hormone on i t s target cell.
Both these
processes are possible targets for contraception. 2.1. Mechanism of Action of Male Sex Hormones
Two types of hormone are concerned in male reproductive regulation; peptide hormones and steroid hormones.
The peptide or protein hormones include luteinis.
ing hormone-releasing hormone (LH-RH), produced by the hypothalamus and two gonadotrophin hormones, luteinising hormone (LH) and f o l l i c l e stimulating hormone (FSH) 6riginating from the anterior p i t u i t a r y .
LH-RH stimulates the synthesis
and release of gonadotrophins from the p i t u i t a r y (Yen et a t . , 1973; Schally, 1977; Guillemin, 1978).
Both gonadal functions, namely spermatogenesis and
androgen secretion, are s t r i c t l y regulated by the p i t u i t a r y gonadotrophins; FSH acts on Sertoli cells where i t is required to i n i t i a t e spermatogenesis and LH regulates testosterone production and secretion from the Leydig cells. I t is noteworthy that human chorionic gonadotrophin (hCG) has similar effects on androgen production by Leydig cells, suggesting the presence of receptor sites for hCG on Leydig cells.
229
230 2.l.l.
P.J. Quinn Peptide homones
The mode of action of LH-RH, LH and FSH on t h e i r target c e l l s is similar to that of most other peptide hormones (see Hsueh, 1978).
Hormone bindinq to specific
receptor sites located on the external surface of the plasma membrane of the target c e l l acts as a positive a l l o s t e r i c modulator of adenylcyclase, the c a t a l y t i c s i t e of which is accessible only from the cytoplasmic surface of the membrane.
These reactions are i l l u s t r a t e d
schematically in Fin. I.
peptidehormone hormone -eceptor
P~ ~ cyclic-AMP~ membrane effects phospho/ diesterase 5-AMP protein synthesis Fig. 1
)
cytoplasm
jr activation ~ inhibition
Schematic diagram of the mechanism of action of peptide hormones acting at s p e c i f i c receptor sites on the outer surface of the plasma membrane of the target c e l l .
This enzyme catalyses the formation of cyclic-AMP from ATP which in turn acts as an a l l o s t e r i c modulator of specific cytoplasmic enzymes.
FSH bindinq to the
plasma membrane of S e r t o l i c e l l s , for example, culminates in a v a r i e t y of c e l l u l a r changes including an elevation in the rate of protein biosynthesis mediated by c-AMP binding proteins and protein kinases.
Analysis of the protein products of
Sertoli c e l l s following FSH stimulation has so far f a i l e d to reveal any selective
Biochemical and Immunological Prospects for Male Contraception
231
synthesis of particular proteins and the effect on protein biosynthesis appears to be of a general nature. production induced by LH.
FHS also exerts a potentiating effect on androgen Binding of LH (or hCG) to the plasma membrane of
Leydig cells ultimately results in release of free cholesterol from an acyl ester located in i n t r a c e l l u l a r fat droplets.
This cholesterol then represents the
precursor of steroidonenesis (Gnodde et a t . , 1979), a pathway that is also believed to be i n d i r e c t l y modulated by synthesis of key regulator enzymes via an increased rate of mRNA transcription stimulated by the action of protein kinases (Rommerts e t a l . ,
1974).
The action of cAMP as a second messenger in these two examples leads to diverse responses in the two d i s t i n c t sets of target cells but i t s role in the p i t u i t a r y is less clear.
For example, both LH and FSH synthesis and release from the
p i t u i t a r y are stimulated by the action of LH-RH presumably by the second messenger cyclic-AHP but the plasma concentrations of each of the gonadotrophins is known to vary under d i f f e r e n t circumstances.
Certainly differences in
response of the p i t u i t a r y to LH-RH are clearly apparent between males and females. I t was o r i g i n a l l y thought that secretion of the nonadotrophins were independently controlled by two hynothalamic releasinn factors acting on two separate 9roups of pituitary cells, one secreting LH and the other FSH.
Studies of Schally et a l . ,
(1971) have tended to support the idea that LH-RH is the only hormone involved and that this stimulates release of LH and FSH from a sinmle pituitary cell type (Tongard et a l . , 1971).
To account for varying levels of the two gonadotrophins
in different stages of sexual maturity and in men with certain spermatonenic defects i t must be assumed that the rate of synthesis of the two hormones within the pituitary cell is independently controlled.
This would result in storane
of differing amounts of hormone in secretory vesicles or selective release of one
or other gonadotrophin upon stimulation with LH-RH.
The concept of a single
hormone responsible for triggering release of two separate products from a target cell other than in tandem i s , however, not completely satisfying. efforts continue to explore the original
Experimental
theory postulating the existence of
separate peptide hormones in the hypothalamus responsible for the release of FSH and LH respectively from the p i t u i t a r y but these have not yet proved successful. The p i t u i t a r i e s of males and females are known to d i f f e r in t h e i r response to LH-RH. This differenceisbelieved to be due to the particular hormonal environments which prevail during development of the brains of each sex (Gorski, 1971). Androgens and oestrogens, for example, are believed to play a direct role in the development of sexually dimorphic characteristics of the brain (Barraclough, 1968) and there is convincing evidence for the presence of both types of steroid
232
P.J. Ouinn
receptors in the hypothalamus of neonatal and prepubertal mice (Fox, 1975; Fox et a l . , 197R). More recently, the existence of androgen receptors has been established throughout the, so-called, c r i t i c a l period of brain sexual differentiation in mice using DNA-cellulose a f f i n i t y chromatrgraphy and velocity sedimentation and by analysis of the androgen-resistant mutant, testicular feminisation (Vito et at., 1979).
The role of steroids has been simply demon-
strated by exposure of the central nervous system of neonatal rats to testosterone. This results in a loss of the capacity of cyclic release of LH in the adult rat but does not affect tonic patterns of hormone secretion (Gorski, Iq68).
Using
sensitive radioimmunoassay methods, Tang (1978) observed the sex differences in synthesis and release of pituitary LH in an in v i t r o system.
I t was found
that LH-RH stimulated release of a siqnificantly greater proportion of total pituitary LH in female glands consistent with the essential mechanism required for the preovulatory LH surge at mid-cycle, whereas a lower response in the male system is geared to the tonic secretion of LH from male pituitary. The most convincing evidence that peptide hormones mediate their effects within the target cell exclusively by binding to a cell surface receptor has been derived by studies of prolactin.
Shiu and Friesen (1976) have succeeded in
purifying the membrane receptors for prolactin by a f f i n i t y chromatography and these were used as antigen to raise specific antibodies to the receptor site. Addition of these antibodies, which are obviously too large to pass across cell membranes, to mammary explants prevented binding of 1251-prolactin to membrane receptors and prolactin-mediated 2.1.2.
incorporation of radiolabelled leucine into casein
Steroid hormones
Unlike the peptide hormones, steroids penetrate cell membranes by a diffusion process and interact with specific receptor molecules located in the cell cytoplasm Despite the fact that some steroid hormone-induced responses can be mediated by effects on cell membranes, such as the well known oestrogen-induced permeability change in uterine cells, the primary mechanism of action is believed to be genome-mediated. The proposed mode of action of steroid hormones is shown diagrammatically in Fig. 2. The process appears to involve the migration of the steroid hormone-receptor protein complex into the cell nucleus where i t interacts with the non-histone acidic proteins associated with the chromosomal DNA.
The
interaction activates the transcriptional apparatus leading to the synthesis of new messenger RNA as well as ribosomal RNA and transfer RNA (O'Malley and Means, 1974).
The RNA then migrates to the cytoplasm where i t directs the translation
of proteins responsible for bringing about the altered function of the target cell.
Biochemical and Immunological Prospects for Male Contraception
testosteron~ e.~
OH
.~hesis / /
{j/
Fig. 2
~
j
/
"
Suggested mechanism of action of steroid hormones. The l i p o p h i l i c character of these hormones enables them to penetrate biological membranes and interact before or after enzymic modification with specific receptor proteins. The binding causes a conformational change in the protein which enters the nucleus and stimulates RNA synthesis. The RNA may be modified by interaction with the steroidcomplex when i t enters the cytoplasm and acts as a template for translation of specific proteins.
233
234
P.J. Quinn
Some steroid hormones l i k e oestradiol and progesterone bind to t h e i r respective receptor proteins in the target c e l l s in a chemically unmodified form.
Testosterone,
however, can be regarded as a prehormone since i t is the binding of a metabolite of testosterone, dihydrotestosterone, which represents the active receptor-protein binding complex.
The conversion of testosterone to dihydrotestosterone in the
target cell is mediated by a s p e c i f i c 5~-reductase enzyme.
Testosterone can also
be converted to oestrogens by both hypothalamus and n e r i f e r a l
f a t tissues where
the product binds to specific oestrogen receptors to modulate events in the cell nucleus.
2.2. Hormonal Regulation of Male Reproduction 2.2.1.
Lutenising Hormone - Releasing Hormone
The synthesis and release of gonadotrophins is u l t i m a t e l y regulated by the central nervous system and biogenic amines play an essential role in the a c t i v i t y of the hypothalamus-hypophysis axis.
A number of studies have shown that LH-RH is
located in neurons within the classical
hypophysiotrophic area of the medial basal
hypothalamus as well as in the medial preoptic area and septum (Barry e£ a t . , Barry and Dubois, 1976; and Silverman et a t . ,
Silverman and Zimmerman, 1978).
1973;
Krey and Silverman (1978)
(1979) have recently assessed the contribution of each of
these cell groups to LH-RH terminals in the median eminence of guinea pig brain usin~ localized lesion techniques.
They found that only destruction of the
arcuate nucleus resulted in diminution
of LH and testosterone levels in the plasma
of male guinea pigs, suggesting that this was the major but not necessarily the only source of LH-RH terminals in the median eminence.
These nerve terminals of
the hypothalamus which are under adrenergic control are believed to regulate the tonic secretion of gonadotrophin.
The role of LH-RH-containing neurons which
project into regions other than the median eminence is unknown but some function in the regulation of sexual behaviour cannot be excluded. As noted above the action of LH-RH on the p i t u i t a r y d i f f e r s in males and females probably as a consequence of the p a r t i c u l a r hormonal environments in each sex during development.
LH-RH, however, seems to influence t e s t i c u l a r function
d i r e c t l y and not e x c l u s i v e l y via stimulating release of gonadotrophin. A profusion of studies have been conducted to synthesize and examine the e f f e c t s on male reproductive capacity of agonists and antagonists of LH-RH following the original
observation of Vale et a t . ,
(1972) who showed that {des His 2) LH-RH is a
potent antagonist of LH-PH (Humphries et a t . , Moersch e t a t . ,
1979;
Schally et a t . ,
Ig76).
1978, 1979; Seprodi e t a t . ,
Ig78a;
The hormone is a decapeptide (see
Table I) featuring a type 116 bend hinged around a glycine residue at position 6
235
Biochemical and Immunological Prospects for Male Contraception TABLE 1
A n t a g o n i s t analogues of LH-~H
Amino 1
2
3
4
acid
5
sequence 6
7
8
9
I0
LH-RH (PyroGlu-
His
-
Trp
Ser -
Tyr -
Gly -
Leu
-
D-Phe-
Phe
D-Phe
-
-
D-Phe
-
D-Leu
-
-
D-Phe
-
D-AIa
-
-
D-Phe-
Pro
D-Trp
-
-
D-Phe-
Leu
D-Trp
-
-
D-Phe- DTrp
D-Phe
-
Arg -
Pro
Gly - HN2)
Antagonist
(I)
de la Cruz e t a l . ,
(2)
V i l c h e z - M a r t i n e z et a l . ,
(3)
Corbin and B e a t t i e (1975)
(4)
Humphries et a l . ,
(5)
Pedroza e t a l . ,
(I) (2) (3) (4) (4) (5)
(1976) (1976)
(1976) (1978)
which i s r e s p o n s i b l e f o r m a i n t a i n i n n the peptide i n an a c t i v e conformation.
The
bend can be c o n s i d e r a b l y s t a b i l i z e d by s u b s t i t u t i n ~ O-amino acids at 9 o s i t i o n 6, particularly
those w i t h bulky side chains.
Such analogues are found to possess
increased potency of gonadotrophin r e l e a s i n g c a g a c i t y . compared the b i o l o g i c a l
activity
Seprodi e t a l . ,
(Ig78b)
o f two l i n e a r LH-RH analogues w i t h t h e i r c y c l i c
c o u n t e r p a r t s and concluded t h a t the r e c e n t o r b i n d i n g conformation of the hormone consisted of a U shaped peptide i n which the C and N t e r m i n i were located in close p r o x i m i t y to each o t h e r . A selected nroup of s y n t h e t i c analogues o f LH-RH, a l l w i t h a n t a g o n i s t a c t i o n , i s presented i n Table I .
S u b s t i t u t i o n o f D-Phe at p o s i t i o n 2 appears to be an
important f a c t o r in antagonism o f LH-RH since replacement w i t h Phe rendered the analogue i n e f f e c t i v e .
The main s i t e o f a c t i o n of these 9eptides appears to be
at the l e v e l of the p i t u i t a r y the t e s t i s be e f f e c t i v e
(see below).
but there i s some evidence f o r a d i r e c t a c t i o n on
For example, some of these pegtides have been shown to
i n p r e v e n t i n g LH release i n men f o l l o w i n g s t i m u l a t i o n by LH-~H
236
P.J. Quinn
(Gonzalez-Barcena et a t . , 1977).
The contraceptive applications of these
peptides, however, may reside in their agonist rather than their antagonist action (Corbin et a t . , 1978).
The effects of long term treatment of male rats with
potent LH-RH agonists has been reported by Sandow et ai.,(1977) and Tcholakian et at.,
(1978).
Both groups reported that LH and FSH levels rise but serum testo-
sterone concentration
contrary to expectations, decrease markedly desnite the
absence of obvious changes in Leydig cell morphology.
The other most notable
feature of chronic LH-RH treatment is a marked decrease in size of the male accessory glands, particularly the seminal vesicles and ventral prostate, as well as the weight of the testes (Vale et a t . , 1977).
I t was suqnested that the
decrease in testosterone levels could be due to either a suppression of certain key steroidogenic enzymes responsible for testosterone biosynthesis or decreased numbers of LH/hCG receptors in the testis.
T h i s process has been considered as
a "desensitization" of the testis to LH following exposure to high levels of LH supported by pharmacological doses of LH-RH (Hsueh et a l . , Ig77).
The possibility
that large chronic doses of LH-RH render the pituitary refractory to further stimulation by LH-RH with a consequent decrease in serum gonadotrophins and atrophy of male reproductive organs has also been considered (Belchez et a t . , 1978; Sandow e t a t . , 1978).
Evidence has recently been presented (Hsueh and Erickson,
1979) to show that LH-RH acts directly on the Leydig cells of the testis to cause a decrease in testicular LH/hCG receptors and testicular steroidogenesis.
Irres-
pective of the precise mode of action of LH-RH analogues the compounds appear to offer, in pharmacological doses, an effective means of regulating male f e r t i l i t y . Another possible site of regulation of LH-RH action is at site of synthesis and storage of the hormone in the hypothalamus.
Active peptidases present in the
hypothalamus have been shown to rapidly degrade LH-RH (Griffiths et a l . , 1974, 1975; Swift and Crighton, 1978, 1979a).
The enzymes appear to be stimulated by
repeated injection of oestradiol and progesterone in the rat (Griffiths and Hooper, 1973).
The action of these steroids is apparently to bind to the
enzymes and thereby to exert a positive allosteric modulation of their activity or alternatively to remove some allosteric inhibitor (Swift and Crighton, 1979b). 2.2.2.
Pituitary Gonadotrophins
Hypophysectomy in men leads to testicular regression which can be restored to some extent by administration of hCG or human menopausal gonadotrophin (MacLeod, 1970). This observation emphasises the degree to which testicular function is controlled by the pituitary.
Russell and Clermont (1977) examined the degenerative changes
in germ cells of the rat during maturation of spermatozoa and compared these with changes associated with hypophysectomy.
~lormally a proportion of germ cells
237
Biochemical and Immunological Prospects for Male Contraception
of mature male rats degenerate at several stages during spermatogenesis includinq Type A spermatogonia, midpachytene spermatocytes, primary and secondary spermatocytes which are particularly vulnerable during their respective maturation divisions and mid and terminal stages of spermiogenesis.
Hypophysectomyappears
to cause a marked increase in degeneration of midpachytene spermatocytes and spermatids in the middle and terminal stages without notably affecting loss of cells at other stages of development.
Administration of FSH does not appear to
influence the course of events associated with hypophysectomy but LH, especially when augmented by FSH, reduces the number of degenerating cells to near normal values. Testicular function is also suppressed by active or passive immunization against gonadotrophins.
Antibodies against LH, for example, cause a reduction in testis
weight in the rat and a reduction and disturbance of spermatogenesis.
Changes in
Sertoli and Leydig cell functions are observed (Madhwa Raj and Dym, 1976) and spermatozoa develop abnormally (Talaat and Laurence, 1971).
In rabbits, maturation
of the gonads is prevented when immature animals are immunized against LH and spermatogonia
arrested at the spermatonia stage (Monastirsky et at., 1971).
Secretion of gonadotrophins is regulated by testosterone in a feedback control process. A diagrammatic representation of feedback control of pituitary gonadotrophins is illustrated in Fig. 3, where the effect of sex steroids on FSH and LH release from the anterior pituitary are summarised.
I t can be seen that while
oestrogens exert stimulatory effects on the release of both gonadotrophins at the pituitary level, androgens inhibit LH but stimulate FSH release.
Studies of
pituitary cells in culture (Drouin and Labrie, 1976) have suggested that androgens have not only specific but in certain circumstances an opposite effect at the pituitary level on the control of FSH and LH secretion.
Thus treatment of cells
with testosterone markedly inhibits LH response to LH-RH but stimulates FSH release.
The feedback control process is best exemplified by studies involvinq
active immunization of animals against testosterone.
Active immunization of
male rabbits, rats and monkeys against testosterone can produce UD to a lO0-fold increase in the total serum testosterone concentration.
The increase in testo-
sterone is accompanied by a parallel increase in serum antibody t i t r e un to about 12 weeks whereupon the steroid level reaches a constant value ( H i l l i e r et a t . , 1975). High t i t r e s of anti testosterone antibodies can also be provoked in castrated individuals but no elevation in total serum testosterone is observed confirming that the steroid is of testicular origin (Wickings, e t a t . , 1977). Although total serum testosterone increases most of this is complexed to antibody and, in fact, the amount of free testosterone decreases slightly in actively
238
P. J, Quinn
immunized animals (Hickings and K'ieschlag, 1978).
Brain
Hypothalamus
+1
I+ -
,
-
.
'
"
/
+
Oestrogens
Anterior pituitary
Gonads
Progestins Fig. 3
The effect of sex steroids on FSH and LH secretion from the anterior p i t u i t a r y .
239
Biochemical and Immunological Prospects for Male Contraception Hyperplasia of the Leydig cell population is a typical
histological
feature of
immunization and is similar to the e f f e c t s observed following hCG administration and blockage of androgen-sensitive receptors of the hypothalamus and p i t u i t a r y by drugs such as cyproterone (Heinert and Taubert, 197R).
As a d i r e c t consequence
of decreased unbound testosterone concentration in the serum, there is an increase in gonadotrophin l e v e l s ; following castration.
the increase, however, is not as marked as that observed Subsequent saturation of antibody-bindinq sites with
exogeneously-administered testosterone can restore LH levels to normal values but, contrary to expectation, FSH levels are unaffected by this procedure (Nieschlag et a t . ,
1975) suggesting that the feedback control of the two 9onadotrophins are
d i f f e r e n t in the way they respond to c i r c u l a t i n g free testosterone. As far as the contraceptive potential of immunization against testosterone is concerned there seems l i t t l e
scope.
