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International Symposium on the Biology of Spermatozoa: Transport, Survival, and Fertilizing Ability
FERTILITY AND STERILITY
Vol. 25, No.9, September 1974 Printed in U.S.A.
Copyright" 1974 The American Fertility Society
Communications and Commentaries INTERNATIONAL SYMPOSIUM ON THE BIOLOGY OF SPERMATOZOA: TRANSPORT, SURVIVAL, AND FERTILIZING ABILITY E. SAAD E. HAFEZ, PH.D. (Cantab.), AND CHARLES G. THIBAULT, PH.D.
i: M;r
Departrrumts of Gynecology-Obstetric~ am! P"lsJ:::~'/;t ~. ~~'tM~'iJ::~Ol, and Growth and Development, Wayne S~te ...Vn~vnalerBltyd RC he A !...~,!!,jn'; Physiologie Animale e ec rc"" nos' v...., , University of Paris VI and Institut lYatio 783501 Jouy-en-Josas, L'ranee
he
An international symposium on The Biology of Spermatozoa was held N 0vember 4 to 7 at the Institute of Agricultural Research in Nouzilly, France. The program dealt with sperm transport in the male and female tract of human, nonhuman primates and other vertebrates, sperm capacitation, sperm acrosomal enzymes and their potential for contraceptive development, and the influence of hormonal and intrauterine contraceptives on human sperm migration in vivo and in vitro. Emphasis was placed on the complex processes of physiological and biochemical mechanisms involved in the transport and maturation of the spermatozoon from the testis to its final destination, and the site of fertilization at the ampullary-isthmic junction of the oviduct. The 31 speakers were from Australia, Belgium, Chile, France, New Zealand, Sweden, United Kingdom, and the United States. The symposium was supported by The French National Institute of Health and Medical Research, The World Health Organization, The French National Institute of Agricultural Research, Schering, Byla-Searle, Syntex, and Roussel. The proceedings, edited by Hafez and Thibault, are being published by S. Karger, Basel, Switzerland. The following is a summary of the symposium papers. Received January 14, 1974.
TESTICULAR FLUIDS
Setchell and Waites reported that fluid collected from the rete testis is a watery, opalescent suspension of living testicular spermatozoa. It differs from fluid collected out of the lumen of seminiferous tubules in its ionic and protein composition and its lower concentration of spermatozoa. Rete testis fluid contains testosterone and fluids differ markedly from blood plasma in many respects. Fluid secretion is probably not involved in the release of spermatozoa from the germinal epithelium, but it is involved in the transport of spermatozoa out of the seminiferous tubules and from the rete testis into the epididymis. The fluid also provides substrates necessary for metabolism of spermatozoa but the spermatozoa may also draw on their endogenous reserves (eg, lipids). Rete testis fl uid and epididymal seminal plasma also contain inhibitors of acrosomal proteinase, and although these inhibitors are not tightly bound to the spermatozoa, they may be important in controlling proteolytic activity during the maturation of epididymal spermatozoa. EPIDIDYMAL SPERMATOZOA
The primary functions of the mammalian epididymis are to serve as a conduit for the transport of spermatozoa from the testis to the ejacula-
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tory duct and to provide a favorable environment for sperm maturation and storage prior to ejaculation. The morphologic and physiologic integrity of the epididymis is androgendependent. Changes in tubular diameter, epithelial height, and histochemical characteristics after castration are repaired or prevented by the administration of exogenous testosterone. The regressive changes in the epididymis following castration or hypophysectomy are associated with a loss of motility and degeneration of spermatozoa contained therein. Sperm maturation in the epididymis (ie, the development of their ability to fertilize) and sperm survival are also controlled by androgens. An early step in the action mechanism of steroid hormones is their interaction with a specific cytoplasmic receptor protein in the target cell. The receptor-hormone complex is subsequently transported into the nucleus where it is associated with chromatin. Danzo and Orgebin-Crist have shown that three androgenbinding proteins are present in the epididymis: (1) an androgen-binding protein present in blood plasma, (2) an androgen-binding protein of testicular origin present in the caput epididymidis, and (3) an androgenbinding protein present in the epididymis of rabbits that have been castrated for 3 or 4 days. The first two binding moieties probably serve as transport proteins for androgens. The target cell receptor, demonstrated in the epididymis of castrated rabbits, may be of primary importance in mediating the androgen-dependent processes that are required for maintaining the many complex morphologic and functional aspects of the epididymis. The role of these androgen-dependent phenomena in the
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economy of the epididymis is not yet understood. Danzo and Orgebin-Crist have advanced the hypothesis that the function of androgens is to act on the epididymis in order to produce secretory products that interact with spermatozoa to bring about changes that are required for their maturation and survival. The observation that androgen-dependent protein synthesis has occurred in the epididymis in organ culture would tend to support this theory. Although the hypothesis may appear simplistic, the complex androgen regulation of epididymal function is directed solely to mediating effects on the tissue itself without affecting the enclosed spermatozoa. The ultrastructural and cytochemical changes in the acrosome of spermatozoa during their transport in the epididymis and female reproductive tract can be studied by a variety of techniques. Such methods include scanning electron microscopy, freeze-etching, and ultrastructural cytochemistry in order to localize glycoproteins and their acidic groups. The last technique involves staining of thin sections in glycolmethylacrylate with phosphotungestic acid, the use of colloidal iron hydroxide, and extraction with pronase and neuraminidase. Flechon observed dense crystalline cores in the corpus epididymidis of the rabbit inside the marginal thickening of the acrosome at the time it becomes rounded and shorter and reaches fertilizing ability. The acrosome is of heterogenous proteins. In ejaculated spermatozoa glycoproteins are located only at a superficial layer of the anterior segment. A relationship between the presence of glycoproteins, crystalline, and amorphous proteins and the localization of various acrosomal enzymes is sug-
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gested. Modifications of comparison and structure during epididymal transport may involve ripening or compact stockage of these enzymes. Rabbit spermatozoa are coated with glycoproteins. In ejaculated spermatozoa, the coat is thin on the head, almost absent on the middlepiece, and thicker on the principal piece, where most of the acidic groups are concentrated. Modifications in glycoproteins and acidic groups of the cell coat occur also in posterior corpus epididymidis where fertilizing ability of spermatozoa is acquired. Flechon has proposed that observed alterations of the plasma membrane during epididymal transport could induce some modifications of the sperm metabolism and surface properties contributing to the acquisition of fertilizing ability. Functional maturity of spermatozoa is attained during the passage of spermatozoa in the avian deferent ducts. The time of sperm transport in the epididymis varies from 1 to 2 days in birds to 11 to 16 days in mammalian species, including humans. In male birds, sperm maturation involves lipomucopolysaccharide complex and glycoproteins observed in the male reproductive tract. While the development of accessory sex organs is controlled by testosterone, this hormone is not necessary for survival of spermatozoa in the male reproductive tract. Data on the extragonadal sperm reserves and the biochemical characteristics of secretions suggest the mammalian epididymis is analogous to the avian deferent ducts.
