Limitations in developing cossypol acetic acid as a male contraceptive

CONTRACEPTION

LIMITATIONS ACETIC

N.K.

ACID

IN

DEVELOPING

AS A

MALE

GOSSYPOL

CONTRACEPTIVE

Lohiya, Kavita Sharma, Mukesh Kumar and Reproductive Physiology Section, Department University of Rajasthan, Jaipur-302004,

Subrat Sharma of Zoology, India

Highly purified gossypol acetic acid (5 mg/day; oral) alone and in combination with potassium chloride (0.25 mglday; oral) was tested in adult male langurs for 120 days to evaluate reversibility of its The treatment resulted antifertility action and possible hypokalemia. Sperm severe impairment of sperm motility. in 01 igosperm ia with activities of morphological defects were evident. The functional libido remained unimpaired. Occurrence of accessory sex glands and hypokalemia was found more pronounced in the gossypol alone group. Extensive renal potassium loss was evident and conversely the renal The activities of serum excretion of sodium decreased markedly. increased significantly. Other parameters of blood and transam i nases of urine did not show any marked alterations. Complete reversal of the above changes was evident following 90 to 105 days of withdrawal of treatment. In conclusion, the oligospermia achieved was reversible and the hypokalemic response of langurs is similar to human and not related to impurity of the drug.

Submitted Accepted

for publication for publication

MAY 1990 VOL. 41 NO. 5

July 19, 1989 January 9, 1990

519

CONTRACEPTION INTRODUCTION has provided a new lead Cossypol in the search for an orally effective male contraceptive. Although it has been considered a very promising agent to induce infertility in many animal species and in human male (l-51, the important concern about hypokalemia and the incidence of considerably delayed recovery of spermatogenesis or irreversibility remains elusive (4-7) and influences the widespread acceptability of gossypol as a male contraceptive. With a view to answer these critical problems, WHO 18) has proposed initiating study in non-human primates in order to expand animal data to the human. Since the toxicity of gossypol has been related with impurities, attempts were made to purify it and carry out pre-cl inical and clinical investigations (8.9). Therefore, the present study was undertaken to assess the effects of gossypol acetic acid alone and in combination with potassium chloride with special attention to reversibility of treatment and evaluation of hypokalemia in male langur monkey. MATERIALS Experimental

AND

METHODS

animal

Adult male langur monkey (Presb tis entellus entellus Dufresne) were caught from junqle around Jalpur, Aia. Fully qrown animals weighing between 15 to 1% kg were selected and housed individually in metallic our primate in seminatural captive cond i t ions in cages and kept facility. Animals were quarantined for two months, during which they were trained for electroejaculation and blood sampling. Animals were fed with controlled diet thrice a day with a free access to water. Veterinary supervision was also provided throughout the study period. Experimental

design

The study period consisted of three (ii) treatment phase; 120 30 days, The animals were allocated 105 days. five animals each: Croup

A:

animals treatment

Group

B:

animals of (5mg/animal

Group

C:

this animals of group (5mg/animal /day; oral ) chloride (0.25mg/animal/day;

Body

this /day;

group oral)

were given as control.

were treated alone during

vehicle

alone

during

with gossypol acetic treatment phase.

acid

acetic acid received gossypol in with potassium combination oral ) during treatment phase.

weight

Body weight of phases of study.

520

of this group phase to serve

phases: (i) pretreatment phase; days, and (iii) recovery phase; into three groups composed of

the

animals

were

recorded

once

a

month

during

all

the

MAY 1990VOL. 41. NO. 5

CONTRACEPTION Semen

analysis

Semen samples were obtained fortnightly (10) and were evaluated macroscopically seminal fluid volume, pH, colour; and density, motility (qua1 ity of progressive motile spermatozoa) ,vitality and morphology Scanning

electron

by penile electroejaculation for semen weight, volume, microscopically for sperm motility and percentage of (11).

microscopy

One or two drops of semen were fixed in 2% glutaraldehyde for 10 min, washed twice for 5 min in O.lM phosphate buffer and finally in double-distilled water, resuspended in double-disti I led water and applied as thin film on a clear coverslip. This sample was first air dried, than sputter coated with silver and observed under SEM. Libido Animals were observation of Biochemical

introduced mounting

analysis

of

to receptive and copulatory seminal

females behaviour

sperm

motility

month

for

plasma

Fructose (13), citric acid (14), lactic phosphatase (16), magnesium (17) and (18) estimations were performed fortnightly. --In vitro

once every (12).