Spermatogenesis, f o r example, does not
appear to be severely impaired by the reduction in free testosterone concentrations, c e r t a i n l y at the levels that can be achieved by active immunization against the steroid.
This might not be unexpected i f testosterone produced l o c a l l y in the
t e s t i s passes d i r e c t l y to the tubules and is not complexed to c i r c u l a t i n g a n t i testosterone antibodies of the serum which presumably do not penetrate the bloodtestis barrier.
Another serious manifestation of active immunization against
testosterone is the marked loss of l i b i d o which f u r t h e r detracts from this approach to contraception. Several drugs are known to suppress p i t u i t a r y function in men and some have been examined as potential contraceptive agents.
Medroxyprogesterone acetate, for
example, is a drug with potent long-acting gestogenic effects but l i k e other s i m i l a r drugs i t s administration is associated with some loss of l i b i d o Kretser, 1976).
(de
Loss of l i b i d o can be counteracted to some extent by combininq
gestogen administration with implants of long-acting testosterone analonues l i k e testosterone oenanthate.
Temporary suppression of spermatoqenesis without loss
of l i b i d o has been claimed from a number of c l i n i c a l combinations (Coutinho and Melo, 1973; et at.,
1978).
Moltz et a t . ,
gestogen, cyproterone acetate.
Frick, 1973;
trials
of androgen/gestogen
de Kretser, 1976; Neumann
(1978) have reported a detailed study of the The drug was found to cause a r e v e r s i b l e o l i q o -
spermia associated with an increase in the proportion of abnormal spermatozoa and a decrease in cell m o t i l i t y which e f f e c t i v e l y reduced the a b i l i t y of spermatozoa to penetrate ovulatory cervical mucus (Kremer t e s t ) .
No loss in l i b i d o was
observed despite a marked reduction in plasma testosterone concentrations which was restored, together with t e s t i c u l a r function, within I? weeks of withdrawal of the drug.
Large-scale c l i n i c a l
trials
of the drug under the auspices of the
P.J. Quinn
240
World Health Organization (7th Annual Report, 1978), however, led to the conclusion that cyproterone acetate has only limited contraceptive potential.
One
reason was that azoospermia is considered a necessary criterion for an effective contraceptive procedure. Drugs that suppress FSH and LH action on the gonads, thereby alterinn gonadal function, have also been investigated.
The contraceptive properties of one such
drug, danazol, an alkylated steroid (see Fin. 4) has been reviewed recently by r)mowski (1979).
Administration of danazol together with testosterone has been
found to cause severe oligospermia or complete azoospermia without reducing libido (Skoglund and Paulsen, 1973; Ulstein e t a t . ,
1975; Paulsen and Leonard, 1976). OH
~~.,i
Fig. 4
c-~CH
Structure of danazol.
A dose of 600 mg of danazol daily together with 200 mg of testosterone oenanthate given intramuscularly once a month was considered a useful contraceptive regimen and in normal men testicular function could be restored to pretreatment levels within 4 months of withdrawal from the drugs. 2.2.3.
Inhibin
Selective destruction of the germinal epithelium by radiation, drugs, testicular hyperthermy and ischemia and immunological aspermatogenesis has often led to elevation of FSH compared to serum LH, suggesting that production of another factor by cells of this tissue acts by a negative feedback process to regulate p i t u i t a r y FSH secretion (Setchell et a l . , 1977). Present evidence suggests that a protein of molecular weight about I0,000 produced by the Sertoli cells, referred to as inhibin, selectively suppresses FSH secretion by p i t u i t a r y cells in tissue culture (Steinberger and Steinberger, 1976) and offers contraceptive potential (Franchimont et a t . ,
1978).
Thus the development of synthetic analogues
of inhibin may suppress spermatogenesis without causing loss of libido (Davies et at.,
1978).
Our present state of knowledge of knowledge of the mechanism of hormonal regulation
Biochemical and Immunological Prospectsfor Male Contraception
241
of male reproduction does not yet provide a s a t i s f a c t o r y means of suppressing spermatogenesis while at the same time maintainin~ l i b i d o .
One of the main
reasons for this is the apparent close interdeDendence of LH and FSH release from the p i t u i t a r y under the influence of LH-RH on the one hand and feedback control by t e s t o s t e r o n e / i n h i b i n on the other.
Nevertheless, the differences
occasionally observed in LH and FSH release are s u f f i c i e n t l y encouraging to maintain current i n t e r e s t in i d e n t i f y i n g separate releasing hormones or factors f o r FSH as well as LH.
Independent regulation of these two gonadotrophins could
prove a useful approach to the development of male hormonal contraceptives,
Chapter 3
Regulation of Testicular Function and Sperm Maturation
Spermatogenesis involves a sequence of events including mitosis, meiosis and morphological changes which culminate in the d i f f e r e n t i a t i o n of highly specialized spermatozoa from the early spermatid progenitor.
Morpholooical features of the
process have been well documented by electron microscopic studies (de Kretser, 1969) and are i l l u s t r a t e d
in Fiq. 5.
Briefly,
the spermatogonia undergo m i t o t i c
d i v i s i o n s to provide groups of primary spermatogonia destined f o r f u r t h e r differentiation
and represent the immediate precursors of snermatocytes.
These
primary spermatocytes subsequently undergo a meiotic d i v i s i o n to form secondary spermatocytes followed by another d i v i s i o n , chromosome number by half.
thereby completing the reduction in
The haploid c e l l s represent the spermatids.
The
nuclei of the spermatids condense and occupy an eccentric position adjacent to the plasma membrane.
The acrosome is d i f f e r e n t i a t e d from elements of the Golgi
complex which become closely applied to the nucleus where i t approaches the plasma membrane.
The axial filament of the t a i l
and develops at the base of the nucleus.
arises from one of the c e n t r i o l e s
The cytoplasm condenses and is
e s s e n t i a l l y extruded from the base of the cell as the so-called residual body or cytoplasmic droplet.
During this process the mitochondria arrange around the
the proximal portion of the t a i l .
At this stage the now mature spermatozoa are
released from the Sertoli c e l l s into the lumen of the seminiferous tubules. Spermatogenesis is a continuous process in sexually mature males and i n t e r r u p t i o n at any stage reduces the rate of gamete production.
E f f e c t i v e contraceptive
intervention in the production of spermatozoa by the t e s t i s aims to eliminate spermatozoa from the ejaculate in such a way that the concentration of c e l l s can be r a p i d l y restored when treatment is discontinued.
By rapid, of course, one
must recognise that, in man, the production of mature spermatozoa requires period approaching 75 days from the nerminal cell stage.
242
Of p a r t i c u l a r concern
Biochemicaland ImmunologicalProspectsfor MaleContraception
243
in this approach, however, is the need to prevent the production of g e n e t i c a l l y abnormal spermatozoa.
spermatogonia I
proliferation
primary I spermatocyte
growth
secondary spermatocyte
maturation
/\
spermatid
1
I
1
transformation
spermatozoon
Fig. 5
Schematic representation of the process of spermatoqenesis
3.1. Inte~erenceof Spermatogenesisby ChemicalAgents A group of compounds that were o r i g i n a l l y developed as anti-cancer agents and bactericides, the n i t r o f u r a n s , were subsequently found to be e f f e c t i v e in r e v e r s i b l y blocking spermatogenesis at the level of the seminiferous tubules independently of the pituitary-gonadal
axis (~!elson et a t . ,
1954).
Hhen adminis-
tered in doses required to suppress sperm counts in men, however, there were marked g a s t r o i n t e s t i n a l
side e f f e c t s that eliminated these anents as e f f e c t i v e
contraceptives (Nelson and Bunge, I~57).
Likewise, diamine compounds are known
244
P.J. Quinn
to reversibly interfere with the function of seminiferous tubules and to lower sperm counts in a number of mammalian species, including primates, with no obvious effects on libido (Drobeck and Koulston, 1962) hut they also act as potent inhibitors of l i v e r enzymes responsible for drug metabolism (Heller et a t . , 1961). Selective damage to spermatogenesis by nitrofurans and manganese, which primarily injure spermatocytes and spermatids, at doses that have no obvious effects on other organs are l i k e l y to exert their effects by an interference with metabolic processes (Paul e t a l . ,
1953;
llematsu, 1966;
Husain e t a l . ,
1976).
Claims have
also been made that a l k y l a t i n g agents and related druQs used to t r e a t neoplasia (Fairley et a l . ,
IQ72;
Hinkes and P l o t k i n , I~73;
Sherins and ne Vita, 197~)
r e v e r s i b l y i n h i b i t spermatogenesis in men but t h e i r potential and usefulness as contraceptive agents have yet to be f u l l y evaluated.
In some experiments,
however, treatment of male mice with the a l k y l a t i n g agent triethylenemelamine (TEM) at a dose of N.Rmg/kg caused developmental retardation of embryos f e r t i l i z e d by defective spermatozoa ejaculated 20 days a f t e r i n t r a p e r i t o n e a l (Hitotsumachi and Kikuchi, 1977).
injection
Chromosomal aberrations such as breaks,
exchanges and premature chromosome condensations give rise to a high incidence of dominant l e t h a l i t y amongst affected embryos especially during the early, preimplantation stages of embryogenesis. damage may remain undetected u n t i l Sheridan, 1978).
Nevertheless TEM-induced chromosomal II
.
l a t e r stages of development (Burkl and
On this basis at least a l k y l a t i n g agents would seem to possess
limited contraceptive p o t e n t i a l . demonstrated a n t i f e r t i l i t y
The structures of some reaoents that have
e f f e c t s in shown in Fig. 6.
Certain metabolic i n h i b i t o r s appear to have selective effects on spermatogenesis. Fluoroacetate, f o r example, which i n h i b i t s the t r i c a r b o x y l i c acid cycle by conversion to f l u o r o c i t r a t e and blocking the enzyme cis-aconitase (Peters, 1952) has been examined in this regard.
The accumulation of c i t r a t e ,
reduction in ATP
concentration and effects of fluoroacetate on other substrate levels in the rat t e s t i s has been reported and found to be associated with tubular degeneration within the organ (Mazzanti et a l . ,
In65).
In a comparative study of the e f f e c t
of chronic exposure of rats to sublethal doses of fluoroacetate on the kidney, l i v e r and t e s t i s i t was concluded that the t e s t i s was the most vulnerable organ e x h i b i t i n g a s i g n i f i c a n t reduction in weight with a marked accumulation of c i t r a t e combined with decreased ATP levels.
~!o such changes were observed in the
other organs at comparable dosages of fluoroacetate.
Other studies have shown
that the p a r t i c u l a r lesion involves loss of t e s t i c u l a r c e l l u l a r elements and decrease of tubular volume resulting from t e s t i c u l a r atrophy with damage to the seminiferous epithelium but not Leydig cells (~azzanti et a l . , 1965).
High doses
of fluoroacetamide (Steinberger and Sud, 1970) and N-methyl-N(l-naphthyl)-fluoro
245
Biochemical and Immunological Prospects for Male Contraception acetamide (Hashimoto et a t . , 1968) cause similar histological
changes in rat
testes. In some instances, withdrawal of these drugs administered at r e l a t i v e l y low dose regimens results in some regeneration of the seminiferous tubules and the development of spermatids and spermatozoa in some tubules but not where residual damage to the tubules remains (Sullivan
a l . , 197g).
fluoroacetyl CoA
CoASH A.
et
FCH2--CO 2
FCH 2 - C - - SCoA
fluoroacetate
O
II
-FC H - - C - - SCoA ""
II
FCH--CO2H
O
I
HO--C-- C02H
I
÷
O=C--CO2H oxaloacetate I H2C--CO2H
citrate synthetase
4-
CH3--C--SCoA -
- -HCH--C--SCoA
II
""
O
I N O - - C -- C 02H
I CH2--CO2H
F C H 2 - C -- N H 2
II fluoroacetamide FCH2--C--N --OH 3
N - methyl - N ( I- naphthyl) fluoroacetamide
Fig. 6
CH2--CO 2
II
0
C,
fluorocitrate
O
acetyl CoA
B.
CH2-- C 0 2 H
The structure and site of action of metabolic i n h i b i t o r s which have a n t i f e r t i l i t y actions in males.
246
P . J . Quinn
Other metabolic i n h i b i t o r s such as thioqlucose (5-thio-D-qlucose), appear to i n h i b i t normal spermatogenesis without a f f e c t i n a Leydiq or S e r t o l i - c e l l of the t e s t i s or decreasina l i b i d o (Zysk e t a l . , High doses of the drug, ~-chlorohydrin, sterility
1975;
populations
Makamura and Hall, Ig77).
have been shown to cause permanent
(Kalla, IQ76) associated with occlusion of the epididymal duct and
subsequent degeneration of the germinal epithelium.
An e f f e c t of ~-chlorohydrin
on t e s t i c u l a r vascular has been suggested as the cause of these changes (Reijonen e t a l . ,
1975).
This is supported by the f a c t that vasoconstrictors
such as noradrenalin potentiate the lesion caused by a larae acute dose of the drug (30mg/kg) in rats whereas the vasodilator, sodium n i t r i t e ,
administered
before, but not a f t e r , a dose of 9hmg/kq ~-chlorohydrin prevented the t e s t i c u l a r epididymal lesion (Kalla and Sinah, 1979).
111trastructural chanaes of the t e s t i s
associated with administration of ~-chlorohydrin in amounts areater than 50mg/kg/ day to adult male rats have been systematically studied by James et a i . , ( 1 9 7 8 ) .
They
concluded that the gonads were the principal organ affected by the drug and that the main morphological changes were the atrophy and/or necrosis of the epididymus and seminiferous epithelium.
An increase in kidney and l i v e r weight did not
appear to be associated with any changes in biochemical functions in contrast to the reproductive system.
Thus, biochemical changes in the epidymus of the rat
especially in the biosynthesis and metabolism of l i p i d s metabolism of t e s t i c u l a r (Edwards et a l . , (!!ohri e t a l . ,
1975) and epididymal spermatozoa
1975) together with changes in the epididymal vasculature
(Reijonen et a l . , antifertility
(Voqlmayr, IO7~) and
1975) appear to underly many of the morphological changes.
action of this drug at the level of spermatozoan metabolism w i l l
The be
considered separately in Section 4.2.3. Other miscellaneous agents l i k e ethionine have also been shown to damage the seminiferous epithelium in a manner resembling gonadotrophin deprivation but the endocrine effects are uncertain (Dunn e t a l . ,
1962).
tadmium salts are known to
damage t e s t i c u l a r and epididymal vasculature but the a n t i f e r t i l i t y
effects
associated with cadmium may r e s u l t from a d i r e c t e f f e c t of the metal ion on isoenzymes of t e s t i c u l a r carbonic anhydrase (Hodgen et a l . , Another recent compound shown to possess a n t i f e r t i l i t y
1970). effects is gossypol, a
yellowish phenolic compound isolated from the seeds, stems and roots of the cotton plant.
Gossypol has been shown to i n h i b i t the f e r t i l i t y
of male rats (see
report of the National Coordinating Group on Male A n t i f e r t i l i t y Ultrastructural
Agents, 1978).
studies have shown that the spermatids are most sensitive to the
drug but with higher doses and prolonged exposure the spermatocytes are also damaged.
Examination of the epididymis revealed e x f o l i a t e d spermatids
247
Biochemical and Immunological Prospects for Male Contraception and spermocytes with numerous dead spermatozoa often with heads separated from tails.
Examination under the electron microscope showed structural
in the acrosomal membranes and mitochondria.
results in a gradual decrease in sperm count u n t i l trial
alterations
Continual exposure to gossypol azoospermia.
In a c l i n i c a l
involving 4~00 healthy male subjects receiving gossypol for a period of more
than six months, the a n t i f e r t i l i t y
e f f e c t evaluated by semen examination showed
the drug to be almost completely e f f e c t i v e .
A low incidence of mild side effects
were reported including the development of hypokalemia but whether this was related to gossypol treatment was uncertain.
Clearly gossypol shows promise as an
e f f e c t i v e male contraceptive and the results of f u r t h e r and more comnrehensive clinical
trials
Hale i n f e r t i l i t y
presently being undertaken are awaited with considerable i n t e r e s t . caused by exposure to certain chemicals in i n d u s t r i a l
have been described.
situations
Thus, azoospermia has been linked with chronic exposure
to reagents l i k e 1,2-dibromo-3-chloropentane which has long been used as an agricultural
nematocide.
The e f f e c t of the drug on suppressing spermatogenesis
has been examined by Potashnik et a l . ,
(1979) who found a selective atrophy of the
germinal epithelium in t e s t i c u l a r biopsy material characterised by i n t a c t Sertoli c e l l s and an increased number of morphologically-normal
Leydig c e l l s .
Hormone
studies showed elevated FSH levels with normal plasma LH and testosterone concentrations.
The use as a male a n t i f e r t i l i t y
agent, however, is precluded
because of the potent carcinogeneicity of the reagent.
3.2. Effects of Temperature and Irradiation I t has lonq been recognised that heating the t e s t i s to temperatures greater than 37°C causes atrophy of the tissues and function is lost.
Not all c e l l s are
affected to the same extent since primary spermatocytes during Dachytene and young spermatids appear to be the most sensitive c e l l s to elevated temperatures. The notion that c r i t i c a l
periods e x i s t in spermatogenesis such as between lepto-
tene and pachytene, another during the maturation d i v i s i o n and a third between the cap-phase and acrosome-phase spermatids, has been proposed by Collins and Lacy (1969) based on experiments involving the exposure of rat testes to a temperature of 43°C for various i n t e r v a l s .
Although the kinetics of spermato-
genesis are not affected by b r i e f periods of heating, populations sensitive to high temperature are depleted and are not replenished u n t i l development of new populations takes place followinQ temperature elevation. The mechanism of thermal s e n s i t i v i t y is unknown but i t has been suggested that increased metabolic a c t i v i t y induced at higher temperatures is not supported by a corresponding supply of oxygen and nutrients from the p e r i f e r a l blood (Setchell, 1978). role of glucose appears to be important because spermatids are remarkably
The
248
P.J. Quinn
dependent on g l y c o ] y t i c metabolism.
Whether g]yco]ysis is required to supply
the energy needed for protein synthesis is unknown but this process is markedly affected by temperature. Protein synthesis, for example, is known to be slower at body temperature (37°C) compared to lower temperatures of the scrotum.
This difference appears to be
largely due to the spermatids (Nakamura and Hail, ]978) since subce]]u]ar systems prepared from these ceils incorporate less amino acid into protein at 37°C compared with 34°C.
Other studies by these authors have shown that the most
temperature sensitive component of the protein synthesizing machine of spermatids are the ribosomes since incubation of a c e i l - f r e e system at temperatures greater than 34°C caused a dissociation of po]ysomes to monosomes.
The effects on polE-
ribosome dissociation could be due to, amongst other things, the reduced synthesis of mRNA strands suitable for translation, f a i l u r e of the i n i t i a t i o n
process or a
change in the s t a b i l i t y of rRNA. As with temperature, ionizing radiations such as X-rays cause selective damage to t e s t i c u l a r tissues.
Type A spermatogonia appear to be the most sensitive to
radiation damage and intermediate and type B spermatogonia are less sensitive. Higher doses are successively lethal to other germinal cells roughly in the order spermatids, spermatocytes and l a s t l y spermatozoa.
Some c e l l s , on the other hand,
are not as severely affected by X-radiation including the peritubular myoid c e l l s , while Sertoli cells are only affected at high doses p a r t i c u l a r l y in immature individuals (Erickson, 1976). Damage caused by ionizing radiations appears to be confined to chromosomal disruption and as a potential contraceptive method i t has severe l i m i t a t i o n s because of the p o s s i b i l i t y of congenital abnormalities.