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Croxato et al; Settlage et aI). Settlage et al reported that spermatozoa were found in the human oviduct within 5 minutes of deposition in the proximal vagina. The numbers of sperm found in the oviduct were directly related to the numbers of sperm in the inseminate during the first 2 hours after insemination. Sperm were distributed throughout the oviduct. The total numbers of sperm present in cervical mucus appeared to be constant until 48 hours later when they were dramatically reduced. On the other hand, there seemed to be a constant level of sperm in the oviduct 15 to 45 minutes after insemination; this was greatly reduced by 6 hours. Endometrial cavity sperm were not found until 90 minutes after insemination. They increased in number for 24 hours and then decreased until they were absent at 48 hours after insemination. Maximal calculated ratios indicate that for every oviductal sperm there are about 500 endometrial sperm, 20,000 cervical mucus sperm, and 10 million motile sperm in the inseminate. Ahlgren et al estimated that some 200 sperm were found in the ampulla of the oviduct of a woman. There are no accurate data on survival time and duration of fertilizing capacity of human spermatozoa. Sperm transport in nonhuman primates. Hafez and Jaszczak described the patterns of sperm transport in nonhuman primates by (1) observing the female reproductive tract by scanning electron microscopy, and (2) taking sperm counts in serial histologic sections at various interPATTERNS OF SPERM TRANSPORT vals from copulation. Sperm transport in women. Three The epithelium of the female retechniques were presented to assess productive tract is made of variable transport in women (Ahlgren et al; proportions of secretory cells with
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microvilli, and ciliated cells with kinocilia beating toward the vagina. Sperm transport was influenced by the rate of coagulation and liquefaction of semen. The number of sperm progressively decreased from the external os of the cervix toward the uterine cavity and oviducts. The mucus in the cervical canal and the uterotubal junctions were considered the main barriers in sperm ascent. When monkeys were mated during midcycle (time of ovulation), sperm ascent was optimal and spermatozoa were often aligned along strands of cervical mucus. These strands of mucus migrated to the cervical crypts or to the uterine cavity. When copulation was performed during the early follicular or luteal phases, sperm ascent was largely or completely inhibited due to the presence of a scant amount of viscous, cellular cervical mucus which showed no ferning or spinnbarkeit. Spermatozoa migrate to the uterine cavity through the longitudinal crypts of the endocervix, as well as the cervical canal. Many more spermatozoa were located in the endometrial glands than in the uterine cavity. Sperm transport in uterine ejaculators. In uterine ejaculators, spermatozoa very rapidly reach the uterotubal junction. The site of fertilization is attained by 2 hours after coitus. The uterotubal junction and isthmus act as very potent barriers against dead sperm and limit the number of sperm moving to the fertilization site. In the sow, there is no resorption of sperm in the uterus, as previously described, since ligature prevents sperm disappearance. Thus, sperm resorption in uterine ejaculators results first from release from the cervix, and second, from phagocytosis after large leukocytic invasion.
September 1974
Sperm transport in ruminants. In cattle, as in sheep (when fixation of the whole genital tract and serial section is used), the first spermatozoa appear to never reach the isthmus before 2 hours. Thibault et al found that the number of spermatozoa present at the uterotubal junction and the isthmus steadily increases from 2 to 18 hours after coitus. However, the maximum number of spermatozoa in the isthmus remains limited to several thousand. The small number of spermatozoa in the ampulla at any time after coitus results from the the efficacy of three barriers: the cervix, the uterotubal junction, and the isthmus. The number of spermatozoa present in the oviduct is not related to the number of spermatozoa inseminated either in the vagina or the uterus. This fact explains the possibility of subnormal fertility in cow inseminated with .0001 of bull ejaculate (375,000 living spermatozoa). PHYSIOLOGIC MECHANISMS OF SPERM TRANSPORT
There are remarkable variations among species in the site of semen deposit (vaginal or uterine), in the volume of accessory fluid, and in the number of spermatozoa ejaculated. However, the number of spermatozoa reaching the oviduct seems to be relatively constant and represents only a very small percentage of that in the ejaculate. Changes in uterine motility and the characteristics of cervical mucus greatly influence the transport of spermatozoa. Both are under the influence of ovarian steroids; maximal responses coincide with production of endogenous estrogen. Uterine motility is depressed by progesterone and enhanced by estrogen. The
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parallel arrangement of macromolecular micelles characterizes the estrogenic phase of the cycle. During the luteal phase, the micelles split up forming a dense network which inhibits sperm penetration. The cervical mucus of postmenopausal women is scant and impenetrable to spermatozoa. Sperm transport is greatly affected when small numbers of spermatozoa are used in insemination. SURVIVAL OF SPERMATOZOA
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Sperm survival in mammals. Sperm survival in the female reproductive tract of common mammals varied from 15 hours in mice and rats to 10 days in the dog. Postcoital tests in monkeys showed that sperm motility in the external cervix lasted less than 36 hours and fertilizing ability was maintained for a shorter period. Ahlgren et al found that the survival time of spermatozoa in the ampulla of the oviduct after coitus was 85 hours. It was not certain whether these spermatozoa were fertile at that time. The fertilizing ability of rabbit spermatozoa was lost before motility. Farris concluded from his studies of artificial insemination with donor sperm that the fertile life of sperm was 24 hours. Ferin's report of 120hour fertilizing capacity for human spermatozoa has not been conflrmed. It is essential that data be obtained from prospective studies on time relations between coitus and ovulation, fertilization, or sperm recovery. Data from retrospective studies have been unreliable. The fertilizing ability of spermatozoa recovered from human oviducts should be tested in vitro. It is doubtful that human spermatozoa were found 2 to 3 weeks after coitus (as reported in previous studies), since the data were based on the recollection of the patient or
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the assumption of the physician when the patient had been in the hospital for some weeks. Reports of such long survival time conflicted with existing data on other mammals (except the hare and the bat). Spermatozoa can survive in the female reproductive tract of the hare for a few weeks and can survive in tropical and temperate bats for a few months. Martinet and Raynaud reported that "superfetation," the simultaneous development of two different generations of eggs, occurs in hare as a result of long-term survival of spermatozoa in the uterus. In some amphibian species, spermatozoa buried in the epithelial cells can remain alive for 2 years. In the cow, spermatozoa survive at least 3 days at the uterotubal junction in some fmger-like folds because of the absence of leukocytes in these folds. Leukocytes invade more or less rapidly the lower parts of the female tract, depending on the animal, and clean the tract of sperm. Little is known about the preferential areas for spermatozoa storage in the uterus or the physiologic interaction between spermatozoa and the secretory cells of the epithelium of the female reproductive tract. Sperm survival in lower forms of life. In reptiles, sperm storage occurs in the vaginal segment of the oviduct, either in light or in the more or less developed seminal receptacles; in lizards these receptacles are linked to the possibility of several successive lays for isolated females. During the weeks preceding ovulation (earlier in snakes than in lizards), spermatozoa reach the precranial segment of the oviduct. They eventually may be stored in seminal receptacles where spermatozoa are protected during lay time. Saint Girons has shown that
