Testicular effects of monensin, a golgi interfering agent in male rats.

OBJECTIVE: The present study was undertaken to explore the effects of monensin, a potent Golgi disturbing agent on male fertility. METHODS: Male Wistar rats were administered monensin at the dose levels of 2.5, 5, and 10 mg/kg b wt. Animals were sacrificed after 67 days of the treatment. The activities of lactate dehydrogenase (LDH), ATPase, acid phosphatase and thiamine pyrophosphatase (TPPase) were measured in the testis. Cytochemical assay of Golgi body marker enzyme, thiamine pyrophosphatase was also performed. Ultrastructural changes in testis were studied by Transmission electron microscopy. Sperm number and motility were also examined. RESULTS AND DISCUSSION: The alterations in the activities of above mentioned enzymes indicate the pronounced effect of the drug on the functioning of spermatogenic cells. The findings from electron microscopy such as membrane disruption, swelling and disintegration of Golgi apparatus strongly suggest the interference of monensin with the functioning of Golgi apparatus in the spermatogenic cells. Data from the sperm number and motility as well as the fertility studies and the resulted litter size further points towards the antifertility effects of monensin in male rats. CONCLUSION: The findings from the present study strongly indicated the effects of monensin on the testis, involving alterations in key enzyme activities and changes at the ultrastructural level.

Effect of monensin on the enzymes of oxidative stress, thiamine pyrophosphatase and DNA integrity in rat testicular cells in vitro.

Monensin, a sodium specific ionophore was evaluated for its in vitro effects on rat testis by studying changes at biochemical parameters as well as at the DNA level. It was observed that monensin produced marked alterations in the activities of various enzymes associated with the testicular functions. The significant inhibition of different enzymes of oxidative defense system points toward the generation of reactive oxygen species (ROS) by monensin treatment. The significant depletion of reduced glutathione and elevation in the level of lipid peroxidation further support the above findings. The significant inhibition of the activities of lactate dehydrogenase and adenosine triphosphatase shows the interference of monensin with the normal energy supply in spermatogenesis. Moreover, the significant increase in the activities of acid phosphatase and thiamine pyrophosphatase demonstrates the interference of monensin with the Golgi-lysosomal complex of the rat testis. Induced DNA fragmentation indicates towards the impact of monensin on the DNA integrity and apoptosis. Further studies are needed to understand the important molecular mechanisms responsible for these effects.

Alterations in oxidative stress-related parameters in rat testis following monensin administration.

Monensin, a carboxylic ionophore, is well known for Na(+)/H(+) exchanger activity across biological membranes. It is also used in the poultry industry for its useful effects as a food additive. The present study has been designed to investigate the effects of monensin on some oxidative stress-related parameters in rat testis. Monensin was administered intratesticularly (5 mug/testis) to both testes by a single dose to Wistar rats for different time periods. After the completion of the respective treatments, various parameters reflecting the antioxidant defense system of the tissue were monitored and marked changes were found in the activities of various enzymes as well as in the levels of reduced glutathione and lipid peroxidation. After 1, 2, 3, and 4 days of monensin treatment, the activity of superoxide dismutase was found to be unaltered. However, after 2 days of monensin treatment, glutathione-S-transferase and catalase showed inhibition in their activities along with the depletion of glutathione (reduced) accompanied by a marked increase in lipid peroxidation. The increase in lipid peroxidation was noticeable even after 1 day of monensin administration. The inhibition in glutathione-S-transferase and glutathione peroxidase activities was also observed along with an increase in lipid peroxidation at the end of the 3-day posttreatment period, while, the 4-day posttreatment schedule caused an increase in the activity of glutathione reductase and glutathione peroxidase that was also accompanied by an inhibition of catalase. The findings of the present study are indicative of the potential of monensin in testicular tissue in contraceptive intervention.

Effect of gossypol on testicular testosterone production in vitro.

Testicular minces were utilized to study the effect of gossypol on testosterone production. Testosterone production was assessed in both control nd gossypol treated groups after 0 to 4 hours incubation in the presence of hCG. Media testosterone was measured by radioimmunoassay. Gossypol did not alter testosterone production when present in incubates at the concentrations of 3.5 x 10(-5) M, 7 x 10(-5) M and 3.5 x 10(-4) M. Preincubation of testis mince with gossypol (7 x 10(-6) M, 7 x 10(-5) M, 3.5 x 10(-4) M) for 1 to 4 hours did not alter subsequent hCG induced testosterone production in mature rats. Testosterone production however, was inhibited in immature rat testis when the whole testis was incubated for 4 hours with different concentrations of gossypol (7 x 10(-6) M, 7 x 10(-5) M, 3.5 x 10(-4) M). In vivo testosterone production was not inhibited in the immature rat testis 24 hours after oral administration of gossypol (100 mg/kg).

