Reversible antispermatogenic e¡ect of gossypolin langur monkeys (Presbytis entellus entellus)

S. SHARMA, M. KUMAR, R.B. GOYAL, B. MANIVANNAN and N.K. LOHIYA*

Department of Zoology, University of Rajasthan, Jaipur ^ 302 004, India*Correspondence

Abstract

The present investigation reports the antispermatogenic e¡ect of the orallyactive highly puri¢ed gossypol acetic acid at 7.5 mg and 10 mg/day for 180 daysin langur monkeys. The results revealed a dose-dependent response in semenanalysis as well as testicular morphology. Uniform severe oligospermia wasobserved in the lower dose (7.5 mg) group, while azoospermia was observed in2 out of 5 animals in the higher dose (10 mg) group and the remaining animalsshowed severe oligospermia. Scanning electron microscopy of spermatozoarevealed deleterious abnormalities in the head and midpiece. Testicularmorphology revealed a decrease in the seminiferous tubule diameter and arrestof spermatogenesis. The lower dose group had a germ cell population up toprimary spermatocytes while the higher dose group had only Sertoli cells andspermatogonia.Withdrawal of treatment for 180 days led to the recovery of allthe parameters studied, to normalcy.

Introduction

Gossypol, a phenolic compound extracted from cotton seeds, has been wellestablished as a potential non-steroidal contraceptive for men [1,2]. Although itsfurther clinical testing has been questioned due to its hypokalemic e¡ect in men [3],studies are encouraging when potassium chloride is used as a supplement [4]. Othermajor areas of concern are: (1) its delayed reversibility or irreversibility, and (2) itsmode of action [2,5,6]. Studies on rats revealed that the drug a¡ects germ cells andthereby spermatogenesis and that the reversal of spermatogenesis may be related tothe dose and duration of drug administration and the degree of the e¡ects on di¡erentgerm cells [7,8]. As it has been shown already that there is species variation inresponse to gossypol [2], a further elaborate investigation was warranted in primates.Since such investigations are less feasible in humans, the present investigation wascarried out in langur monkeys, a primate which is close to humans anatomically andphysiologically [9^11].

Advances in Contraception, 1999; 15: 15^27.# 1999 Kluwer Academic Publishers. Printed in the Netherlands

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Materials and methods

Animals

Fifteen sexually mature and healthy male langur monkeys, Presbytis entellus entellus,6^7 years old and weighing 16^20 kg, were wild-caught from the area around Jaipurand housed in individual metal cages under seminatural captive conditions. Abalanced diet and water were provided ad libitum. The `guidelines for care and useof animals in scienti¢c research' [12] were strictly followed.

Drug

Highly puri¢ed gossypol acetic acid (99.9%) was used throughout.

Experimental design

Prior to the commencement of these experiments, at least three pretreatment semensamples were collected at ten-day intervals, to serve as individual reference controlvalues. The animals were then divided into the following three groups, eachconsisting of ¢ve animals:

Group I: Vehicle-treated controls.Group II: Animals treated orally with gossypol acetic acid at 7.5 mg/animal/day fora period of 180 days.Group III: Animals treated orally with gossypol acetic acid at 10 mg/animal/day fora period of 180 days.

Following 180 days treatment, the treatment was withdrawn from the animalsallowing recovery for a further period of 180 days.

Semen analysis

Semen samples were collected every fortnight by penile electro-ejaculation. Thephysical characteristics were evaluated following the WHO manual [13].

Scanning electron microscopy (SEM)

Spermatozoa were concentrated by mild centrifugation at 300g for 5 min, ¢xed in2.5% glutaraldehyde for 10 min and washed thoroughly in phosphate bu¡er (0.1 mol/L; pH 7.2) and distilled water. A thin ¢lm of spermatozoa was placed on a clearcoverslip, air dried, sputter coated with silver and observed under a scanning electronmicroscope (Philips SEM 501 B).

16 Sharma et al.

Testicular histology

Unilateral testicular biopsy samples were obtained in all the animals (Groups I^III)following treatment and recovery, under anesthesia with sodium thiopentone (25mg/kg body weight, iv) and processed for light microscopy using the routineprocedure.