The incidence of aneuploidy in
surviving human children, for example, is about 0.5% (Nielsen and Sillesen, 1975) although a frequency of 5-10% of aneuploid zygotes at conception has been estimated (Hansmann and Probeck, 1979). disjunction in mammalian spermatogenesis
X - i r r a d i a t i o n appears to induce nonbut an increase in the incidence of
aneuploidies among the F1 progeny is problematical The frequency of X-irradiation-induced
(Szemere and Chandley, 1975).
non-disjunction of sperm chromosomes by
examination of the progeny conceived by irradiated animals, however, tends to give a distorted picture of the total chromosomal aberrations because most irradiated spermatocytes do not mature into spermatozoa.
Many degenerate even after rela-
t i v e l y low doses of X-rays and those that do survive are a highly selected population. Furthermore, not many progeny survive to term because aneuploidy that results from loss or gain of an autosome generally results in early embryonic death.
249
Biochemical and Immunological Prospects for Male Contraception Some disturbance of endocrine function has been reported as a consequence of radiation damage to the t e s t i s .
Testosterone production, however, does not
appear to be affected by moderate doses of X-radiation but when spermatids are depleted from the germinal epithelium, serum LH and FSH become elevated, possibly due to a loss of inhibin production which helps to control p i t u i t a r y gonadotrophin secretion (Setchell e t a l . , Iq77).
Higher doses of X-radiation appear to a l t e r
steroidogenic pathways ( E l l i s , 1970).
3.3. Biochemical and Immunological Aspects of Vasectomy Vasectomy is a widely practiced method of male contraception but i t has the marked disadvantage of rendering the individual p o t e n t i a l l y i r r e v e r s i b l y s t e r i l e . Sterility, libido.
permanent or other wise, nevertheless is achieved with no loss of There are a number of interesting factors associated with b i l a t e r a l
l i g a t i o n of the vas deferencs that may provide a useful basis for the development of more satisfactory male contraceptives. The effect of vasectomy on spermatogenesis has been extensively reviewed (Neaves, 1975;
Heidger and Sawatzke, 1977) and the effect appears to d i f f e r from one
species to another. histological regard.
The effect of vasal obstruction on t e s t i s weight and
features of the seminiferous tubules has been investigated in this
In general, no marked changes in spermatogenesis are observed in the
b u l l , r a t or various primate species that can be attributed d i r e c t l y to vasectomy (Skinner and Rowson, 1968; Howards et a t . ,
1975).
P a l i f l e r and Foote, 1969;
Vare and Bansal, 1973;
Spermatogenesis in the guinea pig i s , however, severely
impaired and changes in the seminiferous epithelium of the rabbit are manifest after about 1 year.
In the dog, histological changes occur as an i n i t i a l
response to vasectomy but the tissue subsequently recovers to return to i t s normal appearance.
Certain other morphological and physiological alterations
in vasectomised individuals have also been observed (MacMillan e t a l . ,
1968;
Igboeli and Rakha, 1970) including changes in the blood-seminiferous tubule barrier that could have repercussions on spermatogenesis and/or sperm maturation (Turner e t a l . , 1979).
Biochemical studies of DNA synthesis in the t e s t i s of
guinea pigs, using t r i t i a t e d thymadine incorporation, has shown that spermatogenesis in vasectomised animals may be altered as a d i r e c t consequence of decreased t e s t i c u l a r DNA synthesis (Howards et a l . ,
1979).
The endocrine function of vasectomised men appears to be normal shortly after vasectomy (Naik e t a l . ,
1976;
Johnsonbaugh e t a l . ,
1977) as well as many years
following operation (Whitby e t a l . , 1979). However, some disturbance in the level of testosterone, dihydrotestosterone, LH, FSH and oestradiol at various intervals after vasectomy have been reported (Smith e t a l . ,
1976;
Purris e t a l . ,
250 1976;
P, J, Ouinn Kobrinsky e t a l . ,
as seriously abnormal.
1976;
Whitby e t a l . ,
1976) but none could be described
No morphological changes of Leydig cells were apparent
at the level of e i t h e r optical or electron microscopy (Kubota, 1969) and no functional
impairment could be detected (Whitby et a l . ,
I t has frequently been reported that low f e r t i l i t y vasectomy (Kar and Phadke, 1975;
Ig79).
is associated with vaso
Schmidt, 1975) and there is some evidence that
the presence of antisperm antibodies in the serum is related to this i n f e r t i l i t y . Antisperm antibodies have been detected in the sera of both sexually mature males and females.
Indeed, a high incidence of such antibodies in the sera of
subjects with otherwise unexplained i n f e r t i l i t y 1954;
Franklin and Dukes, 1964;
antibodies in causing i n f e r t i l i t y
has often been reported (Wilson,
Shulman, 1971).
The precise role of these
is, however, not known (Jones, I~77).
Tests
for detecting antisperm antibodies are f a r from completely r e l i a b l e (see WHO reference bank f o r reproductive immunology, 1977). these antibodies are i l l u s t r a t e d
in Fig. 7.
classes can be i d e n t i f i e d by t h e i r a b i l i t y
Two methods of detecting
Antibodies of the IgM and some IgG to react with antigens located on the
plasma membrane of the c e l l , to f i x complement and lyse the cell - these a n t i bodies are referred to as immobilizing antibodies.
Another method is to observe
spermagglutination which occurs when s p e c i f i c bivalent antibodies cross-link neighbouring c e l l s in a manner similar to haemagqlutination.
The d i f f i c u l t y
about the agglutination tests is that spermatozoa are prone to agglutinate under a v a r i e t y of conditions in the absence of antisperm antibody.
T h u s certain
microorganisms including viruses, some drugs and defined factors present in fresh autologous serum can agglutinate sperm (Boettcher, et a l . ,
1970;
Boettcher, 1974).
Anti-sperm antibodies, nevertheless, can be detected by macroscopic reactions but other factors present in semen are also auto antigenic. and enzymes such as hyaluronidase (Metz, e t a l . ,
Lactoferrin-type proteins
1976) as well as soluble DNA
polymers (Witkin and Higgins, 1978) are included in this category. Antigens of the ABO blood group have been i d e n t i f i e d on sperm (Edwards
et al.,
1964) as have the blood group antigens M, ~! and Tja which, unlike the ABO antigens, are absent from the seminal plasma (Edwards, 1964). Attempts to detect Rh antigens have so f a r proved unsuccessful; i f this were possible then cells from an Rh positive Dd heterozygote genotype could be separated into two populations with obvious advantages.
Histocompatibility antigens (H-2) of mouse sperm have
been observed by fluorescent antibody techniques (Barth and Russell, 1964; Goldberg e t a l . ,
1970) and some of these appear to be products of the H-2 d region
of the major h i s t o c o m p a t i b i l i t y
complex (Vojtiskova e t a l . ,
s p e c i f i c HL-A tissue typing sera and a m i c r o c y t o t o x i c i t y
196~).
The use of
test has indicated that
251
Biochemical and Immunological Prospects for Male Contraception at least some antigens determined by the HI-A locus are present in high conconcentration on human sperm (Fel]ons and Dausset, 1970).
When spermatozoa from
donors heterozygous for certain HL-A antigens were tested with monospecific a n t i serum lysis of about half of the sperm population was observed, suggesting haploid expression of the HL-A antigens.
In other experiments, antigenical]y
d i f f e r e n t types of sperm were detected in semen from a single fonor.
These
observations have yet to be s a t i s f a c t o r i l y explained.
A. acrosome antibody surface
nucleus
sperm head
| 'ane
s-linking pody
midpiece
Fig.7
Tests for serum antisperm antibodies. A. Crosslinking and binding of IgG or IgM antibodies directed against antigens present on the cell surface i n i t i a t e s the classical pathway of complement f i x a t i o n leading ultimately to lysis by terminal complement factor and loss of sperm m o t i l i t y . B. Sperm agglutinating antibodies crosslink antigens on the surface of adjacent cells.
252
P.J. Quinn
Another category of sperm antigens are those associated with subcellular constituents of the spermatozoa.
These include the nucleoprotamines which are
confined to the nuclei of spermatids and spermatozoa c . f . the nucleohistones which are widely distributed in somatic cell nuclei of animals.
They are low-
molecular-weight proteins which are synthesized during the early and middle stages of spermatid d i f f e r e n t i a t i o n when they replace the histones (Louie and Dixon, 1972) Although o r i g i n a l l y believed to be non-immunogenic (they are widely used as heparin antagonists, for example), antibodies to human protamine have recently been observed in the sera of a number of i n f e r t i l e al.,
1974; Kolk and Samuel, 1975;
and vasectomised men (Kolk et
Samuel et a l . , 1975, 1976, 1977; Samuel, 1977)
In other more recent studies, Kay and Alexander (1978) report a change in the antigenic s p e c i f i c i t y of spermatozoa that have been damaged.
T h e y subjected
spermatozoa to mild centrifugation and examined the damaged cells by immunofluorescent methods.
This indicated that new antigens were expressed when the
plasma membrane was stripped off to reveal the underlying subcellular organelles. The t a i l and principal piece region of the cells appeared to be p a r t i c u l a r l y vulnerable to damage and showed a high concentration of the newly-expressed antigens. Numerous studies performed on the serum of normal f e r t i l e men have conclusively shown that the mammalian reproductive system is remarkably unresponsive to autoantigens.
Thus spermatozoa and other products of the male genital t r a c t are
recognised as being f u l l y antigenic in animals (Katsh and Bishop, 1958) and in humans (Mancini, 1974) yet under normal conditions autoimmune responses are rarely detectable.
Similarly many subcellular organelle membranes and soluble
constituents including mitochondrial constituents are antigenic but autoimmunity to such antigens arises only in certain pathological conditions. Antibodies specific to sperm antigens in serum detected by agglutination and immobilization tests, have been associated with vasectomy of various species including men (Ansbacher, 1971, 1973, 1974;
Ansbacher et
al.,
1976;
Gupta et
at.,
1975; Tung, 1975; Linnet and Hjort, 1977; Hellema and R~mke, 1978) guinea-pigs (Alexander, 1973) rabbits (Bigazzi e t a l . , 1976 a and b) rats (R~mke and Titus, 1970; 1977).
Brannen and Coffey, 1974) and monkeys (Alexander
et
a l . , 1974;
Alexander,
I t should be noted that in men, where most of these studies have been
performed, vasectomy is not invariably associated with the presence of antisperm antibodies in the serum.
In general, only 60-70% of vasectomised men e x h i b i t
any appreciable t i t r e s of agglutinating antibodies and somewhat fewer men have sperm immobilizing antibodies.
Typical data is i l l u s t r a t e d in Table 2.
Biochemical and TABLE 2
Immunological Prospects for Male Contraception
253
Number of sera with t i t r e s of antisperm antibodies amongst f e r t i l e and b i l a t e r a l l y vasectomised males. Antisperm antibodies
Group F e r t i l e males (16)
Aqglutinating
ImmobilisinQ
none
none
9
5
Vasectomised males (12)
Numbers in parenthesis r e f e r to the t o t a l number per group In general, sperm immobilizing antibodies are only found in sera which contain sperm agglutins.
Thus in sera from 160 vasectomised men studied by Hallima and
Rumke (1978) only 5 showed sperm immobilizina antibodies, at t i t r e s below that used to detect sperm agglutinating antibodies.
Table 3 presents representative t i t r e s
of d i f f e r e n t antibodies in the serum of vasectomised men. TABLE 3
T i t r e s of sperm aaglutinins and sperm immobilizins in the sera of b i l a t e r a l l y vasectomised men.
Sample Immobilizing
Titre Aqglutinating
Tyne of , agglutination
1
2
32
T
2
0
16
T
3
4
32
T
4
8
256
T
5
0
8
T
6
0
16
7
0
8
8
0
32
T
9
4
16
H
I0
0
16
T
II
32
256
H
0
8
12 * T =
tail-to-tail;
H = head-to-head.
Many studies have established a hiah c o r r e l a t i o n between t i t r e s of sDermagglutinatinq and immobilizing antibodies. detected by an i n d i r e c t immunofluorescence
Antibodies against human protamine, t e s t on swollen sperm heads (Kolk et
254
P.J. Quinn
al.,
1974) are also associated with antibodies specific for antiaens displayed
on the sperm plasma membrane (Samuel et a l . , Ig75) and antibodies directed towards the acrosome, the principal piece and other regions have a high incidence in sera of vasectomised men (Tung, 1975).
The latter study was of particular interest
because the antigen to the principal piece, designated ~P, was reactive only after the cells had been disrupted with detergent, subjected to proteolysis and fixed briefly with methanol suggesting that the antigen was of subcellular origin. Because the antigen was confined to the principal piece of the cell i t was thought to be associated with mitochondrial membranes.
The anti-Mp antibody appeared to
differ from other antibodies directed primarily to the principal piece of the cell since these were accessible to the antigen in live, unfixed cells (Feltkamp et al.,
1965).
Four auto antigens, designated S, P, T and Z have been described in guinea-pig sperm (Voisin and Toullet, 1968).
The f i r s t three have been shown to have
different immunological, physicochemical and pathological al.,
1973; Toullet and Voisin, 1974).
properties (Toullet et
Furthermore, female ouinea-pigs react
against all of these three autoantigens producing antibodies that are
indistino-
uishable from male autoantibodies directed against the same autoantigens. Although the autoantigens of human spermatozoa have not been examined in as great a detail as the guinea-pig, cross-reacting and autoantigens and autoantibodies between these two species have recently been described (D'Almeida and Voisin, 1977) as well as between human, bull and rabbit spermatozoa (Hansen, In72). Attempts to demonstrate cross-reactivity between human and guinea-pio testis have been performed but the results are ambiguous.
Otani and Behrman (1963), for
example, could find no evidence for cross-reactivity although common antigenicity could be observed with tissue extracts (Katsh, 1962). The relationship between f e r t i l i t y and the presence of antisperm antibodies in the serum has been the subject of many investigations.
I t has been noted, for
example, that vasectomised men who have undergone successful reversal of the operation, exhibit poor f e r t i l i t y which appears to be correlated with the t i t r e of sperm agglutinating, but not sperm immobilizing, antibodies in the serum (Sullivan and Howe, 1977). In about half the cases studied by these workers, in which pregnancy was achieved after reanastamosis of the vas deferens, agglutinating antibodies were present suggesting that these antibodies were not primarily responsible for poor f e r t i l i t y of the spermatozoa.
This appears to contrast with
studies by Menge and Protzman (1967) and later by Menge (1971) who observed low conception rates in female rabbits inseminated with semen pretreated with antisperm antibodies.
Normal conception rates were achieved, however, i f antiseminal
255
Biochemical and Immunological Prospects for Male Contraception
plasma antibodies were used to p r e t r e a t the sperm despite the f a c t that about the same extent of agglutination occurred in both qroups. corroborated this finding by performing a r t i f i c i a l
Hetz and Anika (1970)
insemination in the r a b b i t with
sperm pre-treated with univalent antisperm antibody fragments (Fab) agglutinate or immobilize sperm.
which did not
Pates of conception were again low even though
no agglutination or immobilization of the sperm was observed.
I t is apparent
that antibodies directed against other antigens often contribute to poor f e r t i l i t y in post-vasectomised men.
This appears to contrast with e a r l i e r findings that
antisperm antibodies that agglutinate spermatozoa i n t e r f e r e d with the migratory potential of the c e l l s and thus t h e i r a b i l i t y In this regard the a b i l i t y
to reach the ovum (Wilson, 1956).
to migrate through the mucus present in the cervix of
the female reproductive t r a c t would present a formidable b a r r i e r especially i f immobilizing cytotoxic antibodies are also involved (see F j a l l b r a n t ,
1968).
9estruction of the nerve supply to the vas deferens during vasectomy has also been considered as a factor contributing to the low f e r t i l i t y vasectomy (Pabst et a t . ,
observed a f t e r vaso-
1979).
Despite the f a c t that a high c o r r e l a t i o n between spermagglutinin t i t r e s in blood serum and the degree of sperm agglutination in the semen has been claimed (Fjallbrant,
1968;
necessarily
imply that these antibodies are capable of reaching the seminal
fluid,
Ansbacher et a t . ,
1976) high t i t r e s in the serum do not
where they would come into contact with the sperm.
Some types of c i r c u -
l a t i n g sperm autoantibodies, p a r t i c u l a r l y those of the IgH class have not u n t i l recently been detected in semen (Azim e t a t . ,
1978).
The Igr! class of antibodies
is believed to be p r i n c i p a l l y involved in head-to-head agglutination of spermatozoa (Friberg, 1974).
Other classes of serum immunoglobulins such as IgG have been
reported in semen and these presumably arise from transudation from the c i r c u l a t i o n into the secretions of the prostate gland. to cause head-to-tail
agglutination of spermatozoa.
These antibodies are believed IgA has been detected in
semen but the precise source of the immunoglobulin is not known.
Diffusion from
II
blood serum has been suggested (Helleme and Rumke, 1976) while a local secretion into the semen is a mechanism favoured by other workers (Uehling, 1971).
The
l a t t e r mechanism most l i k e l y explains the occasional incidence of t i t r e s higher in the semen than the serum and in which the seminal plasma spermagglutinins belong to the IgA class compared to IgG or IgM spermagglutinins in the blood serum. The presence of sperm agglutins in serum of women has been considered as a cause of human i n f e r t i l i t y .
Remarkable v a r i a b i l i t y
in the presence of these antibodies
and t h e i r t i t r e s have been reported which probably r e f l e c t s
differences in assay
250
P.J. Quinn
sensitivity from one investigator to another.
However, the general trend that
can be discerned from such studies is that relatively hinh frequencies of spermagglutinating sera are associated with certain arouns of i n f e r t i l e women, esoecially those with unexplained i n f e r t i l i t y as compared with non-preanant women with presumably normal f e r t i l i t y or proven f e r t i l i t y (Isojima et a t . , 1974; Shulman et at.,
1975).
A recent detailed study has confirmed that agglutinatinq
antibodies occur in a significantly hiqher frequency and in hiaher t i t r e s in i n f e r t i l e women and the presence of B-spermagglutinins was confined exclusively to this group (Ingersler and Hjort, 197g). The escape of antiaens from the reproductive tract into the host environment and the entry of antibodies and immune cells into the tract is prevented by a system of zona occludens, tight junctions that seal the epithelia lining the male reproductive tubules and ducts. barrier.
The system is referred to as the blood testis
The barrier has been investinated in detail by electron-opaque inter-
cellular tracer studies and high maqnification electron microscopy.
This has
shown a system of tight junctions between adjacent Sertoli cells which subdivide the seminiferous epithelium into two compartments, basal and adluminal (Dym and Fawcett, 1970; Dym, 1973).
Theseunique Sertoli cell junctional complexes are
characterised by bundles of fine filaments adjacent to the plasma membranes and profiles of endoplasmic reticulum next to the filaments (see Nicander, 1967; Flickinger and Fawcett, 1967).
It is the tight junctions unitinq the Sertoli
cells that are believed to represent the structural basis of the blood-testis barrier (Dym and Cavicchia, 1977).
The barrier prevents intercellular diffusion
and serves to protect the individual from sensitisation to his own spermatozoan antigens. The barrier may be breached by disease, trauma or obstruction of the tubules or ducts in which case the incidence of sensitisation to autoantigens may be s i g n i f i cant (Schoenfelt
et
ai.,1976;
Tunn, 1975).
I t should be noted that escape of
the entire or intact spermatozoan from the tract may not be required for the formation of autoantibodies;
presentation of soluble or particulate antigens through
leaky epithelial junctions may be sufficient to sensitise the individual. The detailed mechanism whereby obstruction of the reproductive tract e l i c i t s antisperm autoantibody formation is unknown.
I t seems likely that antigen escapes
from distended ducts into the blood stream where they interact with the host immune system so that the humoral and/or cellular manifestations of sensitisation to spermatozoa are observed.
