Spermatogenic events in rat and mice following intratesticular administration of optical isomers of gossypol

Male rats and mice were administered racemic and dextrorotatory gossypol intratesticularly at the dose of 200 micrograms/testis. In a separate experiment, 100 micrograms of dextrorotatory and levorotatory gossypol was administered to male mice. Animals were sacrificed 72 hours after drug treatment. In another experiment, mice were sacrificed 3, 10, 20, 30 and 60 days after racemic gossypol (200 micrograms/testis) administration. Marked degenerative changes in rat and mice tests were observed in the animals receiving racemic gossypol. Dextrorotatory gossypol had less pronounced effect, both in rat and mice. Stage 7 and 16 spermatids were most susceptible to the deleterious effects of racemic gossypol. In mice, 57.52% and 85.40% decrease in stage 7 and 16 spermatids were observed 72 hrs after drug administration. A progressive recovery was observed after drug treatment; the damage (stage 7 & 16) was reduced to 7.92% and 21.30% after 60 days. Histopathological changes in mice testis following 100 micrograms of levorotatory gossypol were distinctly different from those of the dextrorotatory (100 micrograms/testis) gossypol. On the basis of observations made by us, it can be suggested that mice equally respond to the antifertility effect of gossypol following intratesticular administration. The dextrorotatory gossypol, both in rat and mice, had less pronounced effect on the histoarchitecture of the testis in comparison to racemic and levorotatory gossypol. Our observations further suggest that this animal model provides meaningful information on the mechanism of action of gossypol, and recovery of spermatogenesis is possible after termination of drug treatment.     

Effect of gossypol on domestic fowl, Gallus domesticus

Domestic fowls (Gallus domesticus) were administered gossypol (10 mg/kg body weight/day) by oral intubation for 15 weeks. Drug treatment did not have any effect on body growth rate. The drug treatment, however, caused a marked decrease in the weights of testis and epididymis. Predominant changes in the histoarchitecture of testis (desquamation of germinal epithelium and inhibition of spermatogenesis) were observed following gossypol treatment. Epididymal tubules in gossypol treated animals were devoid of spermatozoa. Gossypol treatment had no effect on hematological parameters (total erythrocyte count, total leucocyte count, hematocrit and hemoglobin) studied in the present investigations.     

Early events in rat testis after gossypol administration

Eighty male rats were grouped into 8 groups of 10 animals each. Animals in groups I-IV were given gossypol (40 mg/kg/day) for 7, 14, 21 and 28 days respectively. Animals of groups V-VIII served as respective controls for groups I-IV. Marked changes in the activities of ATPase and SDH were observed following drug treatment. Decrease in the activity of testis LDH was evident even after 7 days of drug treatment. Activities of B-galactosidase, Glucose-6-phosphatase and Fructose-1-6-diphosphatase were not affected by gossypol treatment. Glycogen contents in testis were not different from those of the controls. A significant decrease in the tubular diameter and germinal height of the seminiferous tubules was observed after 21 days of drug treatment. Quantitative analysis of spermatogenic elements revealed marked decrease in the ratios of resting spermatocyte. A type spermatogonia, pachytene spermatocyte/resting spermatocyte, and stage 19 spermatids/stage 7 spermatids after 7 days of drug treatment. A progressive decrease in the ratios of these cell types was observed as the duration of the drug treatment was extended. Liver enzymes (except SDH and LDH after 28 days of drug treatment) were not affected by gossypol treatment. Our data strongly suggest that degenerative changes in the testis start after one week of drug administration. The histological changes visible at light microscopy level start appearing after 14 days of drug treatment.     

Response of hamster to the antifertility effect of gossypol.

