Antifertility effects of gossypol in male hamsters*t

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1 FERTILITY AND STERILITY Copyright ~ 1982 The American Fertility Society Vol. 37, No.6, May 1982 Printed in U.s.A. Antifertility effects of gossypol in male hamsters*t Shiva K. Saksena, Ph.D.t Rebecca A. Salmonsen, M.A. Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts In the male hamster, administration of 10 or 15 mg/kg/day of gossypol for 5 weeks did not alter the serum concentration of testosterone (T), prolactin (PRL), and luteinizing hormone (LH) or fertility. However, the total sperm population was significantly reduced in the males treated with 15 mg/kg/day of gossypol, and total sterility occurred within 8 weeks. On the other hand, males treated with of gossypol were sterile after 10 weeks of treatment. The T and total sperm population along the reproductive tract in these infertile males was significantly reduced. During the treatment period no change in body weight or weight of testes or seminal vesicles was observed. A full regaining of fertility, serum T level, and sperm population to the normal range were recorded within 8 and 14 weeks (15 and, respectively) after the cessation of treatment. Fertil Steril 37:686, 1982 Oral administration of gossypol, a polyphenolic compound derived from the cotton plant, has been reported to induce temporary infertility in males of several mammalian species. I - 8 Among the various laboratory animal species studied, the incidence of infertility in males was dose-related. 1, 3 The ability of gossypol to cause infertility was attributed to its effects on spermatogenesis and/or widespread damage to epididymal sperm. 2, 9,10 Only minimal side effects both in experimental animals and human beings were observed. However, species and strain differences toward the action of gossypol were also encountered. 5, 7 In a Received November 5, 1981; revised and accepted January 5,1982. *Presented at the Thirty-Eighth Annual Meeting of The American Fertility Society, March 20 to 24,1982, Las Vegas, Nevada. tsupported in part by a subcontract (PARFR-31O) under a Prime Contract from Aid International Development (AID) and a grant from the National Institutes of Health (HD ). :j:reprint requests: Shiva K. Saksena, Ph.D., Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts Saksena and SaImonsen in male hamsters preliminary report where the antifertility effects of orally administered gossypol in male rats, hamsters, and rabbits were studied, it was found that the hamster was more sensitive to the antifertility effects but less sensitive to the toxic effects of gossypol than was the male rat. In contrast, gossypol failed to induce any antifertility effect in the male rabbit. 5 In view of the urgent need for an effective and reversible male antifertility agent, it was our objective to (1) study the dose- and time-related effects of gossypol on sperm population and fertility of male hamsters and (2) monitor the circulating testosterone (T), luteinizing hormone (LH), and prolactin (PRL) levels in gossypol-treated hamsters. ANIMALS MATERIALS AND METHODS Male Syrian hamsters (- 95 gm) purchased from Charles River Breeding colony were housed three animals per cage in an artificially illuminated (14 hours of light) and temperature-controlled (22 ± 1 0 C) room. Purina Chow (Ralston

2 Purina Co., St. Louis, MO) and water were provided ad libitum. After 10 days of acclimatization a male was paired overnight with one proestrous female. Only those males who had successfully sired a litter and attained a weight ranging between 110 to 115 gm were used in this study. EXPERIMENTAL DESIGN acetic acid (G-4382, Sigma Chemical Co., St. Louis, MO) was suspended in steroid suspending vehicle. 