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seminal receptacles do not seem vital for long survival of the spermatozoa since these receptacles are often stored in the light of the vaginal tube. Prostaglandins may improve fertilization in adverse circumstances. However, fertilization is a most efficient process, except in certain exceptional cases (eg, infertile female with hostile cervical mucus or oviductal problems or infertile male with oligospermia, high percentage of abnormal sperm, or sperm of low motility.) In humans, in vitro fertilization is of great clinical significance in solving infertility problems, such as bypassing an irreducible tubal block. Ovulation must be induced in the ovum donor in order to synchronize embryonic development with the maternal environment of the recipient. Fertilizable eggs can be produced after ovulation induction by human menopausal gonadotropin-human chorionic gonadotropin (HMG-HCG) or clomiphene-HCG treatments. The limiting factor of in vitro fertilization no longer is the problem of obtaining capacitated sperm, but the problem of mastering complete control over oocyte maturation in vitro. Soupart has developed an effective, completely controlled, in vitro human fertilization system. This experimental tool is indispensable for the development of new and better contraceptive methods, which should be based on precise inhibition of highly specific molecular mechanisms. Inhibition of fertilization. Tosyllysine chloromethyl ketone (TLCK), nitrophenyl p-guanidino benzoate (NPGB), and to a lesser extent, ethyl p-guanidino benzoate (EPGB) inhibit the in vitro activity of acrosin. Tosylamide phenyl ethyl chloromethyl ketone (TPCK) has no effect on the enzyme. Zaneveld and co-workers
September 1974
have shown that addition of TLCK but not TPCK to spermatozoa in quantities that did not affect their motility, completely inhibited the fertilizing ability of capacitated spermatozoa. TLCK and NPGB, but not EPGB, prevented fertilization if mixed with ejaculated spermatozoa before deposit in the uterus; this shows that these synthetic inhibitors are not removed during capacitation. Zaneveld et al have also shown that TLCK effectively prevents fertilization on vaginal application before coitus and enhances the contraceptive activity of Delfen vaginal cream. Two other synthetic inhibitors, p-amino phenacyl bromide (APB) and p-guanidino-phenacyl bromide (GPB) also result in decreased fertilization if applied vaginally, but are less active than TLCK. DEVELOPMENT OF NEWER CONTRACEPTIVES
Basic and clinical research in the physiology of spermatozoa is needed to develop efficient,reversible, safe, easily administered, low-cost contraceptives for large scale use. Such research should be directed to the development of newer methods which would interfere with transport of spermatozoa in the female reproductive tract; ways should be found to cause the destruction, immotilization, agglutination, or decapacitation of spermatozoa. This can be achieved by intravaginal, intracervical, intrauterine, or intraoviductal devices medicated with potent pharmacologic agents (eg, metallic ions, progestogens, acrosomal enzyme inhibitors, sperm antibodies, and spermicides). Several modes of action can be employed: (1) change the biochemical or biophysical characteristics of the vaginal fluid or cervical mucus (rheo-
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logical properties and micelles formation); (2) interfere with sperm metabolism and survival in the endometrial fluid; (3) interfere with the enzyme and enzyme inhibitor system; or (4) prevent sperm migration through the uterotubal junction to the oviduct. It may be possible to develop and administer systems of medicated slow delivery which would act locally rather than systemically. The vagina is more accessible than the cervix, uterus, or oviduct. Also the cervix and uterotubal junction, because of their narrow canal, provide a better opportunity for interfering with the transport and survival of spermatozoa in vivo. Medicated devices are not related to coitus as the conventional spermicidal foams, creams, and diaphragms. Medicated intravaginal devices could be self-administered, and replaced at frequent intervals. Medicated intracervical devices are not easily self-administered and may have a life span of only 1 year. Medicated intrauterine devices with a definite life span may have inherent problems similar to the intracervical devices. Intraoviductal devices require minor operative procedures, such as hysterectomy, laparoscopy, or culdoscopy. The isthmus of the oviduct and uterotubal junction are rigid canals where contact of spermatozoa with an active agent can be more readily achieved. Recent advances have been made in the use of sperm acrosomal enzyme inhibitors such as contraceptive agents. Enhanced interest in this area, plus the great need for an effective, nonhormonal birth control method, has led to various original concepts and experiments showing the practical application of such antienzymes. Compounds that are com-
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pletely satisfactory have not been found. Even though TLCK appears promising, it may only be useful for vaginal application because of some undesirable properties that inhibit trypsin, plasmin, thrombin, and acrosin. Further studies are needed to elucidate the physiologic and biochemical mechanisms by which such compounds inhibit fertility. Boisseau and Joly discussed the adaptive mechanisms of internal fertilization and sperm storage in amphibians. Among these species, external fertilization occurs in Anura and in two primitive species of Caudata. In female amphibians (Caudata), sperm are stored in the cloacal glands (spermatheca). The tubules of the spermatheca are lined with myoepithelial and secretory cells. Ovarian hormones seem to be essential in maintaining secretory activity of these cells. There seem to be no structural modifications in the regions of contact between the epithelium and sperm. Spermatozoa may actively penetrate outpocketings of the spermatheca, and are evacuated by contractions at the time of ovulation. There are three types of internal fertilization: (1) "cloacal fertilization" occurs in oviparous species where spermatozoa are extruded from the spermatheca onto the eggs just before oviposition; (2) "uterine fertilization" occurs in a viviparous species; and (3) "oviductal fertilization" occurs in ovoviviparous species. CAPACITATION OF SPERMATOZOA
In most species, mammalian spermatozoa must undergo final maturation in the luminal environment of the female reproductive tract before penetrating through the three layers of the egg. Capacitated spermatozoa