Results

Semen analysis

During the course of treatment, a gradual decrease in the sperm count was observedin both dose regimens. Uniform severe oligospermia was observed in all ¢ve animalsin the lower dose group (7.5 mg), while azoospermia was observed in two out of ¢veanimals in the higher dose group (10 mg) and the remaining three animals showedsevere oligospermia between days 120 and 180 of the observation period (Figures 1and 2). Sperm motility also showed a similar trend (Figures 3 and 4).

SEM of spermatozoa

The voided spermatozoa of the treated animals of both the groups showed severedamage in the head and midpiece. The most common abnormalities were round ortapering heads, bent neck, bent midpiece, midpiece swelling and double tails.Occasionally, spermatozoa with normal morphology were also observed. Spermato-zoa obtained during pretreatment, from the control group and after recovery showednormal morphology (Figures 5^7).

Testicular histology

Testicular biopsies from the control/pretreatment samples showed round or oval-shaped seminiferous tubules and intertubular elements with all stages of spermato-genesis. Elongated spermatids were observed in association with Sertoli cells andspermatozoa appeared in the lumen. The Leydig cells appeared normal (Figure 8).

Treatment of gossypol acetic acid at both dose regimens resulted into total arrest ofspermatogenesis. However, a dose-dependent response was observed. In the 7.5-mgdose regimen, the seminiferous tubule diameter decreased (semiquantitatively) andcontained only a few Sertoli cells, spermatogonia and primary spermatocytes. Thegerminal epithelium was disorganized and the lumen contained germinal andphagocytic elements (Figure 9). The 10-mg dose regimen also led to a decrease inthe seminiferous tubule diameter and showed only Sertoli cells and spermatogonia.Phagocytosis in the lumen was evident. The Leydig cells appeared normal (Figure10).

Antispermatogenic E¡ect of Gossypol 17

Recovery of spermatogenesis was evident 180 days following withdrawal oftreatment. The seminiferous tubules attained normalcy and showed evidence ofresumed spermatogenesis with a few elongated spermatids (Figure 11).

Discussion

It has been suggested that the action of gossypol is mediated through the testis [14].The testicular germ cells of the animals treated with gossypol revealed a gradualdamage leading to pyknosis, vacuolization and cytolysis in the spermatids and

Figure 1. Sperm count of control and gossypol at 7.5 mg/day, oral, treated langur monkeysduring the pretreatment, treatment and recovery phases. The values for individual animalshave also been shown

18 Sharma et al.

spermatocytes. Continued treatment resulted in almost complete disappearance ofthe mid and late stages of spermatids and spermatocytes [15^17]. Degenerativechanges in the Sertoli cells have also been reported [18,19]. However, Leydig cellsseem to be una¡ected [14].

It has also been reported that the speci¢c subcellular target for gossypol action isthe mitochondria of the spermatogenic cells [14] and that the spermatozoa derivedfrom the testis of the animals treated with gossypol show remarkable damage in themitochondrial sheath [18,19]. Inhibition of the testicular mitochondrial respiratorychain [20], inhibition of the glycolytic pathway and the citric acid cycle due toinhibition of selective enzymes, particularly the testis and sperm-speci¢c LDH C4

[21^23] and inhibition of mitochondrial ATP production in isolated hamsterspermatids [24] due to gossypol treatment have also been well documented,

Figure 2. Sperm count of control and gossypol at 10 mg/day, oral, treated langur monkeysduring the pretreatment, treatment and recovery phases. The values for individual animalshave also been shown

Antispermatogenic E¡ect of Gossypol 19

suggesting that the mitochondria and their energy production are the speci¢c targetsfor the action of gossypol on spermatogenic cells.

Our earlier studies revealed a dose-dependent response in langur monkeys treatedwith gossypol. The 5-mg dose regimen revealed severe oligospermia, impairment ofsperm motility, morphological defects in the spermatozoa and hypokalemia [6]. Inthe present study, treatment with gossypol (7.5 mg and 10 mg) in langur monkeysrevealed azoospermia in two animals in the higher dose group and severe oligosper-mia with impaired motility in the remaining animals. The voided spermatozoashowed deleterious abnormalities. The morphology of the testis also revealed adose-dependent arrest of spermatogenesis. The former (7.5 mg dose) showed germcells up to primary spermatocytes, while the latter (10 mg dose) showed only thepresence of spermatogonia and Sertoli cells. Recovery of spermatogenesis wasdemonstrated by the testicular morphology and by the increase in sperm count andsperm motility in the ejaculation.