5 Groups of 12 fertile male hamsters were fed daily with 10 or 15 mg/kg/day gossypol in 0.2 ml of vehicle. Another group of 12 hamsters was fed with the vehicle and served as controls. The animals were checked weekly for body weight. Between 9:00 A.M. and 9:30 A.M. on the 34th or 35th day of treatment, six males from the control and treatment groups were decapitated within 10 seconds of removal from the cage for blood collection. Blood was allowed to clot at 4 C, and an aliquot of 0.5 ml serum was stored in glass scintillation vials at - 20 C until assayed for T. The remaining portion of the serum was frozen separately for LH and PRL determinations. Similarly, six hamsters from control and experimental groups were decapitated between 7 and 8 weeks (15 mg/kg/day) and 9 and 12 weeks (10 mg/kg/ day). The time selection was based on the fact that the treated males were found to be sterile during 6 to 7 weeks and 9 to 10 weeks after treatment with 15 and of gossypol, respectively. The blood serum was procured as described earlier. After blood collection, the seminal vesicles, testes, and epididymides were cleaned of adhering tissue, and the weights were recorded. After flushing the vas deferens with 1 ml of saline and macerating the caput and cauda epididymis separately in saline, the sperm population of one side of the reproductive tract was determined with a hemocytometer. 11 FERTILITY TEST Groups composed of five male hamsters of pro v en fertility were fed daily either 10 or 15 mg/kg/ day gossypol in 0.2 ml vehicle. Every 3rd week each treated male was paired with a proestrous female for 8 days, and the female was autopsied 7 days after cohabitation. Males recovered from gossypol-induced sterility were decapitated after siring two litters. The blood was procured for T, PRL, and LH determinations. The organ weights and sperm population along the reproductive tract were also studied. Vol. 37, No.5, May 1982 DETERMINATION OF T For blood serum T concentrations, tritiated T ( dpm in 0.1 ml phosphate buffer) was added to the thawed samples (0.5 ml) for the calculation of recovery losses. Extraction was accomplished by mixing the samples with 8 ml anhydrous diethyl ether. T was separated on microcelite columns and determined by radioimmunoassay (RIA).12 DETERMINATION OF LH AND PRL LH was determined in O.l-ml aliquots of serum by double-antibody RIA, using a modified procedure originally developed by Niswender et al.,13 and a rat LH kit which employed National Institute of Arthritis, Metabolic and Digestive Diseases Rat LH-RP-1 (NIAMDD-Rat LH-RP-1) as the reference standard and antiserum NIAMDD Anti-Rat-LH Serum-S4. PRL RIA was performed on 0.15 ml serum with the use of the same protocol as was used for LH assay. The antiserum employed was NIAMDD-Anti-Rat-Prolactin Serum-2, and the standard curve ranged from 0.25 to 50 ng/tube of NIAMDD-Rat Prolactin-RP-l. RESULTS Oral administration of 10 or 15 mg/kg/day of gossypol for 5 weeks neither induced sterility nor caused any change in the relative organ weights (ROW) of the testis, epididymis, or seminal vesicles in the fertile male Syrian hamster, as compared with the vehicle-treated controls (Table 1). However, after 10 weeks of daily treatment of 10 mg/kg/day of gossypol, the male became sterile and showed a significant decrease (P < 0.05) in the ROW of the epididymis, whereas no significant difference in the weights of testes and seminal vesicles was observed. Males treated with 15 mg/kg/day of gossypol lost their fertility within 7 to 8 weeks of treatment. Again, the ROW of the epididymis was significantly reduced (P < 0.01), while the testis and seminal vesicle weights were not affected. Treatment was terminated when animals were confirmed to be infertile by two sterile matings. Approximately 35 to 40 days after cessation of treatment the group that received regained fertility, but it took 45 to 50 days for the group that received 15 mg/kg/day gossypol to recover. In addition, the ROW of epididymis returned to normal. Interestingly, a significant increase in the weights of seminal vesicles in males treated with 15 mg/kg gossypol was encountered. Saksena and Salmonsen in male hamsters 687

3 Table 1. Relative Weights of Testis, Epididymis, and Seminal Vesicles of Male Syrian Hamsters Treated with Acetic Acid Sacrificed between Reproductive Relative organ weight (mg/ioo gm)a Treatment and dose days of treat- status ment Testis Epididymis Seminal vesicles Control Vehicle Fertile 1257 ± ± ± Fertile 1259 ± ± ± mg/kg/day Fertile 1192 ± ± ± 30 Control Vehicle Fertile 1192 ± ± ± Sterile 1084 ± ± 19 c 917 ± mg/kg/day Sterile 1016 ± ± 31d 952 ± 77 Control Vehicle 125 Fertile 957 ± ± ± 32 b Fertile 1006 ± ± ± mg/kg/day Fertile 1159 ± 25d 295 ± ± 37d aresults of six animals per group, mean ± standard error of the mean. btreatment stopped 70 () and 55 (15 mg/kg/day) days after initiation. CStatistically different from respective controls at P < 0.05, Student's t-test. dstatistically different from respective controls at P < 0.01, Student's t-test. The