In the case of vasectomy, exposure of the immune
system to sperm is likely to occur, in the f i r s t instance, at operation particularly i f this is performed by resection rather than by ligation alone. Post-
257
Biochemical and Immunological Prospects for Male Contraception operatively, exposure through sperm granulomas is orobably the principal route.
The granulomas,which form in a high proportion of vasectomised men (Alexander and Schmidt, 1977) consist of a central mass of packed sperm and cell d6bris bordered by epithelioid histiocytes with an i n f i l t r a t i o n of polymorphonuclear leucocytes. Granulomas are thought to develop as an inflammatory response to extravasated spermatozoa which are surrounded by a wall of leucocytes.
Early granuloma
formation, however, does not appear to be the result of an inflammation associated with an autoimmune reaction (Brannen et a t . , 197~).
No particular type
of antisperm antibody has been shown to be associated exclusively with the presence of a granuloma;
however, more men with granulomas appear to have immobil-
ising antibodies than men in which such antibodies cannot be detected.
The
sequence of exposure to antigen may also be a factor in the antibody response. Exposure of the immune system to antigen at operation could have the effect of e l i c i t i n g a primary antibody immune response.
I t is well known that the uptake
of soluble antigen both through the gastrointestinal (Andr~ et al., 1974) and respiratory (Stokes et a l . , 1975; Thrall et a l . ,
1978) tracts is considerably
reduced in immunised animals and a similar situation may exist with regard to the uptake of soluble antigens derived from spermatozoa or other seminal constituents. Many questions regarding the way sperm antigens interact with the host immune system, however, remain unanswered.
I t is not known, for example, how the
quantity of antigen and the rate of its exnosure influence the nature of the immune response.
The type of exposure may also be an imnortant factor in deter-
mining the expression of immunity, e.g. escape of intact spermatozoa compared with the leakage of soluble antigens.
The immune system may also react to certain
spermatozoan antigens in a tolerant manner but be sensitised by others. A further consideration that must be taken into account in regard to the isolation of the reproductive tissues from the immune system is the possible involvement of phagocytic cells from the host.
The directed migration of polymorphonuclear
leucocytes from the vascular compartment into the tissues is known to be mediated by a variety of humoral agents derived from microorganisms, tissues and plasma proteins including the complement, kinin, f i b r i n o l y t i c and coagulation systems (see review by Hilkinson, 1974).
These chemotactic factors have been identified
i n s i t u in areas of inflammation and in normal biological fluids.
The importance
of the leucocytic influx in the host response to infection is well known. Factors that are released by polymorphonuclear leucocytes that have a toxic effect on adjacent host tissues have also been identified and these are believed to act on spermatozoa in the female reproductive tract.
Muir et a l . (1977) have carried
out a detailed study of the possibility that cell mediated immune reactions may be involved in the abnormalities resulting in the semen of men who had undergone a
258
P, J. Quinn
reversal vasectomy.
Oligospermia with ooor m o t i l i t y and pleomorphism of the
spermatozoa had been suggested e a r l i e r to be due to autoimmune reactions (Shulman, 1971).
An assay for cell mediated immunity to human sperm extracts
was developed using the a b i l i t y
of the antigen to transform peripheral lymphocytes
in c u l t u r e , but they were unable to find any evidence f o r s p e c i f i c stimulation of leucocytes even in patients that had been vasectomised f o r periods of up to one year.
Similarly,
skin tests performed on guinea-pigs using heat-treated homo-
logous epididymal sperm extracts f a i l e d to e l i c i t
a delayed-type h y p e r s e n s i t i v i t y .
I t was concluded that cell-mediated immunity to sperm does not develop in e i t h e r man or guinea-pig following vasectomy despite the obvious humoral response and the c l e a r l y demonstrated cell-mediated immunity to sperm in immunised guinea-pigs (Harcus et a l . , 1973, 197£).
Thesestudies appear to be in complete contrast
to those of Nagarkatti and Pao (1976) who used an inhibition of leucocyte migration test in the presence of sperm antigens as an index of cell mediated immunity.
They reported clear positive results in many males following vasectomy
and an increased incidence with the length of time following operation.
Further-
more, i t has been shown recently that T-cells from long-term vasectomised rhesus monkeys have reduced mitogen reactivity in mixed lymphocyte cultures (Wilson et a t . , 1977) and this appears to be associated with histocompatibility type (Wilson et a l . ,
197q).
Clearly more data is required to determine whether or not cell
mediated immune reactions are a feature of vasectomy in humans.
Chapter 4
Survival of Spermatozoa in the Female Tract and Fertilization
In order that successful f e r t i l i z a t i o n
of the egg may be achieved the inseminated
spermatozoa must be transoorted through the female genital t r a c t to the site of fertilization changes.
and they must have undergone certain certain crucial developmental
Interruption of either processes can provide a potential method of
preventing f e r t i l i z a t i o n .
4.1. Factors Affecting Sperm Viability and Transpo~ Assessment of semen quality a b i l i t y of the spermatozoa.
in
vitro
has been used as a guide to the f e r t i l i z i n g
Spermatozoa that are defective morphologically or
show poor m o t i l i t y are at an obvious disadvantage when compared with normal cells. Furthermore, sperm concentrations appear to be related to f e r t i l i t y .
Other
factors present in the seminal plasma and female t r a c t secretions also assist in sperm transport and maintain v i a b i l i t y of the cells.
Such factors, for example,
have been shown to protect spermatozoa from damage associated with l i p i d autooxidation.
Human spermatozoa have been shown to be p a r t i c u l a r l y susceptible to
damage by oxidative processes. Peroxidation of the membrane phospholipids, p a r t i c u l a r l y docosahexanoic acid associated with plasmalogens, causes a progressive and i r r e v e r s i b l e loss of structural
i n t e g r i t y and v i a b i l i t y manifested by loss
of m o t i l i t y and metabolic a c t i v i t y (Jones and Mann, 1976).
Not only are endogen-
ous l i p i d peroxidation products detrimental to spermatozoa but exogenous f a t t y acid peroxides are potent spermicidal agents (Jones and Mann, 1977).
The
harmful effects of autoxidation processes can be reduced by serum proteins (Stocks e t a l . , 1974) human caeruloplasmin (AI-Timini and Dormandy, 1977) and seminal plasma (Jones et
al.,
1979) presumably by binding the degraded l i p i d s .
The amelioration of these effects by agents present in the female t r a c t is also l i k e l y to be one factor concerned in the v i a b i l i t y of spermatozoa.
259
260
P.J. Quinn
Those factors which are normally present in human semen that may affect f e r t i l i t y w i l l be examined since therapeutic intervention in the male could influence the level of such factors in the eiaculate and hence represent notential male contraceptives. in vitro
I t should be emphasized, however, that postulations based on
studies of spermatozoa should not be regarded as conclusive since
deficiencies recognized in the ejaculate may be compensated by adequate supplies of the substance in secretions from the female t r a c t .
As we shall see, these
secretions appear to provide key substrates for the modification of spermatozoa that are not supplied in the ejaculate. 4.1.1.
The action of prostaglandins on f e r t i l i t y
One factor considered important for transport and v i a b i l i t y of spermatozoa is the presence of prostaglandins in the seminal plasma.
Ejaculates of normally f e r t i l e
men contain about 1 mg of prostaglandin, mainly of the F-tynes includina PGEI , PGE2, 19-OHPGE1 and 19-OHPGE2 (Templeton e t a l . ,
I~7~).
The function of these
prostaglandins is uncertain but a correlation between f e r t i l i t y concentration in semen has been suaaested (Bygdeman et a l . ,
and prostaglandin
I°70).
This should
be considered in conjunction with the fact that no apparent correlation exists between prostaglandin concentrations and other commonly used parameters of semen quality such as sperm count, m o t i l i t y , volume (Asplund, 1947; C o l l i e r et a l . ,
1975).
Bygdeman e t a l . ,
longevity of the spermatozoa or seminal 1970~ Brummer and Gillespie, In72;
This is somewhat surprising considering the fact that
pharmacological doses of prostaglandins affect seminal characteristics of domestic animals.
Thus PGF2~ has been shown to cause an increase in ejaculated
cell numbers in the s t a l l i o n al.,
(Cornwell e t a l . ,
197a), rabbit and bull (Hafs e t
1974) presumably by nromoting smooth muscle contraction of the epididymus
serving to deliver more spermatozoa to the deferent duct.
~nother rather
anomalous situation is the recent report that the prostaglandin content of ejaculates from polyzoospermic men (sperm concentrations qreater than 3xlO 8 c e l l s / ml) are s i g n i f i c a n t l y less than in ejaculates of lower sperm counts (Kelly e t a l . , 1979). Aspirin, in therapeutic doses, has been shown to depress the prostaglandin concentration in semen (Collier and Plower, 1971) but the drua does not appear to reduce fertility,
suggesting that lowered concentrations of prostaglandins are not
primarily responsible for i n f e r t i l i t y .
Prostafllandins, however, are known to
affect sperm metabolism, for example, 19-~H PG~ (but not PGE or PGF) reduce respiration of washed spermatozoa in v i t r o but do not affect alycolysis (Kelly, 1977).
Furthermore, endogenous synthesis of prostaalandins
vesicle is related to the androgen status of the individual.
by the human seminal Testosterone has
261
Biochemical and Immunological Prospects for Male Contraception been shown, for example, to increase the synthesis of 19-NH PGE in hypo~onadal males (Skakkeback e t a t . ,
1976).
Two conceivable mechanisms could be considered
with regard to the action of prostaglandins as male contraceptive aqents.
Firstly,
there is some evidence to suggest that prostanlandins affect sperm transport through the female genital t r a c t by altering tubal or uterine a c t i v i t y .
In this
respect certain prostaglandins have been shown to increase the rate of transport of spermatozoa (Hafs, 1974; Hargrove and E l l i s , 1976) so that removal of prostaglandins from the ejaculate or i n h i b i t i n g t h e i r action on the smooth muscles of the female t r a c t could interfere with sperm transport.
Secondly, by altering the
metabolic a c t i v i t y of spermatozoa the a c t i v i t y of the cells could be impaired to an extent where they may exhibit reduced a b i l i t y to penetrate the cervical mucus and the final barrier to f e r t i l i z a t i o n ,
the investments surrounding the eqg.
This aspect w i l l be dealt with in greater detail in a l a t e r section. Apart from affecting sperm transport and v i a b i l i t y
prostaglandins appear to be
involved in the contraceptive efficiency of intrauterine devices (Thompson, I°73). For example, an increased content of prostaglandins in uteri of rats, hamsters (Saksena e t a l . ,
1974) mice (Lau e t a l . ,
1974) and rabbits (Saksena and Harper,
1974) in which intrauterine devices were inserted has been reported and that administration of indomethacin, a potent i n h i b i t o r of prostaglandin synthesis, suppresses the contraceptive action of the device (Saksena and Harper, 197~).
In
addition, the decidual cell reaction appears to be suppressed by the presence of an intrauterine device and although prostaglandins have been shown to be l e u t o l y t i c (Chatterjee, 1972) i t is not certain whether or not i n h i b i t i o n of decidualization is mediated through alterations in ovarian steroidogenesis alone.
Bronson and
Hamada (1979) have reported experiments on ovariectomised, hormone-replaced pregnant mice treated with PGE2 and PGF2~ to distinguish the extraovarian actions of prostaglandins upon the decidual cell reaction from those mediated by alterations in ovarian steroidogenesis.
They found that the effect of PGF2~ on decidualization
was mediated via an alteration in ovarian steroidogenesis, but that PGE2 acted independently of the ovary.
T h e y suqgested on the basis of these experiments
that the r a t i o of E to F prostaglandins e l i c i t e d within the endometrium by the presence of an intrauterine device i n h i b i t s decidualization at the uterine level possibly mediated by the impairment of histamine release from endometrial mast cells. The prominent i n f l u x of leucocytes into the t r a c t in response to the presence of spermatozoa or other seminal constituents has been repeatedly observed (Mattner, 1969;
Soupart, 1970) and suggests that semen contains leucocyte chemotactic
factors, or agents, that can interact with other proteins to generate chemotactic
262
P.J. Quinn
factors.
Indeed, the
studies of ~laroni and colleagues (~!aroni
in vitro
et al.,
1971, 1972) have demonstrated selective chemotaxis of macrophages towards human and guinea-pig spermatozoa and seminal f l u i d . macrophages there was l i t t l e
Although the spermatozoa attracted
a c t i v i t y for polymorphonuclear leucocytes whereas
seminal f l u i d a c t i v a t i o n of serum or plasma generated chemotactic factors r e l a t i v e l y s p e c i f i c for polymorphs.
The chemotactic factor was not i d e n t i f i e d
but this was l a t e r shown to be due to the i n t e r a c t i o n of both spermatozoa and seminal plasma with the serum complement system to generate a ootent polymorphonuclear leucocyte chemotactic f a c t o r from the f i f t h (Clark and Klebanoff, 1976).
component of complement
The role of prostaglandins in these chemotactic
processes has yet to be assessed. Prostaglandins and p a r t i c u l a r l y the balance between E and F-type prostaglandins are also believed to be responsible for the physiological control of ovum transport in the female t r a c t .
Recent studies of the influence of hCG and steroid treatment
on prostaglandin metabolism by rabbit uterus and oviduct has suggested that changes in metabolism of PGE2 and PGF2m by the oviduct is the primary mechanism regulating ovum transport (Bodkhe and Harper, lq7q).
The use of Drostaglandins
as contraceptive agents has been suggested because they i n t e r f e r e with sperm and ovum transport in the female t r a c t and they could be involved in leucocytic processes.
One of the major d i f f i c u l t i e s
in the use of naturally-occurring
prostaglandins as contraceptives is the high incidence of side effects diarrhea and vomiting and the f a c t that they are not completely r e l i a b l e .
The
quest for synthetic analogues of the prostaglandins which are more e f f e c t i v e and clinically 4.1.2.
acceptable continues. Steroid binding and sperm transport
Specific steroid-binding sites on the surface of human spermatozoa have been identified
(Hyne and Boettcher, 1977).
These include a l o w - a f f i n i t y ,
specific
oestradiol-binding s i t e for which progesterone and a number of s t r u c t u r a l l y related steroids are e f f i c i e n t competitors compared with testosterone. series of binding sites appear to have a high a f f i n i t y
for testosterone.
of sperm migration through cervical mucus or buffered albumin solutions
Another Studies in vltro
have shown that m o b i l i t y is i n h i b i t e d by progesterone or synthetic progestogens (Kesseru e t a l . ,
1975;
Beck e t a l . ,
1976) suggesting that binding of steroids
to spermatozoa may be an e f f e c t i v e method of regulating f e r t i l i t y antifertility
and explain the
e f f e c t s of steroid-releasing i n t r a u t e r i n e (Hagenfeldt, 1976) and
intravaginal devices (Mishell, 1975).
These devices release pharmacological
doses of steroids into the lumen of the female genital t r a c t where they compete for oestradiol-binding sites on the surface of the spermatozoa.
In a detailed
263
Biochemical and Immunological Prospects for Male Contraception study of the binding of steroids to human spermatozoa Hyne and Boettcher (1978) showed that some steroids such as progesterone, lynestrenol and norethynodrel markedly disrupt sperm migration and m o t i l i t y ,
whereas other steroids such as
oestrone have no detectable e f f e c t on these processes.
Some of the steroids
appeared to i n h i b i t oxidative metabolism and t h e i r e f f e c t s may be ascribed to interference with energy requiring steps in f e r t i l i z a t i o n , seemed to i n h i b i t migration and m o t i l i t y d i r e c t l y .
while other steroids
I t was concluded that
synthetic progestogens released from devices within the female t r a c t compete strongly f o r the oestrodiol-binding receptors on the plasma membrane of the spermatozoa where they a l t e r the permeability c h a r a c t e r i s t i c s of the membrane or, in some way, d i s r u p t a membrane function associated with sperm m o t i l i t y .
I t is
noteworthy that the s i t e of action of these steroid hormones is d i f f e r e n t from the i n t r a c e l l u l a r protein-binding sites c h a r a c t e r i s t i c of the mode of action of these hormones as described in Section 2.1.2.
4.2. Developmental AIterations to Spermatozoa in the Female Tract When semen is deposited in the female t r a c t the spermatozoa undergo a series of complex biochemical and morphological changes which are required to render them capable of successfully f e r t i l i z i n g
the egg.
These events are time-dependent
and they appear to rely upon substances provided in secretions of the female t r a c t . The nature of these processes w i l l
be examined in turn and l i k e l y avenues for
contraceptive i n t e r v e n t i o n explored. 4.2.1.
Capacitation and decapacitation processes
Freshly ejaculated spermatozoa from mammalian species are incapable of immediately fertilizing
eggs;
they require a period of incubation e i t h e r i n v l v o or i n v i t r o
during which they acquire the canacity to f e r t i l i z e (see Austin, 196~).
or are said to be capacitated
The process can be reversed at any stage by exposure of the
spermatozoa to fresh seminal plasma or epididymal f l u i d which contains the putative decapacitation f a c t o r whereupon capacitation must recommence from i t s initial
state (Chang, 1957).
The period required to capacitate spermatozoa
varies from one species to another and, for example, a 6-I0 hour incubation is needed to f u l l y capacitate r a b b i t spermatozoa whereas in the mouse only 2 hours is s u f f i c i e n t .
These times r e f e r to the minimum period of incubation under
optimal conditions that fresh epididymal or ejaculated spermatozoa require to complete the changes involved in capacitation.
Since the discovery that the
decapacitation factor could be removed from seminal plasma by u l t r a c e n t r i f u g a t i o n (Bedford and Chang, 1962) a number of studies have been undertaken in an attempt to characterize the f a c t o r or factors.
There is s t i l l
uncertainty about the nature
264
P.J. Quinn
of the decapacitation f a c t o r with some claims that i t is a large protein or elycoprotein constituent of the seminal plasma (mavis, l q 7 l ) , or a ] i n i d - ] i k e (Dukelow e t a l . ,
component
1967) while other workers suggest that i t is a small lysine-
containing peptide (McRorie and Williams,
1974).
There i s , however, general
agreement that the decapacitation factor originates from the male accessory glands as well as the testes and the male genital t r a c t .
Furthermore, the notion that
capacitation of spermatozoa involves the removal or i n a c t i v a t i o n of decapacitation f a c t o r t i g h t l y bound to the plasma membrane of the cell receives popular support (Weinman and Williams,
1964).
A plausible mechanism of action of bound decapacit-
ation f a c t o r has been suggested by Bedford (1977) in which sites of importance in the induction of the next stage of the developmental process, the acrosome reaction, are e f f e c t i v e l y blocked by the factor and u n t i l ment is prevented.
they are unmasked f u r t h e r develop-
In a recent study of the decapacitation f a c t o r in human
seminal plasma (Kanwar e t a l . ,
1979) a macromolecular substance or factors
associated with macromolecules were found to e f f e c t i v e l y i n h i b i t penetration of human spermatozoa into zona-free hamster eggs. The f a c t that seminal plasma contains substances that i n h i b i t capacitation suqgests a number of ways in which contraception might be achieved.
Seminal plasma is
e f f e c t i v e l y separated from spermatozoa in species where semen is deposited in the vagina (e.g. humans and rabbits) by the b a r r i e r provided by the cervix and i t s associated mucus.
I f this b a r r i e r were removed then presumably seminal plasma
would be able to accompany the spermatozoa to the s i t e of f e r t i l i z a t i o n penetration into the egg.
and block
An a l t e r n a t i v e approach could be to attempt to
s t a b i l i z e the i n t e r a c t i o n between the decapacitation factor and i t s receptor site on the surface of the spermatozoa. considerably more information w i l l
Clearly, in order to pursue the second route, be required concerning the chemical nature of
the decapacitation f a c t o r ( s ) and the precise function that the f a c t o r ( s ) 4.2.2.
perform.
The acrosome reaction
When spermatozoa are f u l l y
capacitated they are able to enter the next phase of
development referred to as the acrosome reaction. the acrosome reaction is not r e v e r s i b l e .