Phospholipid analysis in testis-epididymis complex after alpha-chlorohydrin administration.

alpha-Chlorohydrin, 6.5 mg/kg body weight for 14 days, did not alter total phospholipids, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol and sphingomyelin in rat testis and caput and corpus epididymis. However, marked decrease in total phospholipids, phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl inositol in cauda epididymis and vas deferens was observed. p32 incorporation also showed similar changes in phospholipids.

Antifertility effects of an LHRH agonist in male mice.

This study was designed to investigate the effects of repeated high doses of an LHRH agonist on Swiss porton mouse seminiferous epithelium. Seminiferous epithelium showed more pronounced degenerative effects at the higher dose (1000 micrograms/kg wt/day) of the drug, with arrest of spermatogenesis at all stages of differentiation; effects on spermatogenesis become more evident after pachytene spermatocyte stage. The decrease in the testis-specific enzyme LDH-X is accounted for by the decrease in the number of cells of gametogenic origin. Although complete azoospermia was not observed, acute inhibition of motility of spermatozoa may account for severely decreased fertility rates after mating. However, reduced fertility rates due to decreased libido as a consequence of diminished testosterone levels cannot be discounted.

Clinical microdose study of gossypol: effect on sperm motility and renal function.

This study is part of a series designed to identify the smallest effective gossypol dose for male fertility control. Three men, aged 31-35 years, were administered gossypol, 10 mg/day, orally for 3 months. Urinalysis and assays for plasma hormone values, including cortisol, beta 2-microglobulin, potassium, and BUN, showed no changes during treatment. Forward sperm motility, however, was severely affected by the end of the treatment period (less than 4% forward motility). Sperm density also showed a marked decrease. The subjects developed no undue side effects.

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.

Gossypol.

PIP: The male contraceptive gossypol was developed by Chinese workers and tested on 10,000 male volunteers over a period of 5-8 years. It was effective in 99.8% of the cases. It acts also as an antiviral and antifungal agent inhibiting Trypnosone and Plasmodium, which cause Chagas and malaria respectively. This study also supports the efficacy of gossypol. Cellular and molecular lesions in the testis occurred with gossypol. Animal models have shown spermatogenesis after drug withdrawal. Bonnet monkey results support significantly reduced fertility. In vivo and in vitro work has been conducted in order to isolate the differential effects of optical isomers. The current results of unwanted side effects are 1) failure of recovery of spermatogenesis, and 2) hypokalemia. An operational model of action of gossypol on the testis is under development.^ieng

Demonstration of direct effect of estrogen on rat spermatogenesis.

The presence of an estrogen receptor in Leydig cell cytosol suggests that estrogen could have a direct action on Leydig cell function. We have shown earlier the direct effect of estradiol on testosterone biosynthesis. We report in this communication effect of estradiol on spermatogenesis using hypophysectomized rats treated daily for four days with 400 IU hCG/Pregnyl) and 1 IU FSH (Pergonal), a model that eliminates the possibility of feedback effects of estradiol on gonadotropin secretion. Estradiol was administered in subcutaneous silastic capsules. The control animals had empty capsules. The inhibition of spermatogenesis, after estradiol treatment, was marked by the presence of multinucleated giant cells, disorganization of the germinal elements, accumulation of cellular debris and the absence of late spermatid and spermatozoa. These changes in the histoarchitecture of testis were accompanied by the reduction in the diameter of the seminiferous tubules and the thickness of the basement membrane. Morphologically Leydig cells were, however, normal. Inhibition of spermatogenesis was in relation to the amount of estrogen available/administered as estradiol capsule of 0.25 cm did not evoke significant changes in the histology of the testis whereas estradiol capsule of 4 cm caused maximum damage to the spermatogenesis. Similarly progressive damage to the spermatogenesis was quite apparent as the number of days increased after estradiol capsule implantation. Neither high (1600 IU/day/4 days) nor low /5.25 IU/day) doses of hCG synergized the effect of estradiol on spermatogenesis. Testis weight was significantly reduced after estradiol treatment but weight of the epididymis and accessory sex organs did not change. Body weight was also not effected by estradiol treatment.

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.

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 carbon tetrachloride on gonadal physiology in male rats.

Carbon tetrachloride was administered to male albino rats for varied intervals. It was found to have no significant adverse effect on spermatogenesis after 10 and 15 days of treatment. Severe damage to the spermatogenic cycle was observed after 20 days of treatment, leading to exfoliation of the germinal epithelium, depletion of germ cells, and shrinkage of the tubules.

Effect of sulphapyridine on male fertility in the rat.

Male rats administered sulphapyridine (60, 120 and 250 mg/kg) for 60 days demonstrated no change in body weight and testicular weight. However, there was a decrease in the weight of the epididymis. Motility and sperm reserves were reduced and were evident from fewer implantation sites and number of pregnancies. Furthermore, sulphapyridine did not show any effect on the histoarchitecture of the testis or epididymis. Serum levels of testosterone in the treated rats were comparable to their respective controls. Morphological abnormalities as revealed by scanning electron microscopic studies clearly demonstrated the detrimental effects of the drug.