dehydrogenase (1 S), acid glyceryl phosphorylcholine

test

acid on sperm motility were The effects of gossypol acetic --in vitro studied according to the procedure described by Chongthammakun and Dilution of gossypol to 25. 50, 100, 150, 250 and associates (19). 500 uM were made and incubated with sperm at 37’C for 15, 30, 45, 60, 90 and 120 min. Hormone

assay

Testosterone levels in serum were determined in triplicate radioimmunoassay method (20). Binding of antisera specific The intrs+ and inter-assay coefficients of from 50 to 60%. respectively, with 10 pg sensitivity. were 5.98% and 9.82%. Blood/serum

by

using ranged variation

biochemistry

: haemoglobin (21). haematocrit Haematology serum electrolytes: Na and K (24). Cl (23); SCOT and enzymes : and Ca (27); serum and alkaline creatinine phosphokinase (29), analysed every two weeks.

(22). RBC and WBC count (25), PO4 (261, Mg (17) SGPT (28), LDH (15), phosphatase (30); were

Urinalysis Early morning pH, 24-hr urine volume, 24-hr urine protein Mg (17) electrolytes: Na and K (24). Cl (25), PO (261, (27), and creatinine clearance (32), estimation% were performed nightly.

MAY 199OVOL. 41 NO. 5

(31). and Ca fort-

521

CONTRACEPTION Statistics Data are difference

expressed by using

as mean student’s

f SE ‘t’ test.

and

were

analysed

for

statistical

RESULTS Body

weight

No appreciable treatment group

vehicle

changes animals

were when

recorded compared

in the body weight of with the animals that

both received

alone.

Seminology Macroscopic evaluation of semen weight, volume, seminal semen ejaculatory responses were found GROUP

revealed no drug-related fluid volume, colour normal. GROUP

A

REC

1

ohY--%-~ TREA?

DURATION Fig-l.

522

Sperm sperm, groups.

density prior

in The

v.-.*

+ a* .

v

TREAT

changes pH.

8

*P-=0-05 l P
I

and

REC

IN

DAYS

(la), percentage of during treatment to,

live and

(lb) and recovery

motile in all

(1~) three

MAY 1990 VOL. 41. NO. 5

CONTRACEPTION Only a small reduction in sperm density was recorded after 30 days which decreased markedly during subsequent treatment of treatment, achieved after 120 days of drug therapy intervals and 01 igosperm ia decreased significantly as The percentage of live sperm [Fig. la). quality of sperm motility the treatment progressed (Fig. 1 b) . The Only 20-30% of sperm were found motile with declined progressively. poor to non-existent motility after 120 days of treatment (Fig. lc). Following 45 to 60 days of drug exposure, an increase in the number (Fig. 2). These alterations of abnormal sperm f&t-ms was -recorded of recovery. following 90 to 105 days were reversible GROVP h

80

.P<0.05 1 Pc CONTROL

0’

“~‘3~“““~ 90 TREAT

30

. 90 REC

’

sperm in all

‘.“I.’

C 5m~iKKc1

I.‘.‘.

0 30

DURATION

Abnormal recovery

GOSSYPOL

;

*P-z001

0 30

Fig.2.

GROUP

9

5fqldoy;oml

0.02

l

70

GROUP GOSSYFOL

90 TREAT

IN

30

90 REC

u m TREZ

KI

3” REC

DAYS

percentage three groups.

prior

to,

during

treatment

and

Scanning electron microscopic observations revealed damage to sperm deformed and swelled head region; abundance of tail and mid-piece; proximally located cytoplasmic droplets and reduction in the size of These defects were not detected following 90 to 105 sperm (Fig.3). days after cessation of treatment.

Fig.3.

MAY

Scanning electron micrograph and following treatment with days (x 5000) (bar = 1 pm).

1990 VOL.

41 NO.

5

of monkey sperm of gossypol acetic acid

control (b) for

(a) 120

I523

CONTRACEPTION Libido None of treatment Seminal

the with

animals gossypot

plasma

demonstrated acetic acid,

of

change in libido during or in combination with KCI.

biochemistry

Gossypol treatment did not of seminal plasma fructose, phosphatase, magnesium or study period. Effect

any alone

gossypol

on

cause any alterations in lactic dehydrogenase, glyceryl phosphorylcholine

monkey

sperm

in --

the concentrations citric acid, acid throughout the

vitro

At gossypol concentrations of 25 to 50 uM, sperm moti I i ty was reduced significantly during 15 to 120 min of incubation. Sperm were immobi I ized by incubation with 100 u M and 150 u M gossypol for 60 min and within 15 min after incubation with 250 and 500 uM gossypol (Fig.4).

100 -

20 -

OL

0

15 TIME

Fig.4.