One hundred and five sexually mature male hamsters were divided in different groups. In the first experiment hamsters were administered gossypol, 10 mg/kg and 20 mg/kg/body weight/day, for twenty and thirty days. In the second experiment hamsters were administered gossypol, 5, 10 and 20 mg/kg/body weight/day, for sixty days. In the third experiment, hamsters were administered gossypol 5 mg, 10 mg, 20 mg and 40 mg/kg body weight/day for 45 days. Animals in all the groups were given gossypol by oral intubation every day. No significant effect on the body weight of hamsters following gossypol treatment was observed. At low doses the weights of testis and accessory sex organs were not statistically different from those of the controls. A significant decrease in testis and epididymis weight was however observed following high doses of gossypol. Low doses of gossypol treatment did not affect the motility of the vas deferens spermatozoa. The vas deferens spermatozoa were however immotile after 40 mg/kg/day gossypol treatment. Gossypol treatment induced a series of histological changes in the seminiferous epithelium of the hamster testis. The earliest sign of drug effect was seen in spermatids and with the increase in doses the effects became more pronounced and extended to the spermatocytes. At 40 mg/kg dose an almost complete arrest of spermatogenesis was observed. Quantitatively, the ratio of pachytene spermatocytes: resting spermatocytes and step 7 spermatids: pachytene spermatocytes decreased significantly. The step 7 spermatids did not mature to step 19 spermatids at all. Histochemically activities of ATPase, SDH and LDH decreased with the increasing doses of gossypol, the activity of 3B hydroxysteroid dehydrogenase was not affected by gossypol treatment. In testis the glucose-6-phosphatase activity was not affected significantly but the activities of fructose 1, 6-diphosphatase and glucose-6-phosphate isomerase decreased significantly with the increasing doses of gossypol. Amylase activity rose significantly at higher doses. Marked changes in LDH and LDH-X were however observed with the increase in gossypol dose. In liver the activity of glucose-6-phosphatase increased significantly while the activities of fructose 1, 6-diphosphatase, glucose-6-phosphate isomerase and amylase were not affected following gossypol treatment. The glycogen contents however increased significantly following high doses of gossypol. No changes in testosterone production and plasma levels of testosterone were observed following gossypol treatment.     

棉酚对大鼠及豚鼠肾细胞膜Na-K-ATP酶影响的研究

本文对喂服不同剂量棉酚后大鼠和豚鼠肾皮质细胞膜组分中的Na-K-ATP 酶的活性变化进行了分析。大鼠及豚鼠喂服棉酚(5毫克/日×56,10毫克/日×28,15毫克/日×48)后,肾皮质细胞膜组分中与Na-K-ATP 酶特异结合的~3H-哇巴因的量随剂量增加而有减少趋势,但服药组与对照组之间的差别无显著意义。豚鼠喂服不同剂量棉酚和不同饲料(20毫克+低K 饲料,40及60毫克+常规饲料)后,肾细胞膜及匀浆的Na-K-ATP 酶活力随剂量的增加而相应下降,低K 饲料组较常规饲料组为明显,服药组与对照组之间的差异有显著意义。文中讨论了棉酚引起Na-K-ATP 酶活性降减与肾性失钾之间的关系。     

Effect of gossypol on the fertility of male rats.

Thirty male rats were grouped into 5 groups of 6 animals each. Animals in groups II-V were given gossypol at a dose of 5 mg/kg, 10 mg/kg, 20 mg/kg and 40 mg/kg body weight per day for 45 days respectively. Animals of group I served as control. A significant decrease in body weight after administration of 40 mg/kg body weight of gossypol was observed; low doses of gossypol, however did not affect the body weight. Testis, epididymis, prostate and seminal vesicles weights decreased gradually with the increasing doses of gossypol. With the increasing doses of gossypol, a marked decrease in the vas deferens sperm motility was observed. At 40 mg/kg dose there was a total inhibition of sperm motility. Histological studies after 5 mg/kg revealed no apparent sign of degeneration, while after 10 mg/kg dose the changes in the individual cell types were accompanied by overall disorganisation of the germinal epithelium involving displacement of the spermatocytes. The rats treated with 20-40 mg/kg gossypol showed a pronounced deleterious effect on the histological structure of the testis. The drug effect was dose dependent developing sequentially; from the uppermost layer of elongated spermatids affecting round spermatids and finally spermatocytes. Quantitatively the ratios of pachytene spermatocytes: resting spermatocytes, stage 7 spermatids: pachytene spermatocytes, and stage 19 spermatids: stage 7 spermatids and tubular diameter and germinal height decreased significantly. The activities of glucose-6-phosphatase, fructose 1, 6-diphosphatase, glucose-6-phosphate isomerase in testis decreased significantly at high dose (40 mg/kg), while the activity of amylase and glycogen content increased significantly with the increasing doses of gossypol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of mice testis to gossypol acetic acid

Male mice were administered gossypol (2.5, 5.0, 10.0 and 20.0 mg/kg body weight) by oral intubation, subcutaneous and intraperitoneal routes for 30 days. The drug treatment did not have any effect on body weight and organ weights. The motility of cauda epididymis and vas deferens spermatozoa was not affected by gossypol treatment nor there was any effect of gossypol on the histoarchitecture of the testis irrespective of the dose and route of administration. Maximum mortality was observed in mice when animals were given gossypol by intraperitoneal route.     

