Effect of gossypol on spermatozoal lactate dehydrogenase-X (LDH-X) in male rats

The studies in this report evaluate the inhibitory effects of long-term gossypol acetic acid (gossypol) administration on spermatozoal lactate dehydrogenase (LDH-X) activity, fertility and reversibility. Gossypol at 20 mg/kg/day or vehicle only was administered orally to adult male rats for 5 consecutive weeks. Groups of control and gossypol-treated rats were sacrificed at 2-, 4-, and 5-week intervals when LDH-X analysis of spermatozoa were prepared from the tail of the epididymis. An additional group of control and gossypol-treated rats were allowed to recover for up to 8 weeks after withdrawal of the treatment. In the gossypol-treated rats, the spermatozoal LDH-X activity was depressed by 80% of control level after a 5-week treatment period. There was no significant difference in spermatozoal LDH-X activity between the control and treated animals at 2 and 4 weeks. However, the number of ejaculated sperm estimated from the vaginal smear preparations after each mating was significantly less than the control values after 2, 4 and 5 weeks of treatment. At 5 weeks after the cessation of gossypol treatment, the spermatozoal LDH-X activity had only partially recovered but the fertility (number of ejaculated sperm, embryo implantation sites and pups born) had recovered to a level comparable to the control values 5 weeks after the cessation of gossypol treatment. The inconsistencies in the response of spermatozoal LDH-X activity, sperm number and fertility to gossypol may suggest that the antifertility action of gossypol and spermatozoal LDH-X activity in adult male rats may not be directly related as suggested by the results generated from the in vitro studies.     

Antifertility, spermicidal and ultrastructural effects of gossypol and derivatives administered orally and by intratesticular injections

Because there are problems, in men, associated with the use of gossypol related to reversibility and, infrequently, hypokalemia, several laboratories around the world have resorted to the synthesis and evaluation of experimental analogs and optical isomers of gossypol in an attempt to find a compound which retains its pharmacologically desirable antifertility effects while eliminating its suboptimal ones. The present study documents: (a) the effects of fourteen new, orally-administered synthetic analogs of gossypol on testicular ultrastructure and fertility in hamsters, (b) the in vitro effects of these compounds as well as of the optical isomers of gossypol against hamster and human sperm, and (c) the morphological and antifertility effects of intratesticular injections of gossypol-PVP and its optimal isomers in the rat. The results of the study demonstrate that these new analogs are not effective as male antifertility agents and that their in vitro activity is unrelated to their in vivo contraceptive potential. In addition, this report establishes the validity of the intratesticular injection model for the analysis of the mechanism of action of gossypol and its analogs by making these compounds directly available at the testicular site. The significance of these findings is discussed.     

Inhibition by (+) and (-) isomers of gossypol of testosterone release from mouse Leydig cells in vitro

Racemic (+/-) mixtures of gossypol isomers exert an antifertility effect by inhibiting sperm motility and spermatogenesis. Purified (+) gossypol has been shown to be without these actions. In this study pure preparations of both (+) and (-) gossypol were found to inhibit, in a similar manner, both basal and LH-stimulated release of testosterone by isolated Leydig cells at concentrations down to 21 microM. It is possible that use of low doses of pure (-) gossypol could inhibit fertility with less endocrine side effects.     

Studies on mechanism(s) of antifertility action of gossypol in rat and hamster

This study was undertaken with a view to investigate the possible mechanism(s) of antifertility action of gossypol acetate in rats and hamsters. Adult male rats were treated by gavage with 30 mg/kg/day of gossypol for 7 weeks and adult male hamsters were treated similarly with 20 mg/kg/day gossypol for 8 weeks. The treatment caused a marked reduction in the weights of testis and epididymis. Histological examination of the testis in the two species revealed presence of seminiferous tubules showing varying degrees of damage along with a large number of normal tubules. Exfoliation of germ cells and spermatogenic arrest at spermatid stage was a common feature. Leydig cells presented normal morphological features. Though there was a reduction in the diameter of epididymal tubules, the epithelium did not show any morphological alterations. Examination of vasal flushings revealed marked reduction in sperm population and consisted of decapitated and immotile spermatozoa. Gossypol caused a significant reduction in the levels of total protein, RNA and DNA, and a marginal decrease in glycogen content in the testis. This was accompanied by a reduction in the activities of SDH and MDH. Except for LDH activity which showed a marked rise, there was no effect on glycolytic enzymes in the testis. The concentrations of glycerylphosphorylcholine and sialic acid were reduced in the cauda epididymis. The antifertility effects of gossypol appear to be due to its action both on testis as well as on epididymis.     