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can be rendered infertile again ("decapacitated") by incubation with seminal plasma, or certain seminal plasma constituents. Decapacitated spermatozoa have to be "recapacitated" again before they are able to fertilize. Several enzymes in spermatozoa and seminal plasma play a major role in the reproductive process. These enzymes are associated with the acrosome and are used by the spermatozoon to pass through the investments surrounding the ovum. At least three are of major importance: hyaluronidase, corona penetrating enzyme (CPE), and a proteinase. A direct correlation exists between the amount of hyaluronidase and the concentration but not in the morphology or motility of spermatozoa. A hormone-dependent increase in hyaluronidase release from the spermatozoa occurs after they are in the uterus, or when uterine fluid is added in vitro. The increase in sperm hyaluronidase activity during capacitation is not due to the removal of an inhibitor (as in the case with acrosin), since no such inhibitor is present in seminal plasma. CPE obtained from spermatozoa by detergent extraction, can be inhibited by the decapacitation factor (DF). DF, present in seminal plasma in a high molecular weight form, was the first seminal component shown to prevent fertilization in addition to capacitated spermatozoa. The seminal trypsin inhibitors have a similar effect. DF can be reduced to a small peptide that still possesses active antifertility properties. Other enzyme inhibitors with antifertility properties are the glycoproteins, fetuin, and boar Cowper's gland mucin. The sperm neutral proteinase may be considered a model for other acrosomal enzymes in the development
September 1974
of new contraceptives. Zaneveld has shown that the sperm proteinase differs from all other known proteolytic enzymes, although it has many properties in common with trypsin and plasmin. Biochemical and immunologic differences exist between these last two enzymes and the sperm proteinase; like the sperm hyaluronidases, the sperm proteinase appears specifically in the male reproductive tract. The enzyme is given the name "acrosin" as approved by the "Commission on Enzyme Nomenclature." Proteinase inhibitors are present in the seminal plasma of all mammals. No correlation appears to exist between the amount of inhibitor and sperm motility or number of abnormal spermatozoa, although a low concentration of sperm is usually associated with a low inhibitor concentration. Zaneveld divided seminal inhibitors into two groups: those with a rather low molecular weight (5,000 to 15,000) and those with higher molecular weights. The latter are identical to the serum proteinase inhibitors, a l antitrypsin and a1x-antichymotrypsin. The other serum inhibitors, inter-atrypsin inhibitor, u2-macroglobulin, antithrombin III, and the CIa esterase inhibitor, are absent from seminal plasma at least in men. Capacitated sperm possess a much higher acrosin activity than ejaculated spermatozoa, apparently due to the removal of the seminal inhibitors from the sperm during uterine incubation. FERTILIZATION
Mammalian fertilization involves nuclear maturation, cytoplasmic maturation, functional stimulation of cumulus cells, corona radiata, sperm capacitation, acrosome reaction, passage of sperm through the zona
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pellucida, fusion of sperm to oocyte, block to polyspermy, development of male and female pronuclei, DNA replication, chromosome condensation, organization of first cleavage spindle, and first cleavage. Mammalian spermatozoa have several complex enzyme systems which are involved with sperm metabolism (associated with the midpiece), and with fertilization (mostly associated with the sperm head). Most of these enzymes are similar to the ones present in lysosomes and have been thought to originate from the acrosome, a structure that is considered to be a modified lysosome. Hyaluronidase is most likely involved in the penetration of the spermatozoon through the cumulus oophorous of the ovum (the outermost layer); corona penetrating enzyme, through the corona radiata (the second layer); and acrosin, through the zona pellucida (the innermost layer). Fertilization occurs only when a critical number of spermatozoa reach the site of fertilization. Little is known of the physiologic mechanisms by which the spermatozoa encounter the egg in the oviduct. Several mechanisms have been implicated (eg, fluid flow and muscular activity of the oviduct). Harper concluded that an advantage of eliminating dead, abnormal, or immotile sperm on their passage through the reproductive tract is to ensure the greatest viability of the zygote. However, one can hardly envision 99% of ejaculated sperm falling into this category. Another argument against this concept is the considerable success achieved in production of viable fetuses from in vitro fertilization. In polytocous species (especially those with separate uterine horns), fertilization may occur in one oviduct while it does not occur in the other. In a group of
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animals, the fertilization failure rate is 5% at any mating. When the animals of the same group are remated at a different time, the 5% failure of fertilization will usually be among different animals. FUTURE RESEARCH
Basic and applied research on the physiologic mechanisms involved in sperm transport and fertilization is relevant to our social needs in the development of new contraceptive methods. It is hoped that federal and international agencies will give this research priority in the near future. Some of these research areas are: (1) The role of testicular and epididymal secretions in the maturation of spermatozoa in the male reproductive tract. (2) The endocrine, neuroendocrine, and maternal factors which infl uence the rate of transport and survival of spermatozoa in the female reproductive tract. (3) Physiologic significance of spatial separation between spermatozoa and leukocytes in the cervix. (4) Patterns of phagocytosis of spermatozoa in different segments of the female reproductive tract. (5) The role of the cervix and uterotubal junction as "sperm reservoir," sperm barrier, or sperm filter in several mammalian species including nonhuman as well as human primates. (6) The physiologic interaction between sperm heads and the secretory epithelium of the female reproductive tract during short-term and long-term storage. (7) The minimal and optimal number of spermatozoa required for fertilization. The hazards associated with fertilization of aged spermatozoa re-
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quired for fertilization. The hazards associated with fertilization of aged spermatozoa and ova in animals and humans. (8) Mechanisms of sperm transport as affected by structural changes in cervical mucosa and cervical mucus. (9) The function of the cervical crypts in relation to storage or destruction of spermatozoa and the influence of endocrine parameters. (10) The biochemical and physiologic changes in spermatozoa and seminal plasma during coagulation and liquefaction of semen. (11) The proteolytic enzymes present in the sperm, the seminal plasma, and leukocytes in their interaction with the luminal fluids occurring with natural or artificial inhibitors.
September 1974
(12) The immune mechanisms present in the uterus, cervix, and the mucus, which may interact with spermatozoa. (13) The pharmacologic agents which can be administered locally, rather than systemically, with an effect on transport and survival of spermatozoa. (14) Comparative studies on sperm transport in several species of nonhuman primates to establish an experimental model to be used in studies of human reproduction, in order to screen new techniques for fertility regulation. (15) The mechanisms of human fertilization, including complete human oocyte maturation in culture and early embryo cytogenetics.