Effect of optical isomers of gossypol on mammalian spermatozoa: an in vitro study

Human, bull and monkey spermatozoa were treated with different optical isomers of gossypol in vitro. The spermatozoa (concentration 50 x 10(8)) were incubated with gossypol for 15, 30, 45 and 60 min. at 37 degrees C. The concentration of gossypol employed in the experiment was from 5-50 micrograms/ml. A marked inhibition in sperm motility was observed following gossypol treatment; levorotatory gossypol had more pronounced effect on sperm motility in comparison to dextrorotatory and racemic gossypol. Scanning electron microscope study revealed degenerative changes in the sperm head surface. Dextrorotatory gossypol, hitherto known to be non-effective in suppressing the fertility in vivo was found to be equally effective in inhibiting the motility and LDH-X activity of the spermatozoa. Both racemic and dextrorotatory gossypol inhibited fructolysis in bull spermatozoa. Our data suggest that whatever the mechanism of action of enantiomers of gossypol on sperm motility, fructolysis and LDH-X may be, it is evidently clear that dextrorotatory gossypol is equally active in inhibiting the sperm motility and enzyme active in vitro. The action of optical isomers of gossypol on spermatozoa in vitro, appears to be unrelated to the mechanism of orally administered gossypol.     

Effect of gossypol on immature male rats

In immature male rats the body growth rate, the testis and epididymis weight were not affected by gossypol treatment (4, 8 and 40 mg/kg body weight/24 hrs. for 30 days). There was however a marked reduction in the weight of the prostate after high dose gossypol administration. Furthermore gossypol treatment did not show any effect on the histoarchitecture of the testis nor did the drug treatment have any effect on sperm motility. The plasma levels of testosterone, LH and FSH in gossypol treated animals were no different from those on the controls.     

Recovery of normal testis function and sperm motility after cessation of gossypol treatment in hamster

The present investigations evaluate the reversibility of the effect of gossypol on the histoarchitecture of the hamster testis and the motility of the vas deferens spermatozoa. Adult male hamsters of proven fertility were treated orally with gossypol (10 mg/kg/day) for 13 weeks (group III). Another group of animals in the Group IV were given gossypol (10 mg/kg/day) for 13 weeks and were sacrificed 13 weeks after termination of the drug treatment. Animals in groups I and II were given vehicle and served as controls for groups III and IV respectively. To all the animals, gossypol or vehicle was administered by oral intubation. A marked reduction in the spermatozoa motility was observed in the animals of group III. In contrast, the motility of the spermatozoa in the animals of group IV (recovery phase) was in the normal range. A large number of seminiferous tubules were affected by gossypol treatment (group III); these tubules showed degeneration of germ cells, vacuolization and loosening of the germinal epithelium. In the quantitative analysis of the spermatogenic events, the ratio of resting spermatocyte/'A' type spermatogonia was not affected but the ratios of pachytene spermatocytes/resting spermatocytes and stage 19 spermatids/stage 7 spermatids decreased by 21%, 47% and 52% respectively. The tubular diameter and germinal height were decreased by 14% and 33% respectively. In group IV, hamsters were allowed to recover from the gossypol treatment. The seminiferous tubules exhibited normal histoarchitecture and the tubular diameter and germinal height recovered markedly. Recovery in different stages of spermatogenic cycle was evidently clear in this group.(ABSTRACT TRUNCATED AT 250 WORDS)     

棉酚所致低血钾及其与棉酚剂量关系的可能机理

1974年以来,我室试用棉酚男子避孕药255例,发生低血钾性瘫痪24例,这些病例均发生在15mg和20mg的给药组,而当剂量减至12.5mg和10mg时,低血钾不再发生。低血钾性瘫痪病例,用补钾能迅速治愈。通过实验和临床研究表明,部分服醋酸棉酚20mg/天作为起效剂量的对象,服药2个月后开始出现尿NAG和/或β_2M增高以及同时发生肾性失钾。这表明棉酚可能引起近端肾小管损害。作者认为降低棉酚起效剂量或许是一个防止发生低血钾症的途径,而棉酚所致低血钾症是由于肾小管损害所致。     

Effect of gossypol on rats maintained on protein deficient and low potassium diets

Sexually mature male albino rats were divided into four groups of 5 animals each. Animals of group I served as control, whereas animals of group II received gossypol (20 mg/kg body weight/day) for 45 days. Animals of groups III and IV were maintained on protein deficient diet. Animals of group IV received 20 mg/kg gossypol in addition to the protein deficient diet. Animals of group III and IV received protein deficient diet for 45 days before initiating gossypol treatment. The total period of maintaining the animals on protein deficient diet was 90 days. In another experiment, the same experimental protocol was followed except that the animals were maintained on low potassium diet instead of protein deficient diet. A significant decrease in body weight of animals was observed following protein deficient and gossypol (group IV) treatment. Testis weight decreased significantly in the animals of group III (protein deficient) and group IV (protein deficient + gossypol). Similar observations were made in the animals maintained on low potassium diet. In both the experiments, sperm motility was reduced significantly. Histologically, in the testis of animals of group IV (protein deficient + gossypol) almost all the tubules were disorganised and vacuolated and total arrest of spermatogenesis could be observed in majority of the tubules.     