effects of gossypol fed daily for different intervals on PRL, LH, and T and their correlation with sperm population along the reproductive tract are depicted in Table 2. Although male hamsters treated with 10 or 15 mg/kg gossypol for 5 weeks remained fertile, the total sperm population along the reproductive tract was significantly reduced (P < 0.01) in the group given 15 mg/ kg/day (from 297 ± 44 to 74 ± 24 million). No significant changes in the concentrations of PRL, LH, and T were observed. Significant reductions in T (from 3.4 ± 0.4 to 1.8 ± 0.4 and 1.5 ± 0.2 ng/ml) and sperm (from 181 ± 42 to 59 ± 20 and 39 ± 15 million) in hamsters treated with 10 or 15 mg/kg/day gossypol. Approximately 60% of the epididymal sperm were found malformed, with detached heads, and completely immotile. When the gossypol-treated male hamsters regained their fertility, the concentration of T and the sperm population, motility, and morphologic features along the reproductive tract were comparable to vehicle-treated controls. Five weeks of daily feeding of the vehicle did not induce any deleterious effect on the fertility of male hamsters, since 100% (5 of 5) of the females mated to these males were found pregnant, with a number of implantation sites ranging between 10 to 15 per female (Table 3). On the other hand, 3 of 6 and 2 of6 of the females mated to males treated with 10 and 15 mg/kg/day of gossypol, respectively, did conceive. The number of implantation sites in these pregnant animals was between 6 and 13, a range obviously less than those recorded in the normal pregnant females. By 7 to 8 weeks of Table 2. Effects of Daily Oral Administration of Acetic Acid on Serum Prolactin (PRL), Luteinizing Hormone (LH), and Testosterone (T) Concentrations and Sperm Population in Mature Male Syrian Hamsters a Sacrificed between days of Treatment treatment PRL LH ng, RP-lIml ng, LER RP-llml Control Vehicle 9.2 ± 3 49 ± ± 4 48 ± mg/kg/day ± 1 37 ± 13.0 T nglml Total sperm population from one side of tract (in millions) 2.6 ± ± ± ± ± ± 24d Control Vehicle ± 3 51 ± ± 2 82 ± mg/kg/day ± 3 47 ± 19 Control Vehicle ± 2 80 ± 17 b ± 7 64 ± mg/kg/day ± 4 c 84 ± 24.3 aresults of six animals per group. btreatment stopped 70 () and 55 (15 mg/kg/day) days after initiation. CStatistically different from respective controls at P < 0.05, Student's t-test. dstatistically different from respective controls at P < 0.01, Student's t-test. 688 Saksena and Salmonsen in male hamsters 3.4 ± ± ± O.4 c 59 ± 20d 1.5 ± 0.2d 39 ± 15d 4.6 ± ± ± ± ± ± 36.4

4 Table 3. Induction of Sterility by Oral Administration of Acetic Acid and Its Reversal in the Male Syrian Hamster Treatment and dose Test of fertility Females pregnant % Number of viable left for mating embryosc Vehicle Control 5 weeks after 5/ , 14, 12, 10, 14 treatment 3/6 50 6,0,8,0,0,13 15 mg/kg/day 2/ , 9, 0, 0, 0, 0 Vehicle Control 7-8 weeks after 5/ , 13, 11, 9, 10 treatment , 3, 10, 0, 0, 0 15 mg/kg/day 0/6 0 0,0,0,0,0,0 Vehicle Control 10 weeks after 5/ , 15, 13, 11, 14 treatment , 3, 0, 0, 10, 0 15 mg/kg/day 0/6 0 0, 0, 0, 0, 0, 0 b 12 weeks after 0/6 0 0,0,0,0,0,0 15 mg/kg/day" treatment 0/6 0 0, 0, 0, 0, 0, 0 b 16 weeks after 0/6 0 0,0,0,0,0,0 15 mg/kg/day" treatment 0/6 0 0, 0, 0, 0, 0, 0 Vehicle Control 20 weeks after 5/ , 10, 13, 13, 10 b treatment ,13,9,15,9,10 15 mg/kg/day" 3/5 d 60 9,12,0,0,7 Control 22 weeks after 5/ , 10, 11, 13, mg/kg/day" treatment 5/ ,12,7,8,12 "Treatment for 8 weeks. b>freatment terminated in the 10th week. "Females were caged with males individually for 8 days and then sacrificed 7 days later. done male died. treatment all males in the 15 mg/kg/day gossypoltreated group were sterile, so treatment was stopped, while the group still showed 50% fertility. Finally, after 12 weeks of treatment, sterility also occurred in gossypol-treated males, and treatment was therefore terminated. Matings were continued in order to establish the return offertility; and even by the 16th week of the experimental period, males in both treatment groups remained sterile. By the 20th week, the males given of gossypol regained 100% fertility with the normal number of implantation sites (9 to 15 per female), whereas the males treated with 15 mg/kg/day of gossypol impregnated 60% of the females, and the implantation sites ranged between 7 and 12. Twenty-two weeks after initiation of treatment the group given 15 mg/kg/day of