Gossypol: Its toxicological and endocrinological effects in male rabbits

The effects of different doses of orally administered polyphenolic compound ‘Gossypol’ on semen quality, circulating testosterone (T) and fertility of Dutch-belted male rabbits were studied. Bucks fed daily with 80, 40, 20 mg/kg/day gossypol died within 8–17, 23–35 or 35–84 days, respectively, after inicitation of treatment. Following gossypol treatment at 80, 40 or 20 mg/kg/ day, animals lost appetite and body weight, developed hind limb paralysis, breathing difficulties and collapsed while sitting in their cages. At autopsy, the liver and lungs were found congested while the stomach and intestines contained gases. On the other hand, rabbits fed daily with 10 mg/kg/day gossypol exhibited a survival time ranging from 77 to 250 days. Despite the severe side effects resulting in eventual deaths, weekly semen samples from all treated animals did not show any apparent change in sperm motility, morphology or population. Likewise, gossypol-treated males mated to estrous does exhibited a fertility comparable to vehicle-treated controls. Gossypol fed at a dose of 10 mg/kg/day for up to 35 weeks failed to induce sterility. Male rabbits, fed with either 20 or 10 mg/kg/day gossypol, that survived for longer periods of time had substantially reduced T levels by 12–20 weeks depending upon the dose but were fertile at all times. When the $\text{in vitro}$ release of T from the rat testes mince in the presence of hCG and gossypol was evaluated, an inhibition of T release was recorded. It is concluded that although gossypol has been shown to be an effective antifertility agent in several mammalian species, it failed to exhibit such an effect in Dutch-belted rabbits, although serum T levels were reduced. In addition, gossypol exhibited a wide spectrum of toxicity. The $\text{in vitro}$ study demonstrated that high concentration of gossypol can directly inhibit the T synthesis in the testis.     

Involvement of prostaglandin in the antifertility effects of gossypol

This study was undertaken to determine the effects of gossypol alone and gossypol in combination with prostaglandin and aspirin. Rats were administered gossypol (40 mg/kg/day), gossypol and prostaglandin (PGF2 alpha-2 mg/kg/day), gossypol and aspirin (300 mg/kg) for 4 weeks. A marked effect of the gossypol-prostaglandin combination was observed on sperm motility and spermatogenesis. The effect of the gossypol-aspirin combination was less pronounced. The ratio of body weight to testicular and epididymal weights between the different groups showed no marked difference. No effect of drug treatment on plasma testosterone, LH and FSH was observed. The data presented in this paper suggest that prostaglandin plays an important role in the antifertility effects of gossypol.     

Effects of tamoxifen metabolites on fertility of male rat

The effects of chronic oral administration of tamoxifen citrate, at a dose of 0.4 mg/kg/day, were compared to those of subcutaneous (s.c) administration of tamoxifen citrate, 4-hydroxy tamoxifen, N-desmethyl tamoxifen and intermittent oral tamoxifen administration on the fertility of the male rat and its post reversal progeny. The fertility parameters of 120 day-treated male rat sires from all groups and post reversal male F1 progeny of tamoxifen-treated sires were assessed. Chronic tamoxifen treatment via oral or s.c. routes reduced the fertility of the male rat, weights of accessory sex glands, serum luteinizing hormone, and testosterone levels without altering potency or sperm counts. However, antifertility effects of s.c. treatment were comparatively more consistent than those of oral treatment. 4-hydroxy and N-desmethyl tamoxifen failed to produce significant antifertility effects in the male rat. The antifertility effects of intermittent oral treatment were more sustained than those of chronic oral tamoxifen treatment. It is inferred that hepatic metabolism of tamoxifen interferes with its antifertility effects via oral route and that the parameters affected by chronic oral exposure in the male sires are completely reversed in progeny ensuing after an adequate period of drug withdrawal.     