Hnlike the capacitation process
Once spermatozoa have undergone the
acrosome reaction i t is u n l i k e l y that they survive any considerable length of time so that f o r successful f e r t i l i z a t i o n the egg.
the reaction must occur in the proximity of
Indeed the question of whether or not spermatozoa that have undergone
the acrosome reaction in situations remote from the egg are at all capable of subsequently f e r t i l i z i n g
an egg is debatable.
The acrosome reaction involves extensive morphological changes in the head region of the sperm including vesiculation and blebbing of the plasma membrane and the
265
Biochemical and Immunological Prospectsfor Male Contraception outer acrosomal membrane.
This process serves to release factors required to
facilitate
the penetration of the spermatozoa through the i nvesi~ents surrounding
the egg.
Another feature of sperm a c t i v i t y associated with i n i t i a t i o n
of the
acrosome reaction is the development of a pronounced whiplash type of m o t i l i t y (Yanagimachi, 1970, 1972; motility.
Fraser, 1977) often observed as a general a c t i v a t i o n of
I t is possible that such patterns of m o t i l i t y are complementary to the
enzymic attack on the egg investments mounted by enzymes released from the sperm acrosome.
The various impediments to sperm nenetration include the outer cumulus
oophorus and corona radiata layers which envelop the zona pellucida which the egg i t s e l f .
bounds
The structure of these investments are shown schematically in
Fig. 8.
..
'...
.
" d}°" ° o • ', • .u..... " . . . . ' . :........,X..:.~;..O!i~6..:~?,~...: ",. .~J.'.;;~..~7-,~r'~ ~.~.,~r,.,~Jri :.;. •
, •
.-".
: ;'.~t"
'".;:
cm,
°®~'~
",.~'t~,k_).
:-.,~..'/.. - . :..,,.0 : : " ,,..ft.:,.
; ,'...~X'~...~<.
{ :: '.:: ::i .,kI.t z=,. ,,,,u°=
~5 "':
•"
o
~
.' "
Fig. 8
~
~ (.'.
":: , : ~ i i t e l l i l ' ~ -
.,,..
membrane
-v,,.,= :.'..- .,~.: ~ c~";7%'."
Diagram of the investments surrounding the typical mammalian egg.
The factors released from the acrosome are mainly enzymes capable of digesting these layers and allowing spermatozoa to fuse with the egg plasma membrane at the centre. I d e n t i f i c a t i o n of acrosomal enzymes and characterisation of t h e i r putative roles in f e r t i l i z a t i o n
have been undertaken by detergent extraction or sonication pro-
cedures which release the enzymes from the i n t a c t acrosome.
The extracted
enzymes have been p u r i f i e d and t h e i r e f f e c t s on i n t a c t eggs observed,
llnfortun-
a t e l y , these methods do not provide any information concerning the concerted or sequential release of acrosomal enzymes or t h e i r temporal role in the f e r t i l i z a t i o n process.
Moreover, the treatments required to remove "acrosomal components" are
rather drastic and c e r t a i n l y lack s u f f i c i e n t s e l e c t i v i t y to enable precise
266
P.J. Quinn
locations of p a r t i c u l a r enzymes within the structure to be determined.
For
example, contamination of detergent or sonicated extracts with typical mitochondrial enzymes is frequently observed.
~levertheless, a number of enzymes
believed to be released when spermatozoa undergo the acrosome reaction have been i d e n t i f i e d and include 6-glucuronidase, hyaluronidase and other proteases, acid phosphatase, phospholipases and arylsulphatases. Hyaluronidase is one of the more important enzymes involved in the acrosome reaction.
I t is released in some quantity simply by freezing spermatozoa suggest-
ing that i t is r e l a t i v e l y loosely compartmentalized within the c e l l . is responsible f o r penetration of the cumulus oophorus.
The enzyme
I t was o r i g i n a l l y thought
that the entire cumulus layer had to be digested but the evidence was derived mainly from experiments where large quantities of sperm-derived hyaluronidase were employed.
Motile, i n t a c t ejaculated spermatozoa from a v a r i e t y of species have
been shown to approach and adhere to the cumulus but f a i l
to penetrate the layer
unless hyaluronidase is released, a process that is associated with disruption of the plasma and acrosomal membranes (Austin, 1960).
Additional information on
the role of hyaluronidase has been obtained from immunological studies of the r a b b i t system.
I t has been shown, for example, that anti-hyaluronidase antibodies
which bind s p e c i f i c a l l y to the enzyme and block a c t i v i t y prevents f e r t i l i z a t i o n . Another enzyme believed to be released in the acrosome reaction of certain spermatozoa is the corona penetrating enzyme.
I t appears to act in a similar
manner to hyaluronidase but instead causes dispersion of cells present in the corona radiata.
There is no evidence indicating protease a c t i v i t y by the f a c t o r
which may also be present in uterine secretions since the corona is completely removed following f e r t i l i z a t i o n .
Acrosomal enzymes are also present which allow
spermatozoa to penetrate the zona pellucida.
This layer consists mainly of
neutral and acidic glycoproteins linked by disulphide bridges.
Hyaluronic acid
and other glycosidic components have also been detected in this layer.
Since a
v a r i e t y of d i f f e r e n t components are present in the zona pellucida, i t is thought that a combination of enzymes may be required for penetration of the spermatozoa. One enzyme believed to be active in this region has been described in r a b b i t , boar and human spermatozoa and is referred to as acrosin. be present as an inactive zymogen, proacrosin.
This protease is thought to
I t is a calcium-requiring serine
protease which hydrolyses peptide bonds adjacent to h i s t i d i n e residues. Human spermatozoa possess an i n h i b i t o r of acrosin referred to as acrostatin.
It
has been sugqested that acrostatin may be required to prevent l y t i c a c t i v i t y of acrosin when the enzyme is released from spermatozoa during the acrosome reaction since the enzyme would otherwise damage uterine and tubal tissues.
Acrostatin is
267
Biochemical and Immunological Prospects for Male Contraception presumed to be removed from the spermatozoa or i t s e l f
inactivated while present in
the female genital t r a c t , possibly in association with the capacitation process. What r o l e , i f any, secretions from the female t r a c t play in removal of acrostatin is unknown.
The precise locations of acrosin and acrostatin in the sperm head
are also unknown.
Some studies have suggested that acrosin is associated with
the inner acrosomal membrane (Srivastava, 1973;
Brown and Hartree, 1974) while
others provide evidence for a binding s i t e on the acrosome surface (Yanagimachi and Teichman, 1972;
Stambaugh and Buckley, 1972;
Garner et a t . ,
1975).
Any
acrosin that is associated with the outer acrosomal membrane is u n l i k e l y to be involved with penetration of the sperm through the zona pellucida because this membrane is shed before the sperm reaches this layer.
Nevertheless, the p o s s i b i l -
i t y that acrosin may be responsible f o r diaestion of the plasma and outer acrosomal membranes during the acrosome reaction cannot be e n t i r e l y discounted, especially since protease i n h i b i t o r s are known to i n h i b i t the acrosome reaction (Meizel and Lui, 1976).
In a careful detergent-extraction study Bhattacharyya and Zaneveld
(1978) concluded that both acrosin and acrostatin e x i s t at two locations on the spermatozoa; the f i r s t
representina about 10% of the total enzyme and i n h i b i t o r
associated with the outer acrosomal membrane and the remainder at a second s i t e closely adherent to the inner acrosomal membrane.
The acrosin at the l a t t e r s i t e
appears to be predominantly in the proacrosin form. The development of low molecular weight synthetic compounds capable of binding to acrosin and preventing p r o t e o l y t i c action has been suggested as a method which has considerable contraceptive potential
(Zaneveld, 1976).
The compound p - n i t r o p h e n y l
p'-guanidinobenzoate appears to have the most potent i n h i b i t o r y action on human acrosin and to i n h i b i t f e r t i l i z a t i o n at.,
1976).
fertilization
in several snecies tested (Bhattacharyya et
Further agents that i n t e r f e r e with acrosomal factors and block are being a c t i v e l y sought.
Penetration of the zona pellucida must be considered as a more complicated process than simply the enzymic digestion of the zona material.
The existence
of s p e c i f i c sperm receptors, for example, has been suggested on the basis of studies in the hamster.
Addition of plant l e c t i n s such as wheat germ agglutinin
to hamster eggs results in the binding of the l e c t i n to the n-acetyl neuraminic acid residues located in the zona which prevents f e r t i l i z a t i o n .
A neuraminidase
had been characterized in rabbit spermatozoa which is t i g h t l y membrane-bound and removes n-acetyl neuraminic acid residues from the zona. prevents f e r t i l i z a t i o n
Action of the enzyme
and i t s role in penetration of spermatozoa is unknown.
The role of calcium in these processes has been recognised. be required for capacitation (Yanagimachi
and Usui, 1974;
Calcium is known to Rogers and Yanagimachi,
268
P.J. Quinn
1976) and the calcium ionophore, A23187, has been reported to accelerate the capacitation of rabbit spermatozoa i n v i t r o
(Reyes et a l . ,
1978).
Related to
the effect of calcium on capacitation is the concentration of this ion in tubular secretions.
I t has been shown, for example, that calcium concentration increases
in the tubal secretions of the rabbit (Holmdahl and Mastroianni, 1965) and rhesus monkey (Mastroianni et a l . , 4.2.3.
1973) following ovulation.
Control of capacitation and the acrosome reaction
Many studies have been undertaken to investigate the process of capacitation and the acrosome reaction and the development of successful in v l t r o techniques has greatly assisted these studies.
fertilization
One such system has been devised
in the mouse which enables the temporal events in the process to be clearly distinguished (Fraser, 1979).
B r i e f l y the system consists of incubating epidi-
dymal spermatozoa under experimental conditions for a designated time, mixing the spermatozoa with eggs and then f i x i n g the eggs 75 min l a t e r for microscopic examination.
I f spermatozoa are f u l l y capacitated at the time they are added to
the eggs, by the time of f i x a t i o n the f e r t i l i z e d eggs w i l l all have completed meiosis I I , telophase can easily be recognised and the second polar body w i l l observed.
be
Meanwhile, the penetrating spermatozoa shows evidence of usually
complete decondensation with the egg cytoplasm.
I f spermatozoa are not f u l l y
capacitated at the time of addition to the eggs but undergo the acrosome reaction at some later stage, this w i l l be reflected in a correspondignly e a r l i e r stage of development of the f e r t i l i z e d egg.
This technique essentially avoids the need
to assess capacitation on the basis of examination of eggs reaching the two-cell stage 24 hours after adding spermatozoa. Of particular interest in these studies has been the substrate requirements for capacitation and the acrosome reaction.
Despite the fact that fructose is by far
the most abundant hexose substrate in the semen of a wide variety of manTnalian species (Mann, 1964) this sugar does not support capacitation and f e r t i l i z a t i o n those species so far examined.
Thus no f e r t i l i z a t i o n
in
was obtained, for example,
in in vitro systems of mouse (Hoppe, 1976) and rat (Niwa and I r i t a n i , 1978) gametes when the sole exogenous substrate was fructose, although when glucose was present high levels of f e r t i l i z a t i o n were achieved. for
in v i t r o
Most culture media employed
f e r t i l i z a t i o n and preimplantation embryonic development contain
pyruvate, lactate and glucose, and i t was originally suggested that all three substrates were required to support f e r t i l i z a t i o n (Mihamoto and Chang, 1973). Subsequently i t was found that lactate is not an essential ingredient (Hoppe, 1976) A comprehensive study of substrate requirements in the murine system has been reported (Fraser and Quinn, 1980). I t was shown that spermatozoa preincubated
Biochemical and Immunological Prospects for Male Contraception
269
in the absence of exogenous substrates for sufficient time to permit f u l l capacitation in complete medium failed to f e r t i l i z e eggs until supplied with glucose;
f e r t i l i z a t i o n then proceeded immediately and nuclear development advanced
to stages that were indistinguishable from those obtained with glucose present throughout.
Experiments performed under completely anaerobic conditions and in
the presence of inhibitors of oxidative phosphorylation showed that some metabolite of glycolysis was obligatory for i n i t i a t i n g the acrosome reaction and inducing the characteristic whiplash m o t i l i t y .
I t was also confirmed that fructose could
not substitute for glucose in these processes, thereby demonstrating that ejaculated spermatozoa cannot proceed beyond the terminal stages of capacitation without a supply of glucose from female tract secretions. These observations suggest methods for regulating the development of spermatozoa for contraceptive purposes and provide a plausible explanation for the antifertility
action of ~-chlorohydrin.
Metabolic, morphological
and other studies of the effect of various dose levels of
~-chlorohydrin on males of different species have suggested that one possible antifertility
effect of this compound is via an effect on the metabolism of spermatozoa.
Thus when ~-chlorohydrin is administered intravenously i t enters sperm cells and acts as a competitive inhibitor of glycerol phosphorylation (Edwards et a l . , mediated by glycerol kinase (Brooks e t a t . ,
1974).
1976)
The metabolic disposal of
[36CI] and [l~c]-~-chlorohydrin has been investigated in some detail in the rat. Compounds containinq labile halogens are normally detoxified in the l i v e r by conjugation with glutathione and eventually excreted as S-substituted cysteine conjugates in the urine.
Fig. 9 shows the various metabolic pathways delineated for
~-chlorohydrin metabolism.
The phosphorylated product of ~-chlorohydrin is a
potent i n h i b i t o r of glyceraldehyde-3-phosphate al.,
1978).
dehydrogenase (Brown-Woodman et
Irreversible inhibition of this glycolytic enzyme has been shown
to cause a reduction in ATP concentration in the cell (Chulavatnatol et a l . ,
1977).
resulting in a decrease of sperm m o t i l i t y (Brown and White, 1973; Brown-Woodman et al.,
1974).
The effect on general sperm m o t i l i t y , in the l i q h t of studies on
the murine system (Fraser and nuinn, 19nO), may however be secondary to the specific effects that Qlycolytic products exert on the acrosome reaction and whiplash m o t i l i t y . The contraceptive usefulness of ~-chlorohydrin has been shown to be limited by the manifestation of undesirable side effects.
Nevertheless, in the knowledge that
the action of ~-chlorohydrin is to interfere with glycolysis, other strategies seem possible.
The use of less toxic analogues of ~-chlorohydrin or other
reagents that i n h i b i t glyceraldehyde-3-phosphate
dehydrogenase could be developed.
270
P. J. Quinn 3- chloropropan1,2-diol-l-phosphate
CH,CI I CHOH
0
AH 2--0
bl
OH CH,CI I CHOH F
l
7H,CI
co,
I
f
CI
YHOH CO,H
cl-:0 P-chbro lactaldehyde
C02H I Co2H oxalate
p-~ZtYe
or-chlorohydrin CO,H
J CH,- S-glutathibne glycidol
glycerol
-
I CH,OH
CH,OH
I YHPH
glycerolipids
CHOH
I
CH,OH Fig. ?
CH,-S -CH2+H CHOH CH,OH
NH:!-++ excretory pathway as cysteine metabolites
r” ‘t
CO,
Pathways of metabol ism of a-chl orohydrin in mammals
The antibiotic, pentalenolactone (Fig. 10 ) which can be isolated from the fermentation broth of various species of Streptomyces (Aizawa et a~., 197g), for
Fig. 10
Structure of pentalenolactone E.
example, is also known to inhibit glycolysis by forming an irreversible complex with qlyceraldehyde-3-phosphate dehydrogenase (Hartmann et al., 1978).
The anti-
271
Biochemical and Immunological Prospects for Male Contraception b i o t i c i r r e v e r s i b l y i n h i b i t s the enzyme ~rom a variety of sources and when
examined in plant systems, which have three classes of enzyme, there appears to be a high s p e c i f i c i t y for the enzyme concerned with glycolysis (Mann and Mecke, 1977). Another possible contraceptive device may be to supply glucose in the ejaculate, or in the epididymus, with the aim of inducing premature i n i t i a t i o n of the acrosome reaction and whiplash m o t i l i t y , can successfully f e r t i l i z e
the egg.
thereby reducing chances that the spermatozoa Removal of glucose from female t r a c t
secretions would also serve to prevent f e r t i l i z a t i o n
by blocking f u l l
development
of the spermatozoa. Apart from the supply of essential glucose, female t r a c t secretions are believed to contain other factors required to maintain sperm v i a b i l i t y genital t r a c t and assist in the f e r t i l i z a t i o n
process.
in the female
Such factors appear to
assist in the binding and penetration of spermatozoa through the zona nellucida, aid in the dispersal of corona cells and provide immunoloqical protection for the zygote.
Oviduct secretions may also supply enzymes, possibly amylases, respon-
sible for removing decapacitation factor(s) from the seminal plasma.
Other
substances present in these secretions also act to prevent sperm penetration into the egg.
The action of acrosin, for example, can be affected by protease
i n h i b i t o r s which have been shown to be present in tubal secretions of women (~1oghissi, 1970; Hirschhauser et a l . , 1971) and rhesus monkeys (Stambaugh e£ a l . , 1974).
Chapter 5
Conclusions and Future Prospects
In the preceeding sections the hormonal regulation of male reproduction has been outlined and possible sites for developing suitable male contraceptives i d e n t i f i e d . Hany of these offer potential for avoiding the two major hazards associated with contraception in the male, namely, the danger of introducing genetic damage and secondly the loss of l i b i d o . Possibly the most promising area for contraceptive development is that where cooperation between the gametes and t h e i r environment in the female genital t r a c t is required to achieve successful f e r t i l i z a t i o n
and development of the embryo.
Clearly, more research is necessary to i d e n t i f y the molecular bases of these cooperations especially in respect of sperm capacitation and penetration of the egg. Survival of the f e r t i l i z e d
egg is another area receiving special attention with
respect to the development of effective contraceptives.
Amongst the most
promising lines of investigation in this connection is the i d e n t i f i c a t i o n of specific tubal and uterine secretions upon which f e r t i l i z a t i o n
and/or maintenance
of the embryo depend, with the intention of provoking autoimmune responses aimed at blocking t h e i r action. difficulties immunity.
This work, however, is confronted by serious technical
as well as the hazards associated with the development of autoAn alternative approach advocated by Aitken (197ga) is to concentrate
an immune attack on antigens of embryonic origin.
This has the d i s t i n c t
advantage that the respective antigens are exposed only b r i e f l y to the maternal immune system.
No factors known to be released from the developing embryo and to
affect oviduct or uterine secretions have, as yet, been f u l l y characterised.
Other
immunological methods that show promise include the active immunization of women against chorionic gonadotrophin (Talwar et a t . , 1976a & b;
272
Hearn, 1979).
The
273
Biochemical and Immunological Prospects for Male Contraception strategy here has been to design an immunogen that is capable of provoking an
autoantibody reaction against the hormone so that the developino embryo f a i l s to rescue the corpus luteum during early pregnancy.
The use of human chorionic
gonadotrophin and i t s fragments coupled to heterologous proteins as immunogens to produce active immunization in women is currently being investigated, but this approach in all events appears to be nonreversible.
Passive immunization with
gonadotrophin antisera would therefore seem to offer greater prospects.
In this
connection, antibodies against the B-subunit of ovine lutenizing hormone have been shown to cross react strongly with monkey chorionic gonadotrophin and block fertility et at.,
(Thau et a l . ,
1979), i n h i b i t ovulation and terminate pregnancy (Hondgal
1978) in immunized monkeys.
Despite these results major d i f f i c u l t i e s
remain in generating and maintaining antibody t i t r e s of high s p e c i f i c i t y for human chorionic gonadotrophin.