棉酚抑制仓鼠精子顶体酶类活性及其与受精能力的关系

为了预防棉酚引起生育力不可逆的问题,我们研究了棉酚对精子顶体酶类活力与其受精能力之关系.结果表明棉酚对体外仓鼠精子穿透牛宫颈粘液及受精率有明显的抑制作用.喂服棉酚6周后,它可明显地抑制仓鼠精子的受精率,延缓睾丸提取物扩散颗粒细胞的能力,提示透明质酸酶和其他精子顶体酶类可能受到抑制.此外棉酚还可显著降低精子顶体酶和芳香基硫酸酯酶活力.这些结果表明:棉酚抑制仓鼠精子顶体酶活力与其受精率的下降相一致,其抑制作用是可逆的,并与所用棉酚剂量和服药时间呈相关性.据此可得出结论,精子顶体酶活力可用来作为监护棉酚引起不育的指标.     

Distribution of gossypol

Male rabbits and rats were administered gossypol (20 mg/kg/day) for 12 and 7 weeks respectively. Gossypol was estimated in different organs by the aniline method of Smith. Rat and rabbit spleen accumulated the highest level of gossypol. The lowest amount of gossypol was accumulated in the rabbit brain; the level of gossypol in rat brain was below the detectable limit of our method. Although rabbits were administered gossypol for 12 weeks, the accumulation of gossypol in rabbit testis was much lower than that of the rat testis. Our data suggest that non-sensitivity of rabbit to the antifertility effect of gossypol may be due to poor accumulation of gossypol in the testis. Negligible amount of gossypol in the brain rules out the possibility of involvement of hypothalamus-pituitary axis in the mechanism of action of gossypol on the testis.     

Studies on fibrinopeptide A, fibrinopeptide B beta 15-42 and inhibitors of blood coagulation and fibrinolysis during use of oral contraceptives]

To examine a change occurring in the coagulation-fibrinolytic system during oral contraceptive (OC) administration, the author measured fibrinopeptide A and Bbeta15-42 (FPA and FPBbeta15-42) over a long period of time. These are sensitive indicators of coagulation and fibrinolytic activity as well as coagulation fibrinolysis inhibiting factor plasma antithrombin III (AT III) and alpha2 plasmin inhibitor (alpha2 PI). The interesting results are listed as follows. 1) FPA showed almost no change during OC administration, nor was thrombin observed to have been produced. 2) FPB15-42 increased when the drug was administered 3-5 times and 11-12 times (p0.05) but showed significant difference when the drug was administered for the 18th and 24th times. 3) Plasma AT III activity decreased significantly (p0.05) when the drug was given for the 1st time but showed no change when the drug was administered 3-5 times. When administered 11-12 times, AT III was observed to decrease again, but repeat administrations did not cause AT III to decline; this was probably due to inurement. A similar change in immune activity concentration was observed but the change was slight. 4) When the drug was given 3-5 times following the 1st administration, a slight decrease (p0.1) occurred in alpha2 PI levels, but no change worth of mention was noted to occur thereafter. 5) In patients who received norethisterone 5 mg alone, no particular change was seen to occur in FPA, FPBbeta15-42, AT III, or alpha2 PI. Judging from the above results, especially that there was an increase in FPA, the decrease in AT III levels may be attributed not to a reduction in consumption but to a decrease in the AT III itself, due to estrogens. It is possible also that an increase in FPBbeta 15-42 led to plasmin production which was the reason so few thromboses were observed to form. (author's modified)     