Antifertility effect of polyvinylpyrrolidone-gossypol and gossypol in male rats

Thirty-nine adult male Wistar rats were administered 20 mg/kg or 24.3 mg/kg polyvinylpyrrolidone-gossypol (PVP-G) and 20 mg/kg gossypol acetic acid (G-A) 6 times a week for 6 weeks. Mating test was taken to evaluate the antifertility effect. Caudal sperm count, body weight, sex and accessory sex organ weights were recorded. At the end of 6 weeks treatment, the recovery period of 6 weeks was investigated. The results showed that the onset of antifertility action and recovery with PVP-G are faster than with G-A.     

Comparative in vitro spermicidal effects of (+)-gossypol, (+)-gossypol, (−)-gossypol and gossypolone

The comparative $\text{in vitro}$ spermicidal effects of (+)-gossypol, (?)-gossypol and (+)-gossypol were evaluated on the spermatozoa of human, monkey, rabbit, mouse, rat and hamster. The spermicidal effects of gossypol isomers were also compared with those of gossypolone, which is a proposed major metabolite of gossypol. Gossypol isomers and gossypolone were all spermicidal. (+)- and (?)-Gossypol demonstrated spermicidal activities at the same concentration at which (+)-gossypol shows spermicidal effects on the spermatozoa of all species tested. However, gossypolone was less potent than the gossypol isomers. The spermicidal action of gossypol may be a nonspecific effect unrelated to the antifertility mechanism of orally administered gossypol, since (+)-gossypol which is not an effective male antifertility agent also showed the equivalent spermicidal effect to that of (+)-gossypol.     

Mode of action of the antifertility sulphonamides: Lack of effects on folate metabolism

The effects of some antifertility sulphonamides on folate metabolism were investigated in the male rat. Subcutaneous injections of sulphanilamide at a dose of 150 mg/kg/day for 6 weeks produced a marked reduction in fertility of the treated animals. This effect was rapidly recovered by one week after drug withdrawal. Similar treatments with trimethoprim (30 mg/kg/day) or pyrimethamine (8 mg/kg/day) had virtually no effect on fertility. The synergistic effect of trimethoprim or pyrimethamine on the antifertility activity of sulphanilamide was not observed when the drugs were administered in combinations. Treatment with sulphapyridine (450 mg/kg/day for 6 weeks) failed to alter the levels of folate in the blood and the reproductive organs except the testes in which accumulation of folic acid occurred. The results suggest that the antifertility activity of sulphanilamide, sulphapyridine and perhaps some other sulphonamides is not associated with the inhibition of folate metabolism.     

Evaluation of STS-557 as an oral contraceptive in male rat

To examine the nature and site of post-testicular antifertility action of STS-557 (17 alpha-cyanomethyl-17 beta-hydroxy-estra-4,9-dien-3-one), male rats were given the steroid orally daily for 60 days. In doses of 1 and 5 mg per animal per day it had no effect on fertility at the end of 3 weeks of treatment. When the treatment was extended for 60 days, spermatogenic arrest and loss of libido were evident in animals treated with 5 mg dose; animals receiving 1 mg dose of steroid showed no decrease of spermatogenesis or sexual activity and their fertility remained unaffected. In 35-day-old growing rats the steroid produced inconsistent effects on spermatogenesis after a 15-day treatment period at 1 and 5 mg doses. Both in adult and in growing rats the steroid caused a significant reduction in the weights and secretory function of the epididymis and other accessory sex organs; a dose-dependent response was seen in all the sex organs. Evaluation in castrated rat model revealed that STS-557 is a weak anti-androgen. Although this steroid is a potent inhibitor of spermatogenesis, its inhibitory effect on Leydig cell function is a contraindication for its use as a male oral contraceptive.     