Vol. 25, No.9, September 1974 Printed in U.S.A.
Copyright" 1974 The American Fertility Society
Communications and Commentaries INTERNATIONAL SYMPOSIUM ON THE BIOLOGY OF SPERMATOZOA: TRANSPORT, SURVIVAL, AND FERTILIZING ABILITY E. SAAD E. HAFEZ, PH.D. (Cantab.), AND CHARLES G. THIBAULT, PH.D.
i: M;r
Departrrumts of Gynecology-Obstetric~ am! P"lsJ:::~'/;t ~. ~~'tM~'iJ::~Ol, and Growth and Development, Wayne S~te ...Vn~vnalerBltyd RC he A !...~,!!,jn'; Physiologie Animale e ec rc"" nos' v...., , University of Paris VI and Institut lYatio 783501 Jouy-en-Josas, L'ranee
he
An international symposium on The Biology of Spermatozoa was held N 0vember 4 to 7 at the Institute of Agricultural Research in Nouzilly, France. The program dealt with sperm transport in the male and female tract of human, nonhuman primates and other vertebrates, sperm capacitation, sperm acrosomal enzymes and their potential for contraceptive development, and the influence of hormonal and intrauterine contraceptives on human sperm migration in vivo and in vitro. Emphasis was placed on the complex processes of physiological and biochemical mechanisms involved in the transport and maturation of the spermatozoon from the testis to its final destination, and the site of fertilization at the ampullary-isthmic junction of the oviduct. The 31 speakers were from Australia, Belgium, Chile, France, New Zealand, Sweden, United Kingdom, and the United States. The symposium was supported by The French National Institute of Health and Medical Research, The World Health Organization, The French National Institute of Agricultural Research, Schering, Byla-Searle, Syntex, and Roussel. The proceedings, edited by Hafez and Thibault, are being published by S. Karger, Basel, Switzerland. The following is a summary of the symposium papers. Received January 14, 1974.
TESTICULAR FLUIDS
Setchell and Waites reported that fluid collected from the rete testis is a watery, opalescent suspension of living testicular spermatozoa. It differs from fluid collected out of the lumen of seminiferous tubules in its ionic and protein composition and its lower concentration of spermatozoa. Rete testis fluid contains testosterone and fluids differ markedly from blood plasma in many respects. Fluid secretion is probably not involved in the release of spermatozoa from the germinal epithelium, but it is involved in the transport of spermatozoa out of the seminiferous tubules and from the rete testis into the epididymis. The fluid also provides substrates necessary for metabolism of spermatozoa but the spermatozoa may also draw on their endogenous reserves (eg, lipids). Rete testis fl uid and epididymal seminal plasma also contain inhibitors of acrosomal proteinase, and although these inhibitors are not tightly bound to the spermatozoa, they may be important in controlling proteolytic activity during the maturation of epididymal spermatozoa. EPIDIDYMAL SPERMATOZOA
The primary functions of the mammalian epididymis are to serve as a conduit for the transport of spermatozoa from the testis to the ejacula-
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tory duct and to provide a favorable environment for sperm maturation and storage prior to ejaculation. The morphologic and physiologic integrity of the epididymis is androgendependent. Changes in tubular diameter, epithelial height, and histochemical characteristics after castration are repaired or prevented by the administration of exogenous testosterone. The regressive changes in the epididymis following castration or hypophysectomy are associated with a loss of motility and degeneration of spermatozoa contained therein. Sperm maturation in the epididymis (ie, the development of their ability to fertilize) and sperm survival are also controlled by androgens. An early step in the action mechanism of steroid hormones is their interaction with a specific cytoplasmic receptor protein in the target cell. The receptor-hormone complex is subsequently transported into the nucleus where it is associated with chromatin. Danzo and Orgebin-Crist have shown that three androgenbinding proteins are present in the epididymis: (1) an androgen-binding protein present in blood plasma, (2) an androgen-binding protein of testicular origin present in the caput epididymidis, and (3) an androgenbinding protein present in the epididymis of rabbits that have been castrated for 3 or 4 days. The first two binding moieties probably serve as transport proteins for androgens. The target cell receptor, demonstrated in the epididymis of castrated rabbits, may be of primary importance in mediating the androgen-dependent processes that are required for maintaining the many complex morphologic and functional aspects of the epididymis. The role of these androgen-dependent phenomena in the
September 1974
economy of the epididymis is not yet understood. Danzo and Orgebin-Crist have advanced the hypothesis that the function of androgens is to act on the epididymis in order to produce secretory products that interact with spermatozoa to bring about changes that are required for their maturation and survival. The observation that androgen-dependent protein synthesis has occurred in the epididymis in organ culture would tend to support this theory. Although the hypothesis may appear simplistic, the complex androgen regulation of epididymal function is directed solely to mediating effects on the tissue itself without affecting the enclosed spermatozoa. The ultrastructural and cytochemical changes in the acrosome of spermatozoa during their transport in the epididymis and female reproductive tract can be studied by a variety of techniques. Such methods include scanning electron microscopy, freeze-etching, and ultrastructural cytochemistry in order to localize glycoproteins and their acidic groups. The last technique involves staining of thin sections in glycolmethylacrylate with phosphotungestic acid, the use of colloidal iron hydroxide, and extraction with pronase and neuraminidase. Flechon observed dense crystalline cores in the corpus epididymidis of the rabbit inside the marginal thickening of the acrosome at the time it becomes rounded and shorter and reaches fertilizing ability. The acrosome is of heterogenous proteins. In ejaculated spermatozoa glycoproteins are located only at a superficial layer of the anterior segment. A relationship between the presence of glycoproteins, crystalline, and amorphous proteins and the localization of various acrosomal enzymes is sug-
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gested. Modifications of comparison and structure during epididymal transport may involve ripening or compact stockage of these enzymes. Rabbit spermatozoa are coated with glycoproteins. In ejaculated spermatozoa, the coat is thin on the head, almost absent on the middlepiece, and thicker on the principal piece, where most of the acidic groups are concentrated. Modifications in glycoproteins and acidic groups of the cell coat occur also in posterior corpus epididymidis where fertilizing ability of spermatozoa is acquired. Flechon has proposed that observed alterations of the plasma membrane during epididymal transport could induce some modifications of the sperm metabolism and surface properties contributing to the acquisition of fertilizing ability. Functional maturity of spermatozoa is attained during the passage of spermatozoa in the avian deferent ducts. The time of sperm transport in the epididymis varies from 1 to 2 days in birds to 11 to 16 days in mammalian species, including humans. In male birds, sperm maturation involves lipomucopolysaccharide complex and glycoproteins observed in the male reproductive tract. While the development of accessory sex organs is controlled by testosterone, this hormone is not necessary for survival of spermatozoa in the male reproductive tract. Data on the extragonadal sperm reserves and the biochemical characteristics of secretions suggest the mammalian epididymis is analogous to the avian deferent ducts.