F i n a l l y , the presence of autoantibodies directed against
zona pellucida antigens has been detected in some i n f e r t i l e women (Shivers and Dunbar, 1977) and active immunization of mice a~ainst these antiqens leads to a temporary state of i n f e r t i l i t y et at.,
1977).
during which f e r t i l i z a t i o n
is prevented (Gwatkin
Other immunological, hormonal and chemical methods for f e r t i l i t y
control in the female have been reviewed recently by Talwar (1978) and Aitken (1979b). I t may be concluded that despite considerable research experience, acceptable and r e l i a b l e methods og f e r t i l i t y
control in men have not vet been realized.
Nevertheless, a number of approaches appear promisinn and with continued e f f o r t should yield an ef#ective male contracentive in the not too distant future.
Acknowledgement Christine Quinn is thanked for her helpful discussions and for her patience during preparation of the manuscript.
References
Aitken, R.J. (1979a). Tubal and uterine secretions; the possibilities for contraceptive attack. J.Reprod.Fertil., 55, 247-254. Aitken, R.J. (1979b). 35, 199-204.
Contraceptive research and development.
Brit.Med.Bull.,
Aizawa, S., H. Akutsu, T. Satomi, S. Kawatata, and K. Sasaki (1978). AA-57, a new ~ntibiotic related to pentalenolactone. J.Antibiotics, 31, 729-731. Alexander N.J. (1973). Autoimmune hypospermato~enesis in vasectomised ~uinea pi~s. Contraception, 8, 147-164. Alexander N.J. (1977). Fertility in rhesus monkeys after vasovasectomy. Scand.J. Immunol., 6, 675. Alexander N.J., and S.S. Schmidt (1977). Incidence of antisperm antibody levels and granulomas in men. Fertil. Steril., 28, 655-657. Alexander N.J., B.J. Wilson, and G.D. Patterson (1974). Vasectomy: immunologic effects in rhesus monkeys and men. Fertil. Steril., 25, 149-156. AI-Timimi D.J., and T.L. Dormandy (1977). The inhibition of lipid autoxidation by human caeruloplasmin. Biochem. J., 168, 283-288. Andre, C. R. Lambert, H. Bazin and J.F. Heremans (1974). Interference of oral immunization with the intestinal absorption of heterologous albumin. Immunol., 4, 701-704. Ansbacher, R. (1971). Sperm-agglutinating and sperm-immobilizing antibodies in vasectomized men. Fertil. Steril., 22, 629-632. Ansbacher, R. (1973). 792.
Vasectomy:
sperm antibodies.
Ansbacher, R. (1974). 9, 227-237.
Bilateral vas ligation:
274
Fertil. Steril., 24, 788-
sperm antibodies.
Contraception,
Biochemical and Immunological Prospects for Male Contraception
275
Ansbacher, R., P. Hodge, A. Willams and D.M. Mumford (1976). Vas ligation: humoral sperm antibodies. Int. J. Fertil., 21, 258-260. Asplund, J. (1947). A quantitative determination of the content of contraceptive substances in human sperm and their significance for the motility and vitality of the spermatozoa. Acta Physiol. Scand., 13, 103-108. Austin, C.R. (1960). Capacitation and the release of hyaluronidase from spermatozoa. J.Reprod. Fertil., I, 310-311. Austin, C.R. (1969). Sperm capacitation - biological significance in various species. In C.G. Hartman (Ed.), Mechanisms Concerned with Conception, Pergamon, Oxford. pp.285-320. Azim, A.A., S. Fayad, A.A. Fattah, and M. Habeib (1978). male infertility. Fert. Steril., 30, 426-429.
Immunologic studies of
Barraclough, C.A. (1968). In A. McLaren (Ed.), Advances in Reproductive Physiology, Logos, London. pp. Barry, J., and M.P. Dubois (1976). Immunoreactive LFR neurosecretory pathways in mammals. Acta anat., (Basel) 94, 497-503. Barry, J., M.P. Dubois, and P. Poulain (1973). LRF-producing cells of the mammalian hypothalamus. Cell Tiss. Res., 146, 351-366. Barth, R.F., and P.S. Russell (1964). The antigenic specificity of spermatozoa. I. An immunofluorescent study of the histocompatibility antigens of mouse sperm. J. Immunol., 93, 13-19. Beck, K.J., S. Herschel, R. Hungershofer, and E. Schwinger (1976). The effect of steroid hormones on motility and selective migration of X- and Y-bearing human spermatozoa. Fertil. Steril., 27, 407-412. Bedford, J.M. (1977). Sperm/egg interaction: spermatozoa. Anat. Rec., 188, 477-488. Bedford, J.M., and M.C. Chang (1962). Nature, 193, 898-899.
the specificity of human
Fertilization of rabbit ova in vitro.
Belchetz, P.E., T.M. Plant, Y. Nakai, E.J. Keogh, and E. Knobil (1978). Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasin~ hormone. Science, 202, 631-633. Bhattacharyya, A.K., and L.J.D. Zaneveld (1978). Release of acrosin and acrosin inhibitor from human spermatozoa. Fertil. Steril., 30, 70-78. Bhattacharyya, A.K., L.J.D. Zaenveld, B.M. Dragoje, G.F.B. Schumacher, and J. Travin (1976). Inhibition of human sperm acrosin by synthetic agents. J. Reprod. Fertil., 47, 97-1OO. Bi~azzi, P.E., L.L. Kosuda, L.L. Harnick, R.C. Brown, and N.R. Rose (1976a). Antibodies to testicular antigens in vasectomised rabbits. Clin. Immunol. Immunopath., 5, 182-194. Bigazzi, P.E., L.L. Kosuda, K.C. Hsu, and G.A. Andres (1976b). Immune complex orchitis in vasectomised rabbits. J. exp. Med., 143, 382-404.
276
P.J. euinn
Bodkhe, R.R., and M.J.K. Harper (1979). Influence of hCG injection and steroid treatment on prostaglandin metabolism by rabbit uterus and oviduct. J. Reprod. Fert., 57, 101-112. Boettcher, B. (1974). The molecular nature of spermagglutinins and sperm antibodies in human sera. J. Reprod. Fert.,(suppl.) 21, 151-168. Boettcher, B., J. Hay, D.J. Kay, B.A. Baldo, and T.K. Roberts (1970). Spermagglutinating activity in some human sera. Int. J. Fertil., 143-158.
15,
Brannen, G.E., and D.S. Coffey (1974). Immunologic implications of vasectomy: II. Serum-mediated in~nunity. Fertil. Steril., 25, 515-520. Brannen, G.E., J.C. Eggleston, J.S. Adams, and D.S. Coffey (1974). The effects of immuno suppression on sperm granulomas in vasectomised rats. J. Urol., 112, 793-797. Bronson, R., and. Y. Hamada (1978). The .effect of prostaglandins F~Z ~ and E~ on . . . . . pregnancy in mlee during implantation. F e r t l l . S t e r l l . , 30, 354-36~. Brooks, D.E., D.W. Hamilton and A.H. Mallek (1974). Carnitine and glycerylphosphorylcholine in the reproductive tract of the male rat. J. Reprod. Fert., 36, 141-160. Brown, C.R., and E.F. Hartree (1974). Distribution of trypsin-like proteinase in ram spermatozoa. J. Reprod. Fertil., 36, 195-198. Brown, P.D.C., and I.G. Ifhite (1973). Studies on the male anti-fertility druE 3-chloro-l,2-propanediol. J. Reprod. Fert., 32, 337-338. Brown-Woodman, P.D.C., H. Mohri, T. Mohri, D. Suter, and I.G. White (1978). Mode of action of ~-chlorohydrin as a male antifertility agent. Biochem. J., IZ~, 23-37. Brown-Woodman, P.D.C., S. Salamon, and I.G. White (1974). Effect of ~-chlorohydrin on the fertility of rams. Acta. Europ. Fert., 5, 193-197. Brurmner, H.C., and Gillespie, A. (1972). Clin. Endocrin., I, 363-368.
Seminal prostaglandins and fertility.
Burki, K., and W. Sheridan (1978). Expression of TEM-induced damage to postmeiotic stages of spermatogenesis of the mouse during early embryogenesis II cytological investigations. Mut. Res., 52, 107-115. Bygdeman, M., B. Fredricsson, K. Svanborg, and B. Samuelsson (1970). The relation between fertility and prostaglandin content of seminal fluid in man. Fertil. Steril., 21, 622-629. Chatterjee, A. (1972). The possible mode of action of prostaglandins: I. Interruption of decidual reaction by PGF~ and its prevention by using prolactin or progesterone. Acta Endocrlnol. (Kbh), 70, 781-785. Chulavatnatol, M., I. Hasibuan, S. Yindepit, and T. Eksittikul (1977). Lack of effect of ~-chlorohydrin on the ATP content of rat, mouse and human spermatozoa. J. Reprod. Fert., 50, 137-139. Clark, R.A., and S.J. Klebanoff (1976). Generation of a neutrophil chemotactic agent by spermatozoa: role of complement and regulation by seminal plasma factors. J. Immunol., 117, 1378-1386.
Biochemical and Immunological Prospects for Male Contraception
277
Collier, J.G., and R.J. Flower (1971). Effect of aspirin on human seminal prostaglandins. Lancet, 2, 852-853. Collier, J.G., R.J. Flower, and S.L. Stanton (1975). Seminal prostaglandins in infertile men. Fertil. Steril., 26, 868-871. Collins, P., and D. Lacy (1969). Studies on the structure and function of the mammalian testis. II. Cytological and histochemical observations on the testis of the rat after a single exposure to heat applied for different lengths of time. Proc. Roy. Soc., B., 172, 17-38. Corbin, A., C.W. Beattie, J. Tracy, R. Jones, T.J. Foell, J. Yardley, and R.W.A. Rees (1978). The anti-reproductive pharmacology of LH-RH and agonistic analogues. Int. J. Fertil., 23, 81-92. Cornwell, J.C., K.L. Koonce and J.L. Kreider (1974). F2~ on seminal characteristics of the stallion.
Effect of prostaglandin J. Anim. Sci., 38, 226.
Coutinho, E.M., and J.F. Melo (1973). Successful inhibition of spermatogenesis in man without loss of libido: a potential new approach to male contraception. Contraception, 8, 207-217. D'Almeida, M., and G.A. Voisin (1977). Human and guinea-pig cross-reacting spermatozoa autoantigens and autoantibodies. Clin. exp. Immunol., 28, 163-172. Davis, B.K. (1971). Macromolecular inhibitor of fertilization in rabbit seminal plasma. Proc. Natl. Acad. Sci., U.S.A., 68, 951-955. David, S.S., G.R. Huggins, C-R Garcia, and C. Busacca (1979). A synthetic , steroid (R2323) as a once-a-week oral contraceptive. Fert. Steril., 31 278-281. de Kretser, D.M. (1969). Ultrastructural features of human spermJo~enesis. Z. Zellforsch. Mikrosk. Anat., 98, 477-485. de Kretser, D.M. (1976). Towards a pill for men. In R.V. Short and D.T. Baird (Eds.), Contraceptives of the Future, The Royal Society, London. pp.161-174. de la Cruz, A., D.H. Coy, J.A. Vilchez-Martinez, A. Arimura, and A.V. Schally (1976). Blockade of ovulation in rats by inhibitory analogues of luteinizing hormone-releasing hormone. Science, 191, 195-197. Dmowski, ~q.P. (1979). Fertil. Steril.,
Endocrine properties and clinical application of danazol. 31, 237-251.
Drobeck, H.P., and F. Coulston (1962). Inhibition and recovery of spermatogenesis in rats, monkeys and dogs medicated with bis(dichloroacetyl) diamines. Exp. Mol. Pathol., I, 251-258. Drouin, J., and F. Labrie (1976). Selective effect of androgens on LH and FSH release in anterior pituitary cells in culture. Endocrinology, 98, 1528-1534. Dukelow, W.R., H.H. Chernoff, and W.L. Williams (1967). Properties of decapacitation factor and presence in various species. J. Reprod. Fertil.,
1_4, ~93-399. Dunn, M.S., K. Sakamoto, and E.A. Murphy (1962). Effect of ethionine in castrated male rats, with and without testosterone. Proc. Soc. Exp. Biol. Med., IIO, 50~-513.
278
P.J. Quinn
Dym, M. (1973). The fine structure of the monkey (Macaca) Sertoli cell and its role in maintaining the blood-testis barrier. Anat. Rec., 175, 639-656. Dym, M., and J.C. Cavicchia (1977). Further observations on the blood-testis barrier in monkeys. Biol. Reprod., 17, 390-403. Dym, M., and D.W. Fawcett (1970). The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol. Reprod., ~, 308-326. Edwards, E.M., J-L Dacheux, and G.M.H. Waites (1976). Effects of ~-chlorohydrin on the metabolism of testicular and epididymal spermatozoa of rams. J. Reprod. Fertil., 48, 265-270. Edwards, E.M., A.R. Jones and G.M.H. Waites (1975). The entry of ~-chlorohydrin into body fluids of male rats and its effect upon the incorporation of glycerol into lipids. J. Reprod. Fertil., 43, 225-232. Edwards, R.G. (1964. Immunological control of fertility in female mice. Nature, 203, 50-53. Edwards, T.G., L.C. Ferguson, and R.R.A. Coombs (1964). Blood group antigens on human spermatozoa. J. Reprod. Fertil., ~, 153-161. Ellis, L.C. (1970). In A.D. Johnson, W.R. Gomes, and N.L. Van Demark (Eds.), Radiation Effects, Vol. III. Academic Press, New York. pp.333-376. Erickson, B.H. (1976). Effect of 60Co y radiation on the stem and differentiating spermatogonia of the post puberal rat. Radiat. Res., 433-448. Fairley, K.F., J.U. Barrie, and W. Johnson (1972). atrophy related to cyclophosphamide therapy.
68,
Sterility and testicular Lancet, I, 568-569.
Feltkamp, T.E.W., K. Kruyff, N.C.J.J. Ladiges, and P. R~mke (1965). Autospermagglutinins: immunofluorescent studies. Annal. N.Y. Acad. Sci., 124, Fellons, M., and J. Dausset (1970). antigens on human spermatozoa.
Probable haploid expression of HL-A Nature, 225, 191-193.
Fjallbrant, B. (1968). Interrelation between high levels of sperm antibodies, and sterility in men. Acta obstet, gynec. Scand., 47, 102-118. Flickinger, C., and D.W. Fawcett (1967). The junctional specializations of Sertoli cells in the seminiferous epithelium. Anat. Rec., 158, 207-221. Fox, T.O. (1975). 441-444.
Oestradiol receptor of neonatal mouse brain.
Nature,
258,
Estrogen and androgen receptor Fox, T.O., C.C. Vito, and S.J. Wieland (1978). proteins in embryonic and neonatal brain: hypotheses for roles in sexual 18, 525-537. differentiation and behaviour. Am. Zool., Franchimont, P., A. Demoulin, J. Verstraelen-Proyard, M.T. Hazee-Hagelstein, J.S. Walton and G.M.H. Waites (1978). Nature and mechanisms of action of inhibin: perspective in regulation of male fertility. Int. J. Androl., suppl, no.2, pp.69-80.
Biochemical and Immunological Prospects for Male Contraception
279
Franklin, R.R., and C.D. Dukes (1964). Further studies on sperm agglutinating antibody and unexplained infertility. J. Am. Med. Assoc., 190, 682-683. Fraser, L.R. (1977). Motility patterns in mouse spermatozoa before and after capacitation. J. exp. Zool., 202, 439-444. Fraser, L.R. (1979). Rate of fertilization in vitro and subsequent nuclear development as a function of the post-ovulatory age of the mouse egg. J. Reprod. Fertil., 55, 153-160. Fraser, L.R., and P.J. Quinn (1980). A glycolytic product is obligatory for initiation of the acrosome reaction and whiplash motility required for the fertilization of mouse gametes. J. Reprod. Fertil., (in press). Friberg, J. (1974). A simple and sensitive micro-method for demonstration of sperm-agglutinating activity in serum from infertile men and women. Acta obstet, gynec. Scand., (Suppl.) 3-6, 21-29. Frick, J. (1973). Control of spermatogenesis in men by combined administration of progestin and androgen. Contraception, 8, 191-206. Garner, D.L., M.P. Easton, M.E. Munson, and M.A. Doane (1975). Immunofluorescent localization of bovine acrosin. J. Exp. Zool., 191, 127-134. Gorski, R.A. (1968). Influence of age on responses to paranatal administration of a low dose of androgen. Endocrinology, 82, 1001-1004. Gorski, R.A. (1971). Gonadal hormones and the perinatal development of neuroendocrine function. In L. Martini and W.F. Ganong (Eds.), Frontiers of Neuroendocrinology, Oxford Univ. Press, New York. pp. 237-290. Gnodde, H.P., J.A.M.J. Van Dieten, G.P. Van Rees, and I. Rothchild (1979). Conversion of progesterone to testosterone in the rat Leydig cell. Proc. Soc. Exp. Biol. Med., 160, 130-133. Goldberg, E.H., T. Aoki, E.A. Boyse, and D. Bennett (1970). Detection of H-2 antigens on mouse spermatozoa by the cytotoxicity test. Nature, 228, 570-572. Gonzalez-Barcena, D., A.J. Kastin, D.H. Coy, K. Nikoliss, and A.B. Schally (1977). Suppression of gonadotropin release in man by an inhibitory analogue of LH-RH. Lancet, ~, 997-998. Griffiths, E.C., and K.C. Hooper (1973). The stimulatory influence of gonadal steroids and methallibure (ICI 33, 828) on peptidase activity in the female rat hypothalamus. Fertil. Steril., 24, 269-274. Griffiths, E.C., K.C. Hooper, D. Hutson, S.L. Jeffcoate, and D.T. Holland (1975). The presence of peptidases in the rabbit hypothalamus capable of inactivating luteinizing hormone-releasin~ hormone. Fertil. Steril., 26, 802-806. ' ' Griffiths, E.C., K.C. Hooper, S.L. Jeffcoate, and D.T. Holland (1974). The presence of peptidases in the rat hypothalamus inactivating luteinizing hormone-releasing hormone (LH-RH). Acta Endocrinol. , 77, 435-442. Guillemin, R. (1977). Peptides of cerebral origin: their importance in growth physiology and medicine. Neuro-Horm. Hypothal., Masson: Paris. pp.3-16.
280
P. J, Ouinn
Gupta, J., S. Dhawan, G.D. Goel, and K. Saha (1975). Low fertility rate in vasovasostomized males and its possible immunologic mechanism. Int. J. Fert., 20, 183-191. Gwatkin, R.B.L., D.T. Williams and D.J. Carlo (1977). Immunization of mice with heat-solubilized hamster zonae: production of antizona antibody and inhibition of fertility.. Fertil. Steril., 28, 871-887. Hafs, D.H., T.M. Louis, and J.N. Stellflug (1974). Increased sperm numbers in the deferent duct after prostaglandin F2~ in rabbits. Proc. Soc. Exptl. Biol. Med., 145, 1120-1124. Hagenfeldt, D. (1976). The modes of action of medicated intrauterine devices. J. Reprod. Eertil., (Suppl.), 25, 117-132. Hansen, K.B. (1972). Immunofluorescent studies on mammalian spermatozoa: antigenic relationship between human, bull and rabbit spermatozoa. J. Reprod. Fertil., 29, 19-28. Hansmann, I., and H-D. Probeck (1979). The induction of non-disjunction by irradiation in marmnalian oogenesis and spermatogenesis. Mut. Res., 61, 69-76. Hargrove, J.L., and L.C. Ellis (1976). Autonomic nerves versus prostaglandins in the control of rat and rabbit testicular capsular contractions in vivo and in vitro. Biol. Reprod., 14, 651-657. Hartmann, S., J. Neeff, U. Heer, and D. Mecke (1978). Arenaemycin (pentalenolactone): a specific inhibitor of glycolysis. FEBS Lett., 93, 339-342. Hashimoto, Y., T. Makita, H. Miyata, T. Noguchi, and G. Ohta (1968). Acute and subchronic toxicity of a new fluorine pesticide, N-Methyl-N-(l-Naphthyl) fluoroacetamide. Toxicol. appl. Pharmacol., 12, 536-547. Hearn, J.P. (1979). Material Recognition of Pregnancy. (Ciba Foundation Symposium, new series no.64). Excerpta Medica, Amsterdam. pp.353-365. Heidger, P~.M., and C.L. Sawatzke (1977). Fine structural effects of vasectomy. In R.D. Yates and M. Gordon (Eds.), The Male Reproductive System, Masson Inc., New York. pp.131-153. Heinert, G., and H.D. Taubert (1973). Effect of cyproterone and cyproterone acetate on testicular function in the rat: a karyometric study. Endokrinologie, 61, 168-178. Hellema, H.W.J., and P.L. R~mke (1976). Comparison of the tray agglutination technique with the gelatin agglutination technique for the detection of spermagglutinating activity in human sera. Fertil., Steril., 27, 284-292. Hellema, H.W.J., and P. Rumke (1978). Sperm autoantibodies as a consequence of vasectomy. Clin. exp. I~m~unol., 31, 18-29. Heller, C.G., D.J. Moore, and C.A. Paulsen (1961). Suppression of spermatogenesis and chronic toxicity in men by a new series of bis (dichloroacetyl) diamines. Toxicol. Appl. Pharmacol., 3, I-II.