Figure 3. Sperm motility of control and gossypol at 7.5 mg/day, oral, treated langur monkeysduring the pretreatment, treatment and recovery phases, along with the values for individualanimals

20 Sharma et al.

There are two suggested possible mechanisms of action of spermatogenic arrestdue to the treatment used in the present study. The predominant midpiece defects inthe voided spermatozoa of the treated langur monkeys suggest that the selectiveinhibition of mitochondrial activity in the germ cells may inhibit the respiratorychain, generating a cytotoxic e¡ect on the developing germ cells and thus preventingfurther proliferation of germ cells [20]. Secondly, the arrest of spermatogenesis in thetreated testis suggests that there is hormonal mediation, as it is well established thatthe process of spermatogenesis, and more particularly spermiogenesis, is controlledby testosterone [25]. It is also reported that the gossypol inhibits testicularsteroidogenesis and that the oligospermia and azoospermia induced by gossypol

Figure 4. Sperm motility of control and gossypol at 10 mg/day, oral, treated langur monkeysduring the pretreatment, treatment and recovery phases, along with the values for individualanimals

Antispermatogenic E¡ect of Gossypol 21

Figure 5. SEM of the spermatozoa of control langur monkeys showing normal morphology.Magni¢cation 64000

Figure 6. SEM of spermatozoa of langur monkeys treated with gossypol at 7.5 mg/day, oral,showing round and tapering heads and bent midpiece. Magni¢cation 62000

22 Sharma et al.

Figure 7. SEM of spermatozoa of langur monkeys treated with gossypol at 10 mg/day, oral,showing round and amorphous heads, bent midpieces and double tails. Magni¢cation 62000

Figure 8. Histology of the testis of a control langur monkey showing normal spermatogenesis.Magni¢cation 6100

Antispermatogenic E¡ect of Gossypol 23

Figure 9. Histology of the testis of a langur monkey treated with gossypol at 7.5 mg/day for 180days. The seminiferous tubule diameter is decreased. Germinal epithelium shows only Sertolicells, spermatogonia and few spermatocytes. The lumen contains epithelial and phagocyticelements. Leydig cells appear normal. Magni¢cation 6100

Figure 10. Histology of the testis of a langur monkey treated with gossypol at 10 mg/day for180 days. The seminiferous tubule diameter is decreased. Germinal epithelium shows onlySertoli cells and spermatogonia. Leydig cells appear normal. Magni¢cation 6100

24 Sharma et al.

may be secondary to decreased testosterone function [26]. However, the unalteredserum testosterone levels in our earlier studies [6] and the arrest of spermatogonialproliferation in the present study suggest that the former mechanism is likely.

One of the essential criteria of an ideal male contraceptive is its ready reversibility[25]. The major drawback of gossypol as a male contraceptive is its delayedreversibility [2,5,6]. The chances of reversal of gossypol treatment depend on thedose and duration of the treatment. As long as the spermatogonia remain una¡ected,reversal is possible in the subsequent spermatogenic cycle. Once the spermatogoniaare a¡ected, due to overdose or prolonged treatment, permanent sterility may result[2,14,27]. In the present study, the dose chosen, i.e. 7.5 mg and 10 mg, and theduration, 180 days, kept the spermatogonia population una¡ected; hence, reversal ofspermatogenesis was within 180 days after withdrawal of the treatment.

It is suggested that the dose of gossypol should be selected in such a way that itcauses oligospermia with motility impairment. Such a target would provide long-term contraception with ready reversal. Prolonged azoospermia, however, may leadto delayed reversal or permanent sterility. Further studies are in progress tosubstantiate this view.

Figure 11. Histology of the testis of a langur monkey 180 days after withdrawal of treatment.Note the increased seminiferous tubule diameter and normal spermatogenic cells. Magni¢ca-tion 6100

Antispermatogenic E¡ect of Gossypol 25

Acknowledgements

This investigation was ¢nancially supported by the Indian Council of MedicalResearch (ICMR), New Delhi. The samples of highly puri¢ed gossypol acetic acidwere kindly supplied by WHO under its Special Programme of Research in HumanReproduction. The ultrastructural studies were carried out at the sophisticatedinstrumentation facility for Electron Microscopy, All India Institute of MedicalSciences, New Delhi.