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Croxato et al; Settlage et aI). Settlage et al reported that spermatozoa were found in the human oviduct within 5 minutes of deposition in the proximal vagina. The numbers of sperm found in the oviduct were directly related to the numbers of sperm in the inseminate during the first 2 hours after insemination. Sperm were distributed throughout the oviduct. The total numbers of sperm present in cervical mucus appeared to be constant until 48 hours later when they were dramatically reduced. On the other hand, there seemed to be a constant level of sperm in the oviduct 15 to 45 minutes after insemination; this was greatly reduced by 6 hours. Endometrial cavity sperm were not found until 90 minutes after insemination. They increased in number for 24 hours and then decreased until they were absent at 48 hours after insemination. Maximal calculated ratios indicate that for every oviductal sperm there are about 500 endometrial sperm, 20,000 cervical mucus sperm, and 10 million motile sperm in the inseminate. Ahlgren et al estimated that some 200 sperm were found in the ampulla of the oviduct of a woman. There are no accurate data on survival time and duration of fertilizing capacity of human spermatozoa. Sperm transport in nonhuman primates. Hafez and Jaszczak described the patterns of sperm transport in nonhuman primates by (1) observing the female reproductive tract by scanning electron microscopy, and (2) taking sperm counts in serial histologic sections at various interPATTERNS OF SPERM TRANSPORT vals from copulation. Sperm transport in women. Three The epithelium of the female retechniques were presented to assess productive tract is made of variable transport in women (Ahlgren et al; proportions of secretory cells with
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microvilli, and ciliated cells with kinocilia beating toward the vagina. Sperm transport was influenced by the rate of coagulation and liquefaction of semen. The number of sperm progressively decreased from the external os of the cervix toward the uterine cavity and oviducts. The mucus in the cervical canal and the uterotubal junctions were considered the main barriers in sperm ascent. When monkeys were mated during midcycle (time of ovulation), sperm ascent was optimal and spermatozoa were often aligned along strands of cervical mucus. These strands of mucus migrated to the cervical crypts or to the uterine cavity. When copulation was performed during the early follicular or luteal phases, sperm ascent was largely or completely inhibited due to the presence of a scant amount of viscous, cellular cervical mucus which showed no ferning or spinnbarkeit. Spermatozoa migrate to the uterine cavity through the longitudinal crypts of the endocervix, as well as the cervical canal. Many more spermatozoa were located in the endometrial glands than in the uterine cavity. Sperm transport in uterine ejaculators. In uterine ejaculators, spermatozoa very rapidly reach the uterotubal junction. The site of fertilization is attained by 2 hours after coitus. The uterotubal junction and isthmus act as very potent barriers against dead sperm and limit the number of sperm moving to the fertilization site. In the sow, there is no resorption of sperm in the uterus, as previously described, since ligature prevents sperm disappearance. Thus, sperm resorption in uterine ejaculators results first from release from the cervix, and second, from phagocytosis after large leukocytic invasion.
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Sperm transport in ruminants. In cattle, as in sheep (when fixation of the whole genital tract and serial section is used), the first spermatozoa appear to never reach the isthmus before 2 hours. Thibault et al found that the number of spermatozoa present at the uterotubal junction and the isthmus steadily increases from 2 to 18 hours after coitus. However, the maximum number of spermatozoa in the isthmus remains limited to several thousand. The small number of spermatozoa in the ampulla at any time after coitus results from the the efficacy of three barriers: the cervix, the uterotubal junction, and the isthmus. The number of spermatozoa present in the oviduct is not related to the number of spermatozoa inseminated either in the vagina or the uterus. This fact explains the possibility of subnormal fertility in cow inseminated with .0001 of bull ejaculate (375,000 living spermatozoa). PHYSIOLOGIC MECHANISMS OF SPERM TRANSPORT
There are remarkable variations among species in the site of semen deposit (vaginal or uterine), in the volume of accessory fluid, and in the number of spermatozoa ejaculated. However, the number of spermatozoa reaching the oviduct seems to be relatively constant and represents only a very small percentage of that in the ejaculate. Changes in uterine motility and the characteristics of cervical mucus greatly influence the transport of spermatozoa. Both are under the influence of ovarian steroids; maximal responses coincide with production of endogenous estrogen. Uterine motility is depressed by progesterone and enhanced by estrogen. The
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parallel arrangement of macromolecular micelles characterizes the estrogenic phase of the cycle. During the luteal phase, the micelles split up forming a dense network which inhibits sperm penetration. The cervical mucus of postmenopausal women is scant and impenetrable to spermatozoa. Sperm transport is greatly affected when small numbers of spermatozoa are used in insemination. SURVIVAL OF SPERMATOZOA
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Sperm survival in mammals. Sperm survival in the female reproductive tract of common mammals varied from 15 hours in mice and rats to 10 days in the dog. Postcoital tests in monkeys showed that sperm motility in the external cervix lasted less than 36 hours and fertilizing ability was maintained for a shorter period. Ahlgren et al found that the survival time of spermatozoa in the ampulla of the oviduct after coitus was 85 hours. It was not certain whether these spermatozoa were fertile at that time. The fertilizing ability of rabbit spermatozoa was lost before motility. Farris concluded from his studies of artificial insemination with donor sperm that the fertile life of sperm was 24 hours. Ferin's report of 120hour fertilizing capacity for human spermatozoa has not been conflrmed. It is essential that data be obtained from prospective studies on time relations between coitus and ovulation, fertilization, or sperm recovery. Data from retrospective studies have been unreliable. The fertilizing ability of spermatozoa recovered from human oviducts should be tested in vitro. It is doubtful that human spermatozoa were found 2 to 3 weeks after coitus (as reported in previous studies), since the data were based on the recollection of the patient or