gossypol showed 100% fertility. The number of implantation sites in the control group and the group given 15 mg/ kg/day of gossypol was 10 to 13 and 7 to 14, respectively. DISCUSSION, a yellow disequiterpene aldehyde from the cotton plant, has been reported to induce temporary sterility in males.14 The present re- Vol. 37, No.5, May 1982 suits demonstrate that gossypol acetic acid given orally caused a dose- and time-dependent temporary infertility in the male hamster. Daily treatment of of gossypol induced total sterility in hamsters within 10 weeks, whereas at 15 mg/kg/day of gossypol sterility occurred in 7 to 8 weeks. The reversal process, however, was inversely proportional to the dose administered. A period of 5 or 6 to 7 weeks was needed for animals to regain fertility when they had been treated with 10 and 15 mg/kg/day of gossypol, respectively. In all cases, the normal number of pups were sired. Many theories have been put forward to explain gossypol-induced sterility in the male: marked decrease in epididymal sperm,2, 9, 10 decrease in adenosine triphosphatase (ATPase) activity of the human spermatozoa,15 cellular changes and degeneration of spermatids and spermatocytes,14, 16 injury to the seminiferous tubes,14 and chelation of zinc in the male reproductive tract, leading to the inhibition of spermatogenesis.17 The onset of sterility in the present study was associated with a substantial suppression of T levels, a phenomenon observed in rats10 and in rabbits.5 Contrary to these observations, the Chinese researchers reported no apparent changes in the interstitial cells, in histochem- Saksena and Salmonsen in male hamsters 689

5 ical or morphologic appearance in the gossypoltreated male reproductive tract or in the circulating T, 5a-dihydrotestosterone, and luteinizing hormone-releasing hormone (LH-RH) levels.! The results of this study are also in agreement with those reported by Chinese investigators with regard to a decrease in both sperm production and sperm motility after gossypol treatment. Similarly, male rats fed with 15 to 40 mg/kg of gossypol for 2 to 4 weeks produced headless sperm. In addition, spermatids and spermatocytes were found in the cauda epididymis and vas deferens.!4 Since a low number of sperm were observed in the ejaculates 6 and along the reproductive tract of the infertile males (Table 2), it appears that gossypol probably does not completely inhibit spermatogenesis. However, the incidence of low epididymal sperm count, the presence of deformed and nonmotile spermatozoa, and a significant reduction in the epididymal weight imply that gossypol-induced infertility might be a consequence of an array of factors, including both primary and secondary effects of the actions of gossypol. Our results indicate that the levels of LH and PRL in the gossypol-treated infertile hamsters remained comparable to those of the vehicletreated males (Table 2). Also, there was no impairment of weight gain in males treated with 10 mg/kg/day; while one out of six males treated with 15 mg/kg/day gossypol died. However, a significant gain in body weight was evident when the treatment was discontinued. In another study using hamsters, toxicity became evident (40%) as 20 mg/kg/day of gossypol was administered. 7 In spite of a suppressed T, the sterile males induced pseudopregnancy in females, as evidenced by the absence of estrous discharge. This suggests that a suppressed T level after gossypol treatment was adequate to maintain sexual behavior. In view of these diversified results obtained from different laboratories, it is imperative to conclude that more work is needed to improve the treatment regimen of gossypol and to understand its exact mechanism and site(s) of action. However, the substantial reduction in sperm number before suppression of serum T levels does support the suggestion of direct effects of gossypol on sperm production or on epididymal spermatozoa. Likewise, gossypol might exert a direct inhibitory effect on testicular steroidogenesis, 5 because of the lack of an alteration of the LH levels in spite of a reduced T level. Acknowledgments. Thanks are due to Dr. M. C. Chang for his interest in this study and to Ms. Dorothy Jamieson for typing the manuscript. REFERENCES 1. National Coordinating Group of Male Antifertility Agents: : a new antifertility agent for males. 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