References

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2. Prasad MRN, Diczfalusy E. Gossypol. Int J Androl. 1982;5(suppl):53^70.3. Waites GMH. Male fertility regulation. In: Diczfalusy E, Gri¤n PD, Khanna J, eds. Research in

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4. Kumar M, Sharma S, Lohiya NK. Gossypol-induced hypokalemia and role of exogenous potassiumsalt supplementation when used as an antispermatogenic agent in male langur monkey. Contraception.1997;56:251^6.

5. Ho¡er AP, Agrawal A, Meltzer P, Naqvi R, Matlin SA. Antifertility, spermicidal and ultrastructurale¡ects of gossypol and derivatives administered orally and by intratesticular injections. Contraception.1988;37:301^31.

6. Lohiya NK, Sharma K, Kumar M, Sharma S. Limitations in developing gossypol acetic acid as a malecontraceptive. Contraception. 1990;41:519^32.

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18. Ho¡er AP. Ultrastructural studies of spermatozoa and epithelial lining of the epididymis and vasdeferens in rats treated with gossypol. Arch Androl. 1982;8:233^46.

19. Oko R, Hrudka F. E¡ect of gossypol on spermatozoa. Arch Androl. 1982;9:39^43.20. Kim I, Waller DP. Speci¢c inhibition of the testicular mitochondrial respiratory chain in vitro by

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MSreceived 9 Feb. 99.Accepted for publication 22March 99.

Resume

La presente enqueª te porte sur l'e¡et inhibiteur de la spermatogene© se de l'acide acetique gossypol hautementpuri¢e et actif lorsqu'il est administre oralement a© des doses de 7,5 et 10 mg par jour pendant 180 jours a©des singes entelles. Les resultats ont mis en evidence que la reponse, au niveau de l'analyse du sperme et dela morphologie des testicules, dependait de la dose. On a observe une forte oligospermie uniforme dans legroupe a© faible dose (7,5 mg), tandis que dans le groupe a© forte dose (10 mg), deux sujets sur cinqpresentaient une azoospermie et le restant des animaux une forte oligospermie. La microscopie a© balayageelectronique des spermatozo|« des a revele des anomalies delete© res de la teª te et du col. La morphologie destesticules a revele une reduction du diame© tre des tubes seminife© res et l'arreª t de la spermatogene© se. Dans legroupe a© faible dose, on a observe une population de cellules germinales jusqu'au stade de spermatocytesprimaires tandis que le groupe a© forte dose indiquait seulement la presence de cellules Sertoli et despermatogonies. Apre© s l'arreª t du traitement pendant 180 jours, tous les parame© tres etudies s'etaient retablisnormalement.

Resumen

Esta investigacion presenta el efecto antiespermatogenico del acido acetico de gosipol altamente puri¢cadoy oralmente activo a dosis de 7,5 mg y 10 mg diarios durante 180 d|as en monos ``langur''. Los resultadosrevelaron una respuesta relacionada con la dosis en el analisis del semen as| como en la morfolog|atesticular. Se observo una fuerte oligospermia uniforme en el grupo de dosis mas baja (7,5 mg), mientrasque se observo azoospermia en 2 de cada 5 animales del grupo de dosis mas alta (10 mg) y los animalesrestantes presentaron una fuerte oligospermia. La microscopia electronica por barrido de espermatozoidesrevelo anomal|as deletereas en la cabeza y la seccion media. La morfolog|a testicular revelo unadisminucion del diametro de los conductos semin|feros e interrupcion de la espermatogenesis. El grupode dosis mas baja mostro la poblacion de celulas germen hasta espermatocitos primarios, y el grupo dedosis mas alta mostro solo la presencia de celulas de Sertoli y espermatogonia. La interrupcion deltratamiento durante 180 d|as revelo la recuperacion de todos los parametros estudiados, hasta lanormalidad.

Antispermatogenic E¡ect of Gossypol 27