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the assumption of the physician when the patient had been in the hospital for some weeks. Reports of such long survival time conflicted with existing data on other mammals (except the hare and the bat). Spermatozoa can survive in the female reproductive tract of the hare for a few weeks and can survive in tropical and temperate bats for a few months. Martinet and Raynaud reported that "superfetation," the simultaneous development of two different generations of eggs, occurs in hare as a result of long-term survival of spermatozoa in the uterus. In some amphibian species, spermatozoa buried in the epithelial cells can remain alive for 2 years. In the cow, spermatozoa survive at least 3 days at the uterotubal junction in some fmger-like folds because of the absence of leukocytes in these folds. Leukocytes invade more or less rapidly the lower parts of the female tract, depending on the animal, and clean the tract of sperm. Little is known about the preferential areas for spermatozoa storage in the uterus or the physiologic interaction between spermatozoa and the secretory cells of the epithelium of the female reproductive tract. Sperm survival in lower forms of life. In reptiles, sperm storage occurs in the vaginal segment of the oviduct, either in light or in the more or less developed seminal receptacles; in lizards these receptacles are linked to the possibility of several successive lays for isolated females. During the weeks preceding ovulation (earlier in snakes than in lizards), spermatozoa reach the precranial segment of the oviduct. They eventually may be stored in seminal receptacles where spermatozoa are protected during lay time. Saint Girons has shown that
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seminal receptacles do not seem vital for long survival of the spermatozoa since these receptacles are often stored in the light of the vaginal tube. Prostaglandins may improve fertilization in adverse circumstances. However, fertilization is a most efficient process, except in certain exceptional cases (eg, infertile female with hostile cervical mucus or oviductal problems or infertile male with oligospermia, high percentage of abnormal sperm, or sperm of low motility.) In humans, in vitro fertilization is of great clinical significance in solving infertility problems, such as bypassing an irreducible tubal block. Ovulation must be induced in the ovum donor in order to synchronize embryonic development with the maternal environment of the recipient. Fertilizable eggs can be produced after ovulation induction by human menopausal gonadotropin-human chorionic gonadotropin (HMG-HCG) or clomiphene-HCG treatments. The limiting factor of in vitro fertilization no longer is the problem of obtaining capacitated sperm, but the problem of mastering complete control over oocyte maturation in vitro. Soupart has developed an effective, completely controlled, in vitro human fertilization system. This experimental tool is indispensable for the development of new and better contraceptive methods, which should be based on precise inhibition of highly specific molecular mechanisms. Inhibition of fertilization. Tosyllysine chloromethyl ketone (TLCK), nitrophenyl p-guanidino benzoate (NPGB), and to a lesser extent, ethyl p-guanidino benzoate (EPGB) inhibit the in vitro activity of acrosin. Tosylamide phenyl ethyl chloromethyl ketone (TPCK) has no effect on the enzyme. Zaneveld and co-workers
September 1974
have shown that addition of TLCK but not TPCK to spermatozoa in quantities that did not affect their motility, completely inhibited the fertilizing ability of capacitated spermatozoa. TLCK and NPGB, but not EPGB, prevented fertilization if mixed with ejaculated spermatozoa before deposit in the uterus; this shows that these synthetic inhibitors are not removed during capacitation. Zaneveld et al have also shown that TLCK effectively prevents fertilization on vaginal application before coitus and enhances the contraceptive activity of Delfen vaginal cream. Two other synthetic inhibitors, p-amino phenacyl bromide (APB) and p-guanidino-phenacyl bromide (GPB) also result in decreased fertilization if applied vaginally, but are less active than TLCK. DEVELOPMENT OF NEWER CONTRACEPTIVES
Basic and clinical research in the physiology of spermatozoa is needed to develop efficient,reversible, safe, easily administered, low-cost contraceptives for large scale use. Such research should be directed to the development of newer methods which would interfere with transport of spermatozoa in the female reproductive tract; ways should be found to cause the destruction, immotilization, agglutination, or decapacitation of spermatozoa. This can be achieved by intravaginal, intracervical, intrauterine, or intraoviductal devices medicated with potent pharmacologic agents (eg, metallic ions, progestogens, acrosomal enzyme inhibitors, sperm antibodies, and spermicides). Several modes of action can be employed: (1) change the biochemical or biophysical characteristics of the vaginal fluid or cervical mucus (rheo-
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logical properties and micelles formation); (2) interfere with sperm metabolism and survival in the endometrial fluid; (3) interfere with the enzyme and enzyme inhibitor system; or (4) prevent sperm migration through the uterotubal junction to the oviduct. It may be possible to develop and administer systems of medicated slow delivery which would act locally rather than systemically. The vagina is more accessible than the cervix, uterus, or oviduct. Also the cervix and uterotubal junction, because of their narrow canal, provide a better opportunity for interfering with the transport and survival of spermatozoa in vivo. Medicated devices are not related to coitus as the conventional spermicidal foams, creams, and diaphragms. Medicated intravaginal devices could be self-administered, and replaced at frequent intervals. Medicated intracervical devices are not easily self-administered and may have a life span of only 1 year. Medicated intrauterine devices with a definite life span may have inherent problems similar to the intracervical devices. Intraoviductal devices require minor operative procedures, such as hysterectomy, laparoscopy, or culdoscopy. The isthmus of the oviduct and uterotubal junction are rigid canals where contact of spermatozoa with an active agent can be more readily achieved. Recent advances have been made in the use of sperm acrosomal enzyme inhibitors such as contraceptive agents. Enhanced interest in this area, plus the great need for an effective, nonhormonal birth control method, has led to various original concepts and experiments showing the practical application of such antienzymes. Compounds that are com-
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pletely satisfactory have not been found. Even though TLCK appears promising, it may only be useful for vaginal application because of some undesirable properties that inhibit trypsin, plasmin, thrombin, and acrosin. Further studies are needed to elucidate the physiologic and biochemical mechanisms by which such compounds inhibit fertility. Boisseau and Joly discussed the adaptive mechanisms of internal fertilization and sperm storage in amphibians. Among these species, external fertilization occurs in Anura and in two primitive species of Caudata. In female amphibians (Caudata), sperm are stored in the cloacal glands (spermatheca). The tubules of the spermatheca are lined with myoepithelial and secretory cells. Ovarian hormones seem to be essential in maintaining secretory activity of these cells. There seem to be no structural modifications in the regions of contact between the epithelium and sperm. Spermatozoa may actively penetrate outpocketings of the spermatheca, and are evacuated by contractions at the time of ovulation. There are three types of internal fertilization: (1) "cloacal fertilization" occurs in oviparous species where spermatozoa are extruded from the spermatheca onto the eggs just before oviposition; (2) "uterine fertilization" occurs in a viviparous species; and (3) "oviductal fertilization" occurs in ovoviviparous species. CAPACITATION OF SPERMATOZOA
In most species, mammalian spermatozoa must undergo final maturation in the luminal environment of the female reproductive tract before penetrating through the three layers of the egg. Capacitated spermatozoa