30例育龄男性服用棉酚期间的心电图追踪观察

为了解小剂量口服棉酚的安全性，对３０例育龄男性进行了口服小剂量棉酚（１５ｍｇ／ｄ×１２ｗ，继以７．５ｍｇ／ｄ或１０ｍｇ／ｄ×４０ｗ）期间的心电图（ＥＫＧ）随访观察。在整个服药期间有ＥＫＧ改变者中，多数（７７％）为ＰＲ间期延长，平均ＰＲ间期在第４周和第１２周为０．１６６±０．０１７ｓ，显著高于服药前的０．１５９±０．０１４ｓ，差异具统计学的显著性（Ｐ＜０．０５）。ＱＲＳ时限，ＱＴ间期则不见明显变化。本项结果提示小剂量棉酚对心肌和心内传导系统可能有着不同的影响。     

Reproductive endocrine function in gossypol-treated male rats

The mode and sites of antifertility action of gossypol were explored in Long-Evans rats for the purpose of clarifying effects on endocrine function. Mature male rats were treated every five days with gossypol acetic acid (10 mg/kg of body weight, given subcutaneously). Although no conspicuous loss in sperm motility and breeding performance occurred with this dosing regimen, even after prolonged treatment, a statistically significant reduction in serum testosterone (T) and decreases in accessory organ weights were observed after only 3 weeks. Testosterone propionate (500 micrograms daily, subcutaneously) administered concurrently with gossypol to gonadectomized rats prevented the decrease in accessory organ weight, indicating that gossypol probably affects these organs indirectly by altering circulating androgen levels. In another experiment, serum T was measured in gossypol-treated (10 mg/kg/day for 100 days, subcutaneously) and control rats before and after a single challenge dose of 20 IU of hCG. Even though prechallenge serum T levels were suppressed in gossypol-treated rats, postchallenge levels were similar to controls, indicating the absence of significant impairment in interstitial cell response to gonadotropin stimulation. There was also no evidence that the anterior pituitary gland was directly affected by gossypol treatment.     

Antifertility and ultrastructural effects of optical isomers of gossypol administered intratesticularly in rats

This study reports on the effects of optical isomers of gossypol administered intratesticularly on testicular ultrastructure and fertility in rats. Gossypool isomers were administered to adult male rats by daily intratesticular injections for up to two days at a dose of 1200 ugm/testis. The results of this study have demonstrated that the intratesticular injection of racemic and (-) gossypol at a dose of 1200 ugm for 1 or 2 days mimic the effect of gossypol on rat testis after oral administration of 20 to 30 mg dose for 5-7 weeks; (-)-gossypol was found to be 81% effective in inducing mitochondrial sheath damage in the late spermatids of stage VI, VII and VIII tubules compared to 67% for racemic and 7% for (+)-gossypol, the intratesticular injection of (+/-) and (-)-gossypol (1200 ugm/testis, daily for 1 or 2 days) induces complete infertility three weeks after the last injection.     

Gossypol: a male contraceptive with potential?

The serendipitous discovery of gossypol by the Chinese in 1978 may represent the most significant advance ever made in terms of male contraception. Protection from pregnancy has been achieved in 99% of couples who use gossypol. The contraceptive effect seems to be maintainable through a dose of 50 mg/week. Azoospermia can persist for as long as 4 years after gossypol discontinuation. The incidence of overt toxicity in men taking contraceptive doses of gossypol is low, although fatigue, changes in libido, loss of appetite, and headache have been reported. Hypokalemia, the most potentially life-threatening side effect, occurs in an estimated 10% of men who use gossypol for contraception. There is a need for more evaluation of the cardiovascular action of contraceptive doses of gossypol. Animal studies have revealed marked species differences in sensitivity to gossypol's contraceptive action. There is no evidence to date that gossypol has adverse effects on the outcome of subsequent pregnancies or on fetal development, although any drug that interferes with the production and maturation of spermatozoa should be closely monitored from the perspective of reproductive toxicity. It is unlikely that the contraceptive activity of gossypol can be explained by a single molecular event. Development of the contraceptive potential of gossypol is dependent on chemical modifications and the discovery of a derivative with a safe therapeutic ratio. Overall, although gossypol has proven to be an efficient, inexpensive means of fertility control, the side effects of hypokalemia and possibly permanent infertility make it unacceptable at this time.     

Cottonseed--a source of vaginal contraceptive (author's transl)

The antifertility effect of the cottonseed oil, gossypol, had been first observed in China, where it was administered orally to over 40,000 healthy males, with an effectiveness of 99%. The authors of this study have tried gossypol as a vaginal contraceptive. Gossypol acetic acid was dissolved in 0.5 N NaOH, and then diluted. The solution obtained was added to a similar volume of seminal liquid, and sperm motility was carefully measured. The immobilization of spermatozoa was total. The same results were obtained with seminal liquid from rats. One added advantage of vaginal administration of gossypol would be its lubricating quality.