281
Biochemical and Immunological Prospects for Male Contraception Hillier, S.G., G.V. Broom, A.R. Boyns, and E.H.D. Cameron (1975). The active immunisation of intact adult rats against steroid-protein conjugates: effects on circulating hormone levels and related physiological processes. In E.H.D. Cameron, S.G. Hillier and K. Griffiths (Eds.), Steroid Immunoassays, Alpha Omega Publ., Cardiff Wales. pp.97-110. Hinkes, E., and D. Plotkin (1973). patient with acute leukemia.
Reversible drug-induced sterility in a J. Amer. Med. Assoc., 223, 1490-1491.
Hirschhauser, C., V. Kionke, and E. Daume (1971). Trypsin inhibitors in the female genital tract. Acta endocr. Copenh., 68, 413-416. Hitotsumachi, S., and Y. Kikuchi (1977). Chromosome aberrations and dominant lethality of mouse embryos after paternal treatment with triethylenemelamine. Mut. Res., 42, 117-124. Hodgen, G.D., W.R. Gomes, and N.L. Vandemark (1970). In vitro and in vivo effects of cadmium chloride on isoenzymes of carbonic anhydrase in rat testes and erythrocytes. Biol. Reprod., 2, 197-201 Holmdahl, T., and L. Mastroianni (1965). Continuous collection of rabbit oviduct secretions at low temperature. Fertil. Steril., 16, 587-595. Hoppe, P.C. (1976) Glucose requirement for mouse sperm capacitation in vitro. Biol. Reprod., 15, 39-45. Howards, S.S., S.J. Jessee and A.L. Johnson (1975). Micropuncture studies of the blood seminiferous tubule barrier. Biol. Reprod., 14, 264-296. Howards, S.S., C.J. Flickinger, D.W. Holler, and W.B. Panko (1979). The effects of vasectomy on testicular metabolism in the guinea-pig. J. Reprod. Fertil., 55, 401-405. Hsueh, A.J.W. (1978). Current developments in the mechanism of action of reproductive hormones. Clin. Obstet. Gynecol., 21, 53-66. Hsueh, A.J.W., M.L. Dufau, and K.J. Catt (1977). Gonadotropin-induced regulation of luteinizing hormone receptors and desensitization of testicular 3':5'-cyclic AMP and testosterone responses. Proc. Natl. Acad. Sci., U.S.A., 74, 592-595. Hsueh, A.J.W., and G.F. Erickson (1979). Extra-pituitary inhibition of testicular function by luteinising hormone releasin~ hormone. Nature, 66-67.
281,
Humphries, J., J.P. Wan, K. Flokers, and C.Y. Bowers (1976). Presence of proline in position 3 for potent inhibition of the activity of the luteinizing hormone-releasing hormone and of ovulation. Biochem. Biophys. Res. Commun., 72, 939-944. Humphries, J., Y.P. Wan, K. Folkers, and C.Y. Bowers (1978). Inhibitory analogues of the luteinizing hormone-releasing hormone havinB D-aromatic residues in positions 2 and 6 and variation in position 3. J. Med. Chem., 21, 120-126. Humphries, J., Y.P. Wan, T. Wasiak, K. Folkers, and C.Y. Bowers (1979). Structural requirements in positions I, 2, 3 and 6 of the luteinizing hormone-releasin~ hormone (LH-RH) for antiovulatory activity. J. Med. Chem., 22, 774-777.
282
P.J. Quinn
Husain, R., P.K. Seth, and S.V. Chandra (1976). dehydrogenase by manganese in rat gonads. 16, 118-121.
Early inhibition of succinate Bull. Env. Contam. Toxicol.,
Hyne, R.V., and B. Boettcher (1977). The selective binding of steroids by human spermatozoa. Contraception, 15, 163-174. Hyne, R.V., and B. Boettcher (1978). Binding of steroids to human spermatozoa and its possible role in contraception. Fertil. Steril., 30, 322-328. Igboeli, G., and A.M. Rakha (1970). Bull testicular and epididyrnal functions after long-term vasectomy. J. Anim. Sci., 31, 72-75. Ingerslev, H.J., and T. Hjort (1979). Spermagglutinating antibodies and B-spermagglutinins in sera from infertile and fertile women. Fertil. Steril., 31, 496-502. Isojima, S., K. Koyama, and K. Tsuchinya (1974). The effect of infertility in women of circulating antibodies against spermatozoa. J. Reprod. Fertil. (Suppl.), 21, 125-132. James, R.W., R. Heywood, J. Colley, and B. Hunter (1978). The oral toxicity of l-Amino-3-chloro-propanol hydrochloride (cl 88236) in rats. Toxicology, ii, 235-243. Jones, W.R. (1977). Irmnunological aspects of infertility. In J.S. Scott and W.R. Jones (Eds.), Immunology of Human Reproduction, Grune and Stratton, New York. pp. 375-413. Jones, R., and T. Mann (1976). Lipid peroxides in spermatozoa, role of plasmalogen and physiological significance. Proc. Roy. Soc. Lond. B., 193, 317-333. Jones, R., and T. Mann (1977). endogenous phospholipids.
Damage to spermatozoa by peroxidation of J. Reprod. Fertil., 50, 261-268.
Jones, R., T. Mann, and R. Sherins (1979). Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides and protective action of seminal plasma. Fertil. Steril., 31, 531-537. Johnsonbaugh, R.E., C.L. Czerwinski, and M. Edson (1977). Serum hormone levels before and two years after vasectomy. Contraception, 16, 563-568. Kalla, N.R. (1976). Chlorohydrins in male fertility regulation. Reprod., 28, 261-267.
Bibliogr.
Kalla, N.R., and B. Singh (1979). Effect of sodium nitrite on the ~-chlorohydrin-induced lesion of the testis-epididymis complex in the rat. J. Reprod. Fertil., 56, 149-151. Kanwar, K.C., R. Yanagimachi, and A. Lopata (1979). Effects of human seminal plasma on fertilizing capacity of human spermatozoa. Fertil. Steril., 31, 321-327. Kar~ J.K., and A.M. Phadke (1975). 26, 743-756.
Vaso-eDididymal anastomosis.
Fertil. Steril.,
283
Biochemical and Immunological Prospects for Male Contraception Katsh, S. (1962).
Antigenicity of human testis.
J. Urol.,
87,
896-902.
Katsh, S., and D.W. Bishop (1958). The effects of homologous testicular and brain and heterologous testicular homogenates combined with adjuvant upon the testis of guinea pigs. J. Embryol. exp. Morph., 6, 94-104. Kay, R., and N.J. Alexander (1978). Immunofluorescence titres of sperm centrifuged at different speeds. Clin. exp. Immunol., 32, 299-301 Kelly, R.W. (1977). Effect of seminal prostaglandins on the metabolism of human spermatozoa. J. Reprod. Fertil., 50, 217-222. Kelly, R.W., I. Cooper and A.A. Templeton (1979) Reduced prostaglandin levels in the semen of men with very high sperm concentrations. J. Reprod. Fertil., 56, 195-199.
Kesseru, E., P. C~acho-Ortega, G. Landahn, and G. S c h o p f l l n (1975). In v i t r o a c t i o n of progestogens on sperm m i g r a t i o n i n human c e r v i c a l mucus. Fertil. Steril., 26, 57-61. Kobrinsky, N.L., J.S. Winter, F.I. Reyes, and C. Faiman (1976). Endocrine effects of vasectomy in man. Fertil. Steril., 27, 152-156. Kolk, A.H.J., and T. Samuel (1975). Isolation, chemical and immunological characterization of two strongly basic nuclear proteins from human spermatozoa. Biochim. Biophys. Acta, 393, 307-319. Kolk, A.H.J., T. Samuel and P. Rumke (1974). Auto-antigens of human spermatozoa. I. Sohbilization of a new auto-antigen detected on swollen sperm heads. Clin. exp. Immunol., 16, 63-76. Krey, L.C., and A.J. Silverman (1978). The luteinizing hormone-releasing hormone (LH-RH) neuronal networks of the guinea-pig brain: II. The regulation on gonadotropin secretion and the origin of terminals in the median eminence. Brain Res., 157, 247-256. Kubota, R. (1969). Electron microscopic studies of the testis after vasectomy in rats and men. Jap. J. Urol., 60, 373-388. Lau, I.F., S. K. Saksena, and M.C. Chang (1974). Prostaglandin F in the uterine horns of mice with intrauterine devices. J. Reprod. Fert., 429-432.
37,
Linnet, L., and T. Hjort (1977). Sperm agglutinins in seminal plasma and serum after vasectomy; correlation between immunological and clinical findings. Clin. exp. Immunol., 30, 413-420. Louie, A.J., and G.H. Dixon (1972). Trout testis cells. II. Synthesis and phosphorylation of histones and protamines in different cell types. J. biol. Chem., 247, 5498-5505. MacLeod, J. (1970). The effects of urinary gonadotrophins following hypophysectomy and in hypogonadotrophic eunchoidism. Adv. Explt. Med. Biol., IO, 577-586. MacMillan, K.L., C. Desjardins, K.T. Kirton, and H.D. Hafs (1968). Gonadal and extragonadal sperm reserves after unilateral vasoligation in rabbits. Fertil. Steril., 19, 982-990.
284
P.J. Quinn
Madhwa Raj, H.G., and M. Dym (1976). The effects of selective withdrawal of FSH or LH on spermatogenesis in the immature rat. Biol. Reprod., 14, 489-494. Mancini, R.E. (1974). Immunologic and testicular response to a damage induced in the contralateral gland. In R.E. Mancini and L. Martin (Eds.), Male Fertility and Sterility. Academic Press, London. pp.271-3OO. Main, S.J., R.V. Davies, and B.P. Setchell (1978). Feedback control by the testis of gonadotropin secretion: an examination of the inhibin hypothesis. J. Endocrin., 79, 255-270. Mann, K., and D. Mecke (1979). Inhibition of spinach glyceraldehyde-3-phosphate dehydrogenases by pentalenolactone. Nature, 282, 535-536. Mann, T. (1964). The Biochemistry of Semen and the Male Reproductive Tract. Methuen, London. Marcus, Z.H., J.H. Freisheim, J.L. Houk, J.H. Herman, and E.V. Hess (1978). In vitro studies in reproductive immunology. I. Suppression of cellmedicated immune response by human spermatozoa and fractions isolated from human seminal plasma. Clin. Immunol. Immunopath., 9, 318-326. Marcus, Z.H., Y. Soffer, A. Ben-David, S. Peleg, and L. Nebel (1973). Studies on sperm antigenicity. I. Delayed hypersensitivity to spermatozoa. Europ. J. Immunol., 3, 75-78. Maroni, E.S., D.N.K. Symon, and P.C. Wilkinson (1972). Chemotaxis of neutrophil leucocytes towards spermatozoa and seminal fluid. J. Reprod. Fertil., 28, 359-368. Maroni, E.S., and P.C. Wilkinson (1971). Selective chemotaxis of macrophages towards human and guinea-pi~ spermatozoa. J. Reprod. Fertil., 27, 149-152. Mastroianni, L., M. Urzua, and R. Stambaugh (1973). The internal environmental fluids of the oviduct. In S.J. Segal, R. Crozier, P.A. Corfman, and P.G. Condliff (Eds.), The Regulation of Mammalian Reproduction, Charles C. Thomas, Springfield, Ill. pp.376-384. Mattner, P.E. (1969). Differential leucocytic responses to spermatozoa in the cervic and the uterus in ewes. J. Reprod. Fertil., 18, 297-303. Mazzanti, L., M. Lopez, and M.G. Verti (1965). Atrofia del testicolo prodotta dal monofluoroacetato sodico nel ratto albino. Experentia, 21, 446-447. McRorie, R.A., and W.L. Williams (1974). Biochemistry of mammalian fertilization. Ann. Rev. Biochem., 43, 777-833. Meizel, S., and C.W. Lui (1976). Evidence for the role of a trypsin-like enzyme in the sperm acrosome reaction. J. Exp. Zool., 195, 137-140. Menge, A.C. (1971). Antiserum inhibition of rabbit spermatozoal adherence to ova. Proc. Soc. exp. Biol. Med., 138, 98-102. Menge, A.C., and W.P. Protzman (1967). Origin of the antigens in rabbit semen which induce antifertility antibodies. J. Reprod. Fertil., 13, 31-40.
Biochemical and Immunological Prospects for Male Contraception
285
Menge, A.C., and W.P. Protzman (1967). Origin of the antigens in rabbit semen which induce antifertility antibodies. J. Reprod. Fertil., 13, 31-40. Metz, C.B., and J. Anika (1970). Failure of conception in rabbits inseminated with nonagglutinating, univalent antibody-treated semen. Biol. Reprod., 2, 284-290. Metz, C.B., M.G. O'Rand, and C.T. Cordle (1976). Agglutination and immobiliza6on of human spermatozoa. In E.S.E. Hafez (Ed.), Fertility Regulation in Men, C.V. Mosby Co., St. Louis, pp.276-286. Mishell, D.R. (1975). Intravaginal rings for contraceptive use: comment. Contraception, 12, 249-252.
an editorial
Miyamoto, H., and M.C. Chang (1973). The importance of serum albumin and metabolic intermediates for capacitation o~ spermatozoa and fertilization of mouse e~gs in vitro. J. Reprod. Fertil., 32, 193-205. Moersch, G.W., M.C. Rebstock, E.Lo Wittle, F.J. Tinney, E.D. Nicolaides, M.P. Hutt, T.E. Mich, J.M. Vandenbelt, R.E. Edgren, J.R. Reel, W.C. Dermody, and R.R. Humphrey (1979). Synthesis and biological activity of peptide antagonists of Luliberin (Luteinizing hormone-releasing hormone). J. Med. Chem., 22, 935-943. Moghissi, K.S. (1970). Human Fallopian tube fluid. Fert. Steril., 21, 821-829.
I. Protein composition.
Mohri, H., D.A.I. Suter, P.D.C. Brown-Woodman, I.G. White, and D.D. Ridley (1975). Identification of the biochemical lesion produced by ~-chlorohydrin in spermatozoa. Nature, 255, 75-77. Moltz, L., A. Roemmler, U. Schwartz, and J. Hammerstein (1978). Effects of cyproterone acetate (CPA) on pituitary ~onadotropin release and on androgen secretion before and after LH-RH double stimulation in men. Int. J. Androl. (suppl) 2, 713-719. Monastirsky, R., K.A. Laurence, and E. Tovar (1971). The effects of gonadotrophin immunization of prepubertal rabbits on gonadal development. Fertil. Steril., 22, 381-325. Moudgal, R.N., V.R. Mukku, S. Prahalada, G.S.R.C. Murty, and C.H. Li (197B). Passive immunization with an antibody to the 6-subunit of ovine luteinizin~ hormone as a method of early abortion - a feasibility study in monkeys (Macaca radiata). Fertil. Steril., 30, 223-229. Muir, V.V., J.L. Turk, and H.G. Hanley (1977). Comparison of allergic aspermatogenesis with that induced by vasectomy. II in vitro studies of cell-mediated immunity to sperm after vasectomy in man and guinea-pi~. Clin. exp. Immunol., 28, 461-466. Nagarkatti, S., and S.S. Rao (1976). Cell mediated ir~nunity to homologous spermatozoa followin~ vasectomy in the human male. Clin. exp. Immunol., 26, 239-242. Naik, V.K., A.N. Thaleur, A.R. Sheth, U.M. Joshi, S.S. Rao, D.S. Pardanani, J.K. Kulsreshtla, and R.K. Handa (1976). The effect of vasectomy on pituitary-gonadal function in men. J. Reprod. Fertil., 48, 441-448.
286
P.J. Quinn
Nakamura, M., and P.F. Hall (1977). Effect of 5-thio-D-glucose on protein synthesis in vitro by various types of cell from rat testes. J. Reprod. Fertil., 49, 395-397. Nakamura, M., and P.F. Hall (1978). The influence of temperature upon polysomes of spermatids of rat testes. Biochem. Biophys. Research Commun., 85, 756-761. National Coordinating Group on Male Antifertility Agents (1978). Gossypol: new antifertility agent for males. Chin. Med. J. (Engl.Ed.), i, 417-428. Neaves, W.B. (1975). Biological aspects of vasectory. ~, 7 pp.382-404.
In Handbook of Physiology
Nelson, W.O., and R.G. Bunge (1957). The effect of therapeutic doses of nitrofurantoin (Furadantin) upon spermatogenesis in man. J. Urol., 275-282.
7--7,
Nelson, W.O., E. Stemberger, and A. Boccabella (1954). Recovery of spermatogenesis in rats treated with nitrofurans. Anat. Rec., 118, 333-338. Neumann, F., R.C. Nicholson, B. Schenck, and H. Steinbeck (1978). The physiological action of progesterone and the pharmacological effects of progestogens - a short review. Int. J. Androl., suppl, no.2 , pp.147-154. Nicander, L. (1967). An electron microscopical study of cell contacts in the seminiferous tubules of some mammals. Z.Zellforsch.Mikroskop.Anat., 83, 375-397. Nielsen, J., and J. Sillesen (1975). among 11,148 newborn children.
Incidence of chromosome aberrations Hum. Genet., 30, 1-12.
Nieschlag, E., K-H. Usadel, E.J. Wickings, H.K. Kley, and W. Wuttke (1975). Effects of immunization with steroids on endocrine functions in male animals. In E. Nieschlag (Ed.), Immunization with Hormones in Reproductive Research, North Holland, Amsterdam. pp.155-170. Niwa, K., and A. Iritani (1978). Effect of various hexoses on sperm capacitation and penetration of rat eggs in vitro. J. Reprod. Fertil., 53, 267-271. O'Malley, B.W., and A.R. Means (1974). Female steroid hormones and target cell nuclei. Science, 183, 610-620. Otani, Y., and J. Behrman (1963). homologous testis and sperm.
Immunization of the guinea pig with Fertil. Steril., 14, 456-467.
Pabst, R., O. Martin, and H. Lippert (1979). Is the low fertility rate after vasectomy caused by nerve resection during vasectomy? Fertil. Steril., 13, 316-320. Paufler, S.K., and R.H. Foote (1969). Spermatogenesis in the rabbit following ligation of the epididymidls at different levels. Anat. Rec., 164, 339-348. Paul, H.E., M.F. Paul, F. Kopko, R.C. Bender, and G. Everett (1953). Carbohydrate metabolism studies on the testis of rats fed certain nitrofurans. Endocrinology, 53, 585-592.