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can be rendered infertile again ("decapacitated") by incubation with seminal plasma, or certain seminal plasma constituents. Decapacitated spermatozoa have to be "recapacitated" again before they are able to fertilize. Several enzymes in spermatozoa and seminal plasma play a major role in the reproductive process. These enzymes are associated with the acrosome and are used by the spermatozoon to pass through the investments surrounding the ovum. At least three are of major importance: hyaluronidase, corona penetrating enzyme (CPE), and a proteinase. A direct correlation exists between the amount of hyaluronidase and the concentration but not in the morphology or motility of spermatozoa. A hormone-dependent increase in hyaluronidase release from the spermatozoa occurs after they are in the uterus, or when uterine fluid is added in vitro. The increase in sperm hyaluronidase activity during capacitation is not due to the removal of an inhibitor (as in the case with acrosin), since no such inhibitor is present in seminal plasma. CPE obtained from spermatozoa by detergent extraction, can be inhibited by the decapacitation factor (DF). DF, present in seminal plasma in a high molecular weight form, was the first seminal component shown to prevent fertilization in addition to capacitated spermatozoa. The seminal trypsin inhibitors have a similar effect. DF can be reduced to a small peptide that still possesses active antifertility properties. Other enzyme inhibitors with antifertility properties are the glycoproteins, fetuin, and boar Cowper's gland mucin. The sperm neutral proteinase may be considered a model for other acrosomal enzymes in the development
September 1974
of new contraceptives. Zaneveld has shown that the sperm proteinase differs from all other known proteolytic enzymes, although it has many properties in common with trypsin and plasmin. Biochemical and immunologic differences exist between these last two enzymes and the sperm proteinase; like the sperm hyaluronidases, the sperm proteinase appears specifically in the male reproductive tract. The enzyme is given the name "acrosin" as approved by the "Commission on Enzyme Nomenclature." Proteinase inhibitors are present in the seminal plasma of all mammals. No correlation appears to exist between the amount of inhibitor and sperm motility or number of abnormal spermatozoa, although a low concentration of sperm is usually associated with a low inhibitor concentration. Zaneveld divided seminal inhibitors into two groups: those with a rather low molecular weight (5,000 to 15,000) and those with higher molecular weights. The latter are identical to the serum proteinase inhibitors, a l antitrypsin and a1x-antichymotrypsin. The other serum inhibitors, inter-atrypsin inhibitor, u2-macroglobulin, antithrombin III, and the CIa esterase inhibitor, are absent from seminal plasma at least in men. Capacitated sperm possess a much higher acrosin activity than ejaculated spermatozoa, apparently due to the removal of the seminal inhibitors from the sperm during uterine incubation. FERTILIZATION
Mammalian fertilization involves nuclear maturation, cytoplasmic maturation, functional stimulation of cumulus cells, corona radiata, sperm capacitation, acrosome reaction, passage of sperm through the zona
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pellucida, fusion of sperm to oocyte, block to polyspermy, development of male and female pronuclei, DNA replication, chromosome condensation, organization of first cleavage spindle, and first cleavage. Mammalian spermatozoa have several complex enzyme systems which are involved with sperm metabolism (associated with the midpiece), and with fertilization (mostly associated with the sperm head). Most of these enzymes are similar to the ones present in lysosomes and have been thought to originate from the acrosome, a structure that is considered to be a modified lysosome. Hyaluronidase is most likely involved in the penetration of the spermatozoon through the cumulus oophorous of the ovum (the outermost layer); corona penetrating enzyme, through the corona radiata (the second layer); and acrosin, through the zona pellucida (the innermost layer). Fertilization occurs only when a critical number of spermatozoa reach the site of fertilization. Little is known of the physiologic mechanisms by which the spermatozoa encounter the egg in the oviduct. Several mechanisms have been implicated (eg, fluid flow and muscular activity of the oviduct). Harper concluded that an advantage of eliminating dead, abnormal, or immotile sperm on their passage through the reproductive tract is to ensure the greatest viability of the zygote. However, one can hardly envision 99% of ejaculated sperm falling into this category. Another argument against this concept is the considerable success achieved in production of viable fetuses from in vitro fertilization. In polytocous species (especially those with separate uterine horns), fertilization may occur in one oviduct while it does not occur in the other. In a group of
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animals, the fertilization failure rate is 5% at any mating. When the animals of the same group are remated at a different time, the 5% failure of fertilization will usually be among different animals. FUTURE RESEARCH
Basic and applied research on the physiologic mechanisms involved in sperm transport and fertilization is relevant to our social needs in the development of new contraceptive methods. It is hoped that federal and international agencies will give this research priority in the near future. Some of these research areas are: (1) The role of testicular and epididymal secretions in the maturation of spermatozoa in the male reproductive tract. (2) The endocrine, neuroendocrine, and maternal factors which infl uence the rate of transport and survival of spermatozoa in the female reproductive tract. (3) Physiologic significance of spatial separation between spermatozoa and leukocytes in the cervix. (4) Patterns of phagocytosis of spermatozoa in different segments of the female reproductive tract. (5) The role of the cervix and uterotubal junction as "sperm reservoir," sperm barrier, or sperm filter in several mammalian species including nonhuman as well as human primates. (6) The physiologic interaction between sperm heads and the secretory epithelium of the female reproductive tract during short-term and long-term storage. (7) The minimal and optimal number of spermatozoa required for fertilization. The hazards associated with fertilization of aged spermatozoa re-
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quired for fertilization. The hazards associated with fertilization of aged spermatozoa and ova in animals and humans. (8) Mechanisms of sperm transport as affected by structural changes in cervical mucosa and cervical mucus. (9) The function of the cervical crypts in relation to storage or destruction of spermatozoa and the influence of endocrine parameters. (10) The biochemical and physiologic changes in spermatozoa and seminal plasma during coagulation and liquefaction of semen. (11) The proteolytic enzymes present in the sperm, the seminal plasma, and leukocytes in their interaction with the luminal fluids occurring with natural or artificial inhibitors.
September 1974
(12) The immune mechanisms present in the uterus, cervix, and the mucus, which may interact with spermatozoa. (13) The pharmacologic agents which can be administered locally, rather than systemically, with an effect on transport and survival of spermatozoa. (14) Comparative studies on sperm transport in several species of nonhuman primates to establish an experimental model to be used in studies of human reproduction, in order to screen new techniques for fertility regulation. (15) The mechanisms of human fertilization, including complete human oocyte maturation in culture and early embryo cytogenetics.