The per gossypol

Testosterone

cent motility at concentrations

AFTER

of

45

90

INCIJEATION

monkey ranging

90 (min

sperm after from 25 PM

120

1

to

incubation 500 PM.

with

levels

Circulating levels of testosterone the administration of gossypol KCI at all the intervals studied

524

30

were within alone or with (Fig.5).

control range following the supplementation of

MAY 1990 VOL. 41. NO. 5

CONTRACEPTION

, B r

GRouP

A ( CO+JTR~L;VWIICLE

GROUP

B (St-q

GOSSYPOL

ACETIC

ACID/

GROUP

C (5n-q

GOSSYPOL

ACETIC

AClD425mg

0

50

of Circulating levels during pre-treatment, represents mean + SE.

90

120

TREATMENT DURATION

5.

1 DAY;ORAL)

I

30

PT

Fig.

TREATED

30

so

90

RECOVERY IN

DAYS

testosterone of groups treatment and recovery.

A.

B and C Each value

Toxicology remarkable 96 days of medication, no initial 30 to During the levels in both treatment alterations were recorded in serum potassium serum potassium levels of the gossypol B and C. Thereafter, groups; alone group (B) declined more rapidly. After 120 days of drug hypokalemia was found more pronounced in the gossypol exposure, alone group than that of the combination group (Fig. 6). kaliuresis accompanied by retention of sodium, Renal potassium loss, of sodium decreased markedly with the as the urinary excretion in kal iuresis were observed both advancement of sever i ty of activity of serum transaminases was treatment groups (Fig . 7). The in both significantly following 60 days of medication increased These effects of gossypol treatment were treatment groups (Fig. 8). fully reversible following 90-105 days of recovery.

MAY

1990 VOL.

41 NO. 5

525

CONTRACEPTION

GKW-A

(‘.X+ITROL;VEHICLE TREATED) GOSSYPOL ACETIC ACID/@AYj~L, GOSSYPOL ACETIC AClD+O25mq KClIDAY.ORAL)

DURATION 6.

Serum potassium treatment groups recovery .

and (B

;:gg!

1

COVERY

TREATMENT

Fig.

:

sodium and C)

IN DAY? levels prior

of control to, during

(A) as well treatment

as and

0 GROUP-AICONTROL;VEHlCLE TREATED) m GROUP-Bmlq GGsYPoL ACETIC AclD/Dnu,oRnL, m GROUP-C(5m.1GOSMFU dCETlC ACID+0 25qKCLIDI\YjORAL, l 0

TREATMENT

RECOVERY

DURATION

Fig.

526

7.

Urinary treatment recovery.

levels of potassium groups (B and C)

PC005 P-ZOO2

and prior

IN

DAYS

sodium to,

of control (A) during treatment

and and

MAY 1990 VOL. 41. NO. 5

CONTRACEPTION

GROUP-A CONTROL

GROUP-B

GROUP-C

GOssmxtimg/doy,aal

i!!zz$z D2Smg/dayi

w

I_..I.I.I.I.I, OS0

30

TREAT

DURATION Fig.8.

Serum transaminase and treatment groups recovery.

m-01

30

90

030

TREAT

REC

IN

REC

DAYS

(SCOT and SCPT) (I3 and C) prior

activity in control to, during treatment

(A) and

serum and urinary electrolytes other than Na Haematological values, CPK and alkaline phosphatase levels fluctuated within and K, LDH, Early morning pH of urine, 24-hr control and/or pretreatment range. urine protein and creatinine clearance did not urine volume, 24-hr show any marked alterations. DISCUSSION Administration of gossypol is known to induce infertility but delayed in important gossypol irreversibility issue recovery or is an have in investigation this The results obta i ned contraception. evidenced by inhibition of spermatogenesis as revealed reversible There are species differences induction of reversible oligospermia. attributed to difference in sensitivity in terms of antifertility effect, to gossypol damage (1, 33-35). Several mechanisms of gossypol (i) interference with certain antiferti I ity effects have been proposed: thereby decreasing the incorporation metabolic steps in mitochondria, of amino acids with concomitant selective damage to target germ cell (36); (ii) inhibition of germ cell differentiation; and (iii) induction of gonadal prostaglandin biosynthesis (1, 36-40).