Biochemical and Immunological Prospects for Male Contraception
287
Paulsen, C.A., and J.M. Leonard (1976). Clinical trials in reversible male contraception. I. Combination of danazol plus testosterone. In C.H. Spilman, T.J. Lobl and K.T. Kirton (Eds.), Regulatory Mechanisms of Male Reproductive Physiology, Excerpta Medica, Amsterdam. pp.197-210. Pedroza, E., J.A. Vilchez-Martinez, D.H. Coy, A. Arimura, and A.V. Schally (1978). Correlation between in vivo inhibition of gonadotropin release induced by LH-RH and the blockade of ovulation by synthetic analogues of LH-RH. Int. J. Fertil., 23, 294-299. Perrenoud, A. (1974). Malthusianisme et Protestantisme; Weberien. Annal. E.C.S., 29, 983-985. Peters, R.A. (1952).
Lethal synthesis.
un modNle d~mographique
Proc.Roy. Soc., B,
139, 143-170.
Potashnik, G., N. Ben-Aderet, R. Israeli, I. Yanai-Inbar, and I. Sober (1979). Suppressive effect of 1,2-dibromo-3-chloropropane on human spermatogenesis. Fertil. Steril., 30, 444-447. Purvis, K., S.K. Saksena, Z. Cekan, E. Diczfalusy, and J. Giner (1976). Endocrine effects of vasectomy. Clin. Endocrinol., 5, 263-269. Reijonen, K., M. Kormano, and R.J. Ericsson (1975). Studies on the rat epididymal blood vessels following alpha-chlorohydrin administration. Biol. Reprod., 12, 483-490. Reyes, A., B. Goicoechea, and A. Rogado (1978). Calcium ion requirement for rabbit spermatozoal capacitation and enhancement of fertilizing ability by ionophore A23187 and cyclic 3':5'-monophosphate. Fert Steril., 29, 451-455. Rogers, B.J., and R. Yanagimachi (1976). Competitive effect of magnesium on the calcium-dependent acrosome reaction in guinea-pig spermatozoa. Biol. Reprod., 15, 614-619. Rommerts, F.F.G., B.A. Cooke, and H.J. van der Molen (1974). The role of cyclic AMP in the regulation of steroid biosynthesis in testis tissue. J. Steroid Biochem., 5, 279-285. It
Rumke, P., and M. Titus (1970). Spermagglutinin formation in male rats by subcutaneously injected syngeneic epididymal spermatozoa and by vasoligation or vasectomy. J. Reprod. Fertil., 21, 69-79. Russell, L.D, and Y. Clermont (1977). Degeneration of germ cells in normal hypophysectomised and hormone treated hypophysectomised rats. Anat. Rec., 187, 347-366. Saksena, S.K., and M.J.K. Harper (1974). Prostaglandin-mediated action of the intrauterine devices: F-prostaglandins in the uterine horns of pregnant rabbits with unilateral intrauterine devices. Fertil. Steril., 25, 121-126. Saksena, S.K., I.F. Lau, and V.D. Castrachane (1974). Prostaglandin mediated action of IUD's. II. F-prostaglandins (PGF) in the uterine horn of pregnant rats and hamsters with intrauterine devices. Prostaglandins, 97-106. Samuel, T. (1977). Antibodies reacting with salmon and human protamines in sera from infertile men and from vasectomized men and monkeys. Clin. exp. Immunol., 30, 181-187.
,
288
P.J. Quinn II
Samuel, T., N.J. Alexander, and P. Rumke (1977). Reproducibility of the indirect immunofluorescence test on swollen human spermheads. Clin. exp. Immunol., 28, 223-225. Samuel, T., A.H.J. Kolk, P. Rumke, L.A. Aarden, and M. Bustin (1976). and DNA detection in swollen spermatozoa and somatic cells, by immunofluorescence. Clin. exp. Immunol., 24, 63-71.
Histone
Samuel, T., A.H.J. Kolk, P. Rumke, and J.M.J. Van Lis (1975). Autoimmunity to sperm antigens in vasectomized men. Clin. exp. Immunol., 21, 65-74. Sandow, J., W. Von Rechenber~, and G. Jerzabek (1977). Endocrine effects o£ chronic treatment with the LH-RH analogue D-SER (TBU)6 ethylamide. Acta Endocrinol., 84, 33-41. Sandow, J., W. Von Rechenberg, G. Jerzabek, and W. Stoll (1978). Pituitary gonadotropin inhibition by a highly active analog of luteinizing hormonereleasing hormone. Fert. Steril., 30, 205-209. Schally, A.V. (1977). Aspects of hypothalamic regulation of the pituitary gland with major emphasis on its implications for the control of the reproductive processes. In: Prix Nobel pp.201-234. Schally, A.V., A. Arimura, A.J. Kastin, H. Matsuo, Y. Baba, R.W. Redding, R.M.G. Nair, and L. Debeljuk (1971). Gonadotropin-releasing hormone: one polypeptide regulates secretion of luteinizing and folliclestimulating hormones. Science, 173, 1036-1037. Schally, A.V., A.J. Kastin and D.H. Coy (1976). LH-releasing hormone and its analogues: recent basic and clinical investigations. Int. J. Fertil., 21, 1-30. Schmidt, S.S. (1975). 47, 309-314.
Vas anastomosis:
a return to simplicity.
Brit. J. Urol.,
Schoenfeld, C., R.D. Amelar, and L. Dubin (1976). Clinical experience with sperm antibody testing. Fertil. Steril., 27, 1199-1203. Seprodi, J., D.H. Coy, J.A. Vilchez-Martinez, E. Pedroza, and A.V. Schally (1978a) Branched-chain analogues of luteinizing hormone-releasing hormone. J. Med. Chem., 21, 276-280. Seprodi, J., D.H. Coy, J.A. Vilchez-Martinez, E. Pedroza, W-Y. Huang, and A.V. Schally (1978b). Cyclic analogues of luteinizing hormone-releasing hormone with significant biological activities. J. Med. Chem., 21, 993-995. Setchell, B.P. (1978).
The Mammalian Testis, Paul Elek, London.
Setchell, B.P., R.V. Davies, and S.J. Main (1977). Inhibin. In A.D. Johnson and W.R. Comes (Eds.), The Testis, Vol. IV. Academic Press, New York. pp. 189-238. Sherins, R.J., and V.T. De Vita (1973). Effect of drug treatment for lymphoma on male reproductive capacity. Studies of men in remission after therapy. Ann. Intern. Med., 79, 216-219.
289
Biochemical and Immunological Prospects for Male Contraception Shiu, R.P.C., and H.G. Friesen (1976). Blockade of prolactin action by an antiserum to its receptors. Science, 192, 259-261. Shivers, C.A.,and B.S. Dunbar (1977). Autoantibodies to zona pellucida: possible cause of infertility in women. Science, 197, 1082-1084.
a
Shulman, S. (1971). Antigenicity and autoimmunity in sexual reproduction: a review. Clin. exp. Immunol., 9, 267-288. Shulman, S,, H. Jackson, and M.L. Stone (1975). Comparative studies of sperm agglutinating activity in groups of infertile and fertile women. Am. J. Obstet. Gynecol., 125, 139-145. Silverman, A.J., L.C. Krey, and E.A. Zimmerman (1979). A comparative study of the luteinizing hormone releasing hormone (LH-RH) neuronal networks in mammals. Biol. Reprod., 20, 98-110. Silverman, A.J., and E.A. Zimmerman (1978). Pathways containing luteinizing hormone-releasing hormone (LH-RH) in the mammalian brain. In D.E. Scott (Ed.), Neural Hormones and Reproduction, Third Brain-Endocrine Interaction Symposlum. Karger, Basel. pp. 83-96. Skakkebaek, N.E., R.W. Kelly, and C.S. Corker (1976). Prostaglandin concentrations in the semen of hypogonadal men during treatment with testosterone. J. Reprod. Fertil., 47, 119-121. Skinner, J.D., and L.E.A. Rowson (1968). Some effects of unilateral cryptorchism and vasectomy on sexual development of the pubescent ram and bull. J. Endocrin., 42, 311-321. Skoglund, R.D., and C.A. Paulsen (1973). Danazol-testosterone combination: a potentially effective means of reversible male contraception, a preliminary report. Contraception, 2, 357-361. Smith, K.D., R.K. Tcholakian, M. Chowdhury, and E. Steinberger (1976). An investigation of plasma hormone levels before and after vasectomy. Fertil. Steril., 27, 144-151. Soupart, P. (1970). Leukocytes and sperm capacitation in the rabbit uterus. Fertil. Steril., 21, 724-756. Srivastava, P.N. (1973).
Location of zona lysin.
Biol. Reprod.
9,
84.
Stambaugh, R., and J. Buckley (1972). Histochemical subcellular localization of the acrosome proteinase effecting dissolution of the zona pellucida using fluorescein-labelled inhibitors. Fertil. Steril., 23, 348-352. Stambaugh, R., H.M. Seitz, and L. Mastroianni (1974). Acrosomal proteinase inhibitor in rhesus monkey oviduct fluid. Pert. Steril., 25, 352-357. Steinberger A., and E. Steinberger (1976). Secretion of an FSH-inhibiting factor by cultured Sertoli cells. Endocrinology, 99, 918-921. Steinberger, E., and B.N. Sud (1970). Specific effect of fluoroacetamide on spermatogenesis. Biol. Reprod., 2, 369-375. Stocks, J., J.M.C. Gutteridge, R.J. Sharp and T.L. Dormandy (1974). The inhibition of lipid autoxidation by human serum proteins and ~-tocopherol. Clin. Sci. Mol. Med., 47, 223-230
290
P.J. Quinn
Stokes, C.R., J.F. Soothill, and M.W. Turner (1975). function of I g A . Nature, 255, 745-746.
Irmnune exclusion is a
Sullivan, M.J.,and G.E. Howe (1977). Correlation of circulatin~ antisperm antibodies to functional success in vasovasectomy. J. Urol., 117, 189-191. Sullivan, J.L., F.A. Smith, and R.H. Garman (1979). Effects of fluoroacetate on the testis of the rat. J. Reprod. Fertil., 56, 201-207. Swift, A.D., and D.B. Crighton (1978). Relative activity, plasma elimination and hypothalamic and pituitary degradation of synthetic luteinizing hormone releasing hormone and certain of its analogues. J. Endocrinol., 77, 35P. Swift, A.D., and D.B. Crighton (1979a). Relative activity, plasma elimination and tissue degradation of synthetic luteinizing hormone releasin~ hormone and certain of its analogues. J. Endocrinol., 80, 141-152. Swift, A.D., and D.B. Crighton (1979b). The effects of certain steroid hormones on the activity of ovine hypothalamic luteinizin~ hormone-releasing hormone (LH-RH)-degrading enzymes. FEBS Letts., IO0, 110-112. Szemere, G., and A.C. Chandley (1975). Trisomy and Triploidy induced by X-irradiation of mouse spermatocytes. Mut. Res., 3_33, 229-238. Talaat, M., and K.A. Laurence (1971). Impairment of spermatogenesis and libido through antibodies to luteinizing hormone. Fertil. Steril., 113-118. Talwar, G.P. (1978). contraception.
2--2,
The present and future of immunologic approaches to Int. J. Gynaecol. Obstet., 15, 410-414.
Talwar, G.P., N.C. Sharma, S.K. Dubey, M. Salahuddin, C. Das, S. Ramakrishnan, S. Kumar, and V. Hingorani (1976a). Isoin~nunization against human chorionic gonadotropin with conjugates of processed 6-subunit of the hormone and tetanus toxoid. Proc. Natl. Acad. Sci. U.S.A., 73, 218-222. Talwar, G.P., N.C. Sharma, S.K. Dubey, M. Salahuddin, N. Shastri, and S. Ramakrishnan (1976b). Processing of the preparations of 6-subunit of human chorionic gonadotropin for minimization of cross-reactivity with human luteinizing hormone. Contraception, 13, 131-139. Tang, L.K.L. (1978). Sex difference in LH response to LHRH and DBcAMp and effect of testosterone. Amer. J. Physiol., 235, E291-E294. Tcholakian, R.D., A. de la Cruz, A.K. Chowdhury, M. Chowdhury, A.V. Schally, and A. Steinberger (1978). Effects of an LH-RH analogue in male rats pretreated with oestradiol benzoate. J. Reprod. Fert., 54, 441-445. Templeton, A.A., I. Cooper, and R.W. Kelly (1978). Prostaglandin concentrations in the semen of fertile men. J. Reprod. Fert., 52, 147-150. Thau, R.B., K. Sundaram, Y.S. Thornton, and L.S. Seidman (1979). Effects of immunization with the 6-subunit of ovine luteinizing hormone on corpus luteum function in the rhesus monkey. Fert. Steril., 31, 200-204.
291
Biochemical and Immunological Prospects for Male Contraception Thompson, I.E. (1973). The IUD and prostaglandins: Obstet. Gynaecol., 42, 617-620.
a review of the evidence.
Thrall, R.S., L.B. Peterson, J.H. Lineham, P. Abramoff, and V.L. Moore (1978). The effect of immunization on the uptake of intratracheally administered antigen. Clin. Immunol. Ir~nunopath., IO, 136-147. Tougard, C., B. Kerdelhue, A. Tixier-Vidal, and M. Jutisz (1971). Localisation par cyto-immunoenzymologie de la LH, de ses sous-unit~s ~ et B e t de la F SH dans l'ad~nohypophyse de la ratte castr~e. Comp. Rend. Acad. Sci. Paris, S~rie D. 273, 897-900. Toullet, F., and G.A. Voisin (1974). Spermatotoxic, spermagglutinating and cytotoxicactivities of guinea-pig autoantibodies to sperm autoantigen T. J. Reprod. Fertil., 37, 299-313. Toullet, F., G.A. Voisin, and M. Nemirovsky (1973). Histoimmunochemical localization of three guinea-pig spermatozoal autoantigens. Immunology, 24, 635-653. Tung, K.S.K. (1975). Human sperm antigens and antisperm antibodies. on vasectomy patients. Clin. exp. Immunol., 20, 93-104.
I. Studies
Turner, T.T., D.A. D'Addario, and S.S. Howards (1979). Effects of vasectomy on the blood-testis barrier of the hamster. J. Reprod. Fertil., 55, 323-328. Uehling, D.T. 769-773.
(1971).
Secretory IgA in seminal fluid.
Fertil. Steril.,
22,
Uematsu, K. (1966). Testicular changes of rats induced by nitrofurazone; a light and electron microscopic study. Med. J. Osaka Univ., 16, 287-306. Ulstein, M., N. Netto, J. Leonard, and C.A. Paulsen (1975). Changes in the sperm morphology in normal men treated with danazol and testosterone. Contraception, 12, 437-444. Vale, W., G. Grant, M. Amoss, R. Blackwell, and R. Guillemin (1972). of enzymatically dispersed anterior pituitary cells. Functional validation of a method. Endocrinology, 91, 562-572.
Culture
Vale, W., C. Rivier, M. Brown and J. Rivier (1977). Pharmacology of thyrotropin releasing factor (TRF), luteinizing hormone releasing factor (LRF) and somatostatin. Adv. exp. med. Biol., 87, 123-156. Vare, A.M., and P.C. Bansal (1973). Changes in the canine testes after bilateral vasectomy - an experimental study. Fertil. Steril., 24, 793-797. Vilchez-Martinez, J.A., D.H. Coy, E.J. Coy, A. Arimura, and A.V. Schally (1976). Comparison of the anti-LH/FSH-RH and anti-ovulatory activities of [D-Phe2,D-Leu 6] - LH-RH and [D-Phe2,D,AIa 6] - LH-RH. Endocr. Res. Commun., ~, 231-238. Vito, C.C., S.J. Wieland, and T.O. Fox (1979). Androgen receptors exist throughout the 'critical period' of brain sexual differentiation. Nature, 282, 308-310.
292
P.J. Ouinn
Volgmayr, J.K. (1974). Alpha-chlorohydrin-induced changes in the distribution of free myo-inositol and prostaglandin F2~ , and synthesis of phosphatidylinositol in the rat epididymis. Biol. Reprod., II, 593-600. Voisin, G.A., and F. Toullet (1968). Etude sur l'orchide aspermatogenetique autoimmune et les autoantigenes de spermatozoides chez le cobaye. Ann. Inst. Pasteur, Paris, 114, 727-755. Vojtigkov~, M., M. Pol~kova and Z. Pokornfi (1969). Histocompatibility on mouse spermatozoa. Folia Biol.(Prague) 15, 322-332.
antigens
Weinman, D.E., and W.L. Williams (1964). Mechanism of capacitation of rabbit spermatozoa. Nature, 203, 423-424. Whitby, R.M., R.D. Gordon and B.R. Blair (1979). vasectomy: a prospective five-year study. ~0
The endocrine effects of Fertil. Steril., 21, 518-520.
Reference Bank for Reproductive Immunology (1977). Auto- and iso-antibodies to antigens of the human reproductive system. Clin. exp. Immunol., 30, 173-180.
Wickings, E.J., and E. Nieschlag (1978). The effects of active immunization with testosterone on pituitary-gonadal feedback in the male rhesus monkey (Macaca mulatta). Biol. Reprod., 18, 602-607. Wickings, E.J., C.A. Tafurt, H. Hoogen, and E. Nieschlag (1977). of titre and specificity of antibodies to testosterone. J. Steroid Biochem., 8, 147-150.
The development
Wilson, L. (1954). Sperm agglutinins in human semen and blood. Biol. Med., 85, 652-655.
Proc. Soc. exp.
Wilson, L. (1956). ~, 262-267.
Sperm agglutination due to autoantibodies.
Fertil. Steril.,
Wilson, B.J., N.J. Alexander, G. Porter, and D.L. Fulgham (1977). Cell-mediated immunity in vasectomised rhesus monkeys. Fertil. Steril., 28, 1349-1355. Wilson, B.J., G. Porter, and A. Goldstein (1979). Reduced T cell reactivity in vasectomised rhesus monkeys: association with histocompatibility type. Pertil. Steril., 31, 434-440. Wilkinson, P.C. (1974). Edinburgh,
Chemotaxis and Inflammation.
Churchill Livingstone,
Witkin, S.S., and P.J. Higgins (1978). Effects of vasectomy and antisperm antibodies on human seminal fluid deoxyribonucleic acid polymerase activity. Fertil. Steril., 29, 314-319. Yanagimachi, R. (1970). The movement of golden hamster spermatozoa before and after capacitation. J. Reprod. Fert., 23, 193-196. Yanagimachi, R. (1972). Penetration of guinea-pig spermatozoa into hamster eggs in vitro. J. Reprod. Fertil., 28, 477-480. Yanagimachi, R., and R.J. Teichman (1972). Cytochemical demonstration of aerosomal proteinase in mammalian and avian spermatozoa by a silver proteinate method. Biol. Reprod., 6, 87-97.
Biochemical and Immunological Prospects for Male Contraception Yanagimachi, R., and N. Usui (1974). Calcium dependence of the acrosome reaction and activation of guinea-pig spermatozoa. Exptl. Cell Res., 161-174.
293 89,
Yen, S.S.C., R. Rebar, G. Vandenberg, F. Naftolin, H. Judd, Y. Ehara, K.J. Ryan, J. Rivier, M. Amoss, and R. Guillemin (1973). Clinical studies with synthetic LRF (luteinizing hormone-releasing factor). In C° Gual and E. Rosemberg (Eds.), Hypothalamus Hypophysiotropic Hormones. Excerpta Medica, Amsterdam. pp.217-229. Zaneveld, L.J.D. (1976). Sperm enzyme inhibitors as antifertility agents. In E.S.E. Hafez (Ed.), Human Semen and Fertility Regulation in Men. C.V. Mosby Co., St. Lou'is. pp.570-582. Zysk, J.R., A.A. Bushway, R.L. Whistler, and W.W. Carlton (1975). Temporary sterility produced in male mice by 5-thio-D-glucose. J. Reprod. Fertil., 45, 69-72.