Synthesis and assessment of fertility-regulating potential of 2-(2'-chloroacetamidobenzyl)-3-(3'-indolyl) quinoline in adult rats as a male contraceptive agent

BACKGROUND: The purpose of this study was to investigate the fertility-regulating potential of the compound 2-(2'-chloroacetamidobenzyl)-3-(3'-indolyl) quinoline in male rats. STUDY DESIGN: Rats of proven fertility were treated with the compound by oral gavage for 1 to 8 consecutive weeks. Functional fertility, testicular, epididymal and seminal vesicular weight, epididymal sperm count and spermatogenesis were quantitated. Reproductive hormones and some biochemical parameters were measured. RESULTS: Functional fertility was reduced significantly as revealed by a fall in fertility and pregnancy rate. The weight of the reproductive organs was reduced significantly. A reduction of sperm count and number of different types of testicular cells was observed. The treatment with the compound resulted in decline of testosterone and an increase of FSH hormone levels. The compound effectively reduced testicular protein, glycogen and epididymal glyceryl phosphorylcholine. Increase in testicular alkaline phosphatase and cholesterol was also observed. Fertility and other effects were regained gradually after cessation of treatment. CONCLUSION: The results revealed from the study indicate that the compound has reversible antifertility activity and can be explored as male contraceptive agent.     

Inhibition of lactate dehydrogenase-X by imino-derivatives of gossypol:structure activity relationship

Six imino-derivatives (II, III, IV, V, VI, VII) of gossypol (I) have been synthesized, and their effects were evaluated on the purified mouse lactate dehydrogenase-X. Three of these derivatives (V, VI, VII) with aldehyde groups substituted with hydrophobic functionalities showed equivalent or more inhibitory effects on lactate dehydrogenase-X than gossypol, whereas three other derivatives (II, III, IV) with aldehyde groups substituted with hydrophilic functional groups lost the ability to inhibit lactate dehydrogenase-X. It is suggested that two aldehyde groups of gossypol are not essential to inhibit lactate dehydrogenase-X. Furthermore, the hydrophobic property of the gossypol molecule seems to play a more important role in inhibiting lactate dehydrogenase-X. Therefore, lactate dehydrogenase-X inhibition by gossypol may not be associated with its antifertility mechanism, because the aldehyde group of gossypol is known to be required for its antifertility effect.     

Combined administration of low-dose gossypol acetic acid with desogestrel/mini-dose ethinylestradiol/testosterone undecanoate as an oral contraceptive for men

To evaluate the efficacy and feasibility of a new regimen of low-dose gossypol acetic acid (GA) combined with desogestrel/ethinylestradiol and testosterone undecanoate (DSG/E/TU) as a male contraceptive, adult male rats were fed orally with GA (12.5 mg/kg/day) and DSG (0.125 mg/kg)/E (0.025 mg/kg)/TU (100 mg/kg) daily for 8 weeks as loading dose until infertility, and a similar low dose of GA alone for infertility maintenance. Control animals were administered a single low dose of GA (12.5 mg/kg/day) or DSG (0.125 mg/kg)/E (0.025 mg/kg)/TU (100 mg/kg), and vehicle, respectively. Results demonstrated that the combined dosage regimen could damage epididymal sperm motility and density, and induce infertility within 8 weeks in rats; the infertility could be consistently sustained by giving single GA (12.5 mg/kg/day), and was reversible in about 8 weeks following withdrawal of gossypol. The regimen rendered treated male rats with spermiation failure within a period of 6-20 weeks of treatment. Also, the serum luteinizing hormone, follicle-stimulating hormone and testicular interstitial fluid testosterone levels showed a transient decrease at the end of 6 or 8 weeks, which returned to control levels after 8 weeks of recovery phase. No hypokalemia or other adverse effects in viscera were observed. These results provide a promising approach to using the new regimen for the development of an effective and reversible oral male contraceptive.     

Antifertility effect of sulfasalazine in the male rat

Sulfasalazine, which has been used for treatment of ulcerative colitis in man, caused a dose-dependent and reversible reduction in fertility of the male rat. By five weeks after forced feeding with sulfasalazine in corn oil at daily doses of 300, 450 and 600 mg/kg, the fertility decreased to 60.9, 35.5 and 26.8% of the control rats, respectively. Besides, the number of cohabited female being successfully inseminated was significantly reduced especially at high dose. However, complete recoveries of the mating behavior and fertility were evident by three weeks after drug withdrawal. Number of spermatozoa in the caput, the corpus and the cauda epididymides were not changed, but motility of spermatozoa collected from the cauda epididymides was significantly decreased. The body weight and the weights of testes, epididymides, seminal vesicles, prostate glands and coagulating glands as well as the concentration of plasma testosterone were not altered by five weeks after drug treatment at a dose of 450 mg/kg. This study shows that sulfasalazine also has an antifertility action in the male rat without affecting the blood androgens as previously reported in male patients.     