MAY 1990VOL. 41 NO. 5

I527

CONTRACEPTION In addition to antispermatogenic effect, gossypol has been shown to have a direct inhibitory action on sperm motility, evidenced by decreased percentage of motile sperm in the ejaculates of gossypoltreated animal as well as inhibition of sperm motility in vitro. The mechanisms by which gossypol disrupts functional activ>yTsperm involve: [i) the uncoupling of oxidative phosphorylation at the may (41-44); mitochondrial level (ii) inhibition of sperm specific isoenzyme LDH-X (45-48) ; and (iii) impairment of the ATPase activity (42,43.49). Morphological defects in sperm during gossypol treatment supports these mechanisms (50). Cossypol did not exert any effect on the functioning of accessory glands, as evidenced by constituents in the seminal plasma circulating levels of testosterone (13.51); hence, may reflect normal functioning of Leydig cells. It may be concluded administration of gossypol does not exert inhibitory effects steroidogenesis (36.38.52.53).

sex and the that on

The occurrence of hypokalemia in monkey, an eftect not observed in other animal species, is an important finding of the present study and be explained by: (i) species specific tolerance; (ii) may inhibition of Na-K-ATPase activity; (iii) damage to the renal tubules; (iv) modification of membrane permeability; (v) loss of potassium from muscles; and (vi) stimulation of prostaglandin biosynthesis responsible for renal potassium loss (7,4,54,55). The most likely possible mechanism for gossypol-induced hypokalemia in human has been suggested as a direct effect on renal tubules resulting in increased urinary potassium loss (9). Orally administered gossypol was found accumulated in large amounts in the liver. Correlation between accumulation and primary site of The activities of glutamicgossypol toxicity has been reported (56). and glutamic-pyruvic transaminases in blood oxaloacetic increased serum (56,57) indicating some degree of hepatic damage. Gossypol the activity with the alter of these enzymes by reacting m=y substrate blocking the action of the enzyme and/or by combining with the enzyme itself (58). Our study has shown that gossypol has satisfactory antifertility properties. Its side effects, though not so profound, are quite evident and are not related with impurities. Hence, if the toxicity can be overcomed, gossypol can be used as a male contraceptive. should be Further studies performed to establish an adequate supplementation dose of potassium chloride and finally to study langur monkey can be used as an ideal animal model. hypokalemia;

MAY

1990 VOL.

41. NO.

5

CONTRACEPTION ACKNOWLEDGEMENTS This study was sponsored by the Indian Council of Medical Research New Delhi. Samples of highly purified gossypol acetic acid IlCMR), (99.9% purified) were supplied by WHO. The authors are also grateful Waites for suggestions and his keen interest. The to Dr. C.M.H. comments received from the Steering Committee of the WHO Task Force on Methods for the Regulation of Male Ferti I i ty are greatly appreciated.

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MAY

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58.

Olgiati, K.L., Toscano, D.G., Atkins, W.M. and Toscano, W.A. inhibition of adeny late cyclase. Arch. Biochem. cossypol Biophys. 231 : 411-415 (1984). Nadakavukaren, M-J., Sorensen, R.H. and Tone, J.N. Effect of gossypol on the ultrastructure of rat spermatozoa. Cell Tissue Res. 204 : 293-296 (1979). M.R.N. Dinakar. N., Arora. R. and Prasad, Effects of microquantities of testosterone on the epididymis and accessory glands of the castrated rhesus monkey, Macaca mulatta. J. -(1974). Endocr. 60 : 399-408 Cu, Z.P., Guang, Y., Sang. G.W., Wang, W.C., Wang, Y-X., Zhoa, X. J., Cheng, Z.X., Ge, J.L. and Liu, X.H. The effects of gossypol on reproductive endocrine function in man. Advances in Fertility Regulation in the Male (Qian Shao-Zhen and Geoffrey M.H. Waites, Editors). The People’s Medical Publishing House, Beijing, 1985. p.33-37. biochemical and endocrine studies Ultrastructural, Hoffer, A.P. its isomeric derivatives on the on the effects of gossypol and male reproductive tract. In: Cossypol, a potential contraceptive for men (S. Segal, Editor). Plenum Press, New York, t985, p-143-186. R.T.T. hypokalemia. Wang, C. and Yeung , Gos5ypol and Contraception 32 : 237-252 (1985). Charles, Y.S. Potassium deficiency. A review of its causes, consequences and diagnosis. Advances in Fertility Regulation in the Male (Sian Shao-Zhen and Geoffrey M.H. Waites, Editors). The People’s Medical Publishing House, Beijing, 1985, p-15-19. Abou-Donia, M.B. Physiological metabolism of effects and gossypol. Residue Rev. 61 : 125-160 (1976). Effects of different levels of Braham, J.E. and Bressani. R. gossypol on transaminase activity, on non-essential to essential amino acid ratio, on iron and nitrogen retention in rats. J. Nutr. 105 : 348-355 (1975). and Abou-Donia. M.B. Toxicological effects of Nomeir, A.A. gossypol. In: Male Fertitity and its Regulation (T.J. Lob1 and E.S.E. Hafez, Editors). M.T.P. Press Ltd., Lancaster, 1985, p.lll-133.

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