Effects of multiglycosides of Tripterygium wilfordii (GTW) on rat fertility and Leydig and Sertoli cells.

Primary cultures of rat Leydig and Sertoli cells were used to evaluate the direct effects of GTW on testicular cells and to compare these to the effects of gossypol acetate. Both GTW and gossypol acetate can affect the survival of Leydig and Sertoli cells. But Sertoli cells are much more sensitive than Leydig cells, either to gossypol acetate or GTW. Leydig and Sertoli cells all died when they were exposed to gossypol acetate or GTW at a dose of 3.0 micrograms/ml or 30 micrograms/ml, respectively, for 24 hours. The cell survival-time course demonstrated that the cell numbers were decreased after 2 hours, and especially so after 8 hours. No significant changes were observed in testosterone production in Leydig cells after 24 hours of exposure to 1.0-20 micrograms/ml GTW. The forward motility of epididymal spermatozoa was completely lost and fertility of rat was significantly inhibited after the treatment of GTW in vivo. It is concluded that GTW does affect the fertility of rat and viability of cultured rat Leydig and Sertoli cells.     

口服低剂量醋酸棉酚对男性抗生育效果的临床研究

目的:评估口服低剂量醋酸棉酚对男性抗生育效果的安全性和可逆性。方法:招募30名健康有生育力的男性志愿者进入研究,随机分为两组,首先均口服醋酸棉酚15mg/d达到生精抑制,然后两组分别口服醋酸棉酚7.5mg/d(A组)或10.0mg/d(B组)的维持剂量,共用药52周后停药观察52周,检测其精液参数、血钾含量、生殖激素水平和血液生化指标。另10名男性作为血钾对照组。结果:达到生精抑制时间为17.05±0.62周,抑制有效率为76%,效果维持率72.22%;停药52周88.89%生精功能完全恢复;A、B组比较均无统计学差异。用药期间A、B两研究组血钾值有波动,但无因低血钾而退出者。结论:口服低剂量醋酸棉酚的男性抗生育效果肯定,未发生低血钾麻痹,停药后生精功能恢复较好。     

Gossypol does not affect testicular fluid secretion in rats

Male rats fed with gossypol acetic acid at a dose rate of 20 mg/kg/day for 16 weeks were rendered infertile. Spermatozoa flushed out from the cauda epididymides completely lost their capacity to initiate forward motility. However, the rate of testicular fluid secretion measured by the efferent duct ligation technique was not affected by gossypol treatment. The sodium and potassium concentrations of the secreted fluid were found to be unchanged. It is concluded that at low antifertility doses, gossypol disrupts spermatogenic elements in the testis without affecting fluid secretion by the Sertoli cells.     

Effects of gossypol acetate on pituitary-adrenal axis in male albino rats.

Histopathological effects of gossypol acetate (GA) on pituitary-adrenal axis in male Wistar rats were investigated. Sexually mature rats of proven fertility were administered orally with 10 and 25 mg/kg for 4 and 5 weeks, respectively. In both experimental groups, corticotrophs (ACTH cells) of anterior pituitary showed progressive regression. At the 10 mg low-dose treatment for 4 weeks, no significant changes were observed in all the zones of the adrenal gland, while at the same dose for 5 weeks of treatment, cells of zona glomerulosa showed hypertrophy and degranulation as compared to that of control. Cells of zona fasciculata and reticularis showed no significant changes. In the medulla, the thickly granulated cells were degranulated and reduced in number. At the 25 mg treatment, the cells of the zona fasciculata showed hypertrophy and degranulation. The possible mechanism of action of GA is discussed.     

(-)-Gossypol: an active male antifertility agent

The enantiomers of gossypol have been resolved by preparative HPLC of diastereomeric Schiff's base derivatives on a chiral bonded phase. Whereas (+)-gossypol has previously been reported to be inactive, (-)-gossypol is now shown to be active as a male oral antifertility agent in hamsters.