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.

I. The modulatory effect in genotoxic responses due to age and duration of PHT-therapy in epileptic patients.

Sister chromatid exchange (SCE) frequency has been studied from the peripheral blood lymphocyte cultures of 42 epileptic patients on the anticonvulsant drug phenytoin (PHT) for 3 months and their follow-up (6 and 9 months), of 33 epileptics who had not started therapy (PHT-untreated), and of 40 normal healthy controls, all in the same age group, i.e., 10-30 years. PHT-treated epileptic patients at all three durations of therapy (3, 6, and 9 months) showed higher SCE frequency (P < 0.001) than healthy controls and PHT-untreated patients. There was no significant difference in SCE frequency between control and PHT-untreated patients, suggesting that disease is not associated with an increased frequency of SCEs. The frequency of SCEs seems to be influenced by an age factor, when older treated patients (21-30 years) showed higher SCE frequencies at 3 and 6 months (P < 0.001) and 9 months (P < 0.05) than the younger age group (10-20 years). SCE frequency increased linearly with the duration of therapy, i.e., from 3 months to 9 months. No correlation was found between SCE frequency and sex with respect to controls, PHT-untreated, and PHT-treated subjects. In conclusion, the modulating effect on SCE frequencies elicited by age and duration of therapy has been clearly demonstrated by SCE mean analysis. Teratogenesis Carcinog. Mutagen. 21:135-149, 2001.

II. An altered proliferation response due to the anticonvulsant phenytoin (PHT) in epileptic patients.

Lymphocyte proliferation kinetics (LPK) is an end point used in genetic toxicology that was proposed as an alternative for the screening of anticonvulsant drugs. The effect of phenytoin (PHT) was investigated on the mitotic and proliferation indices in cultured blood lymphocytes of 33 sporadically collected untreated and 42 PHT-treated epileptics, where the duration of treatment was 3, 6, and 9 months, and 40 control subjects (age range 10-30 years). PHT induced mitotic delays and decreased the mitotic index. A significant heterogeneity of the first, second and the third metaphases between treated and untreated groups was revealed. A reduction of the proliferation index (P < 0.001) and proliferation delay per cycle (P < 0.001) was also observed. There was little variation between the controls and untreated patients (P > 0.05). The results have confirmed that PHT can affect responses leading to genotoxicity. Teratogenesis Carcinog. Mutagen. 21:151-164, 2001.

[A pill for the man? Current status of fertility control in the man]. / Pille für den Mann? Aktueller Stand der Fertilitätskontrolle beim Mann.

Taking all facts into consideration from animal experiments and clinical studies with regard to the development of a male contraceptive you must be aware that the 'pill for men' will hardly be available in this century. Because of the increasing interest of the industry and the effort of the WHO and other similar institutions, like the Population Council of New York, to develop a male pill the stagnation of the past 20 years could be overcome, and it may be possible to have an adequate method in 2005. In all probability this will be a combination of hormones either from a gestagen plus testosterone preparation or a potent LHRH agonist and/or antagonist, also in combination with a long-acting testosterone preparation, with testosterone levels within the normal range. Nowadays it cannot be said which role gossypol will finally play. There are studies going on with gossypol with some promising results.

alpha-Glucosidase activity in the rat epididymis under different physiological conditions.

The estimation of alpha-glucosidase activity in semen is widely used as a marker of epididymal function. In the present studies, glucosidase activity was evaluated in the different segments of the rat epididymis under various physiological conditions. In addition, the effect of two known male antifertility agents, gossypol and alpha-chlorohydrin, on enzyme activity was evaluated. Enzyme activity was absent from the epididymis of rats aged 10 and 20 days but became detectable at 30 days of age when the adult pattern of distribution (highest activity in the caput epididymis) was established. Enzyme activity was reduced significantly in all segments of the epididymis at 7 days after castration and a significant decrease in activity was also observed following the administration of either gossypol or alpha-chlorohydrin. These findings are consistent with a role for alpha-glucosidase in sperm maturation in the epididymis.

Regulation of male fertility by pyrimethamine in adult mice.

Studies were carried out to determine the antifertility and reversibility effect of pyrimethamine (PYR) in adult male mice. The parameters mainly included sperm count and motility, fertility, histoarchitecture of testis and testicular cell kinetics quantitatively following oral administration of PYR (50 mg/kg body weight per day) for 30 days. The same parameters were also studied in PYR-treated animals which were allowed to recover for 45 days (recovery group). The results suggest that sperm motility as well as counts were significantly decreased in PYR-treated animals, and the fertility rate fell to zero. Testicular histology as well as germ cell kinetics were altered. However, in the animals of the recovery group, all the parameters studied were more or less similar to those of control animals. The study demonstrates the antifertility as well as reversible efficacy of PYR.

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.

Spermatozoal abnormalities and male infertility in the rat following sulfasalazine treatment.

OBJECTIVE: To determine the (reversible) antifertility effect in the male rat of sulfasalazine, a common therapy for inflammatory bowel disease. METHODS: Sulfasalazine (120, 250 and 500 mg/kg) was administered for 60 days by oral gavage to sexually mature male rats. RESULTS: Both motility of sperm and fertility of animals was significantly altered at the highest dose (500 mg/kg). Morphological abnormalities included alteration in the normal profile of the spermatozoa and presence of lesions on the sperm head. CONCLUSIONS: Results suggest that sulfasalazine brings about its antifertility effects by altering sperm motility and number of spermatozoa in addition to increased surface abnormalities, which render the spermatozoa infertile.

Effect of human gamma interferon on mice testis: a quantitative analysis of the spermatogenic cells.

Effect of Human Gamma Interferon (Hu-IFN-gamma) on the testicular histology was studied in mice. Male mice were administered Hu-IFN-gamma intratesticularly at the doses of 2, 10 and 20 micrograms/testis in a volume of 1.0 microliter isotonic normal saline. Contralateral testis served as control and was administered same amount of vehicle. All the animals were sacrificed 7 days after drug administration. Body weight and the weights of testis and epididymis were not affected by IFN treatment nor was there any effect of the drug on the motility of the vas deferens spermatozoa. Low dose of IFN (2 micrograms) did not have significant effect on the histoarchitecture of the testis and various spermatogenic elements, a progressive damage was however observed with the increasing doses of IFN. Pronounced deleterious effect of IFN on the testis leading to desquamation of the germinal epithelium, reduction in the germinal cell height and tubular diameter was observed with 20 micrograms dose. Quantitative studies on seminiferous epithelium showed a significant decrease in the number of Sertoli cells, stage-7 spermatids and stage-16 spermatozoa. The ratios of resting type spermatocyte: type A spermatogonia and stage-7 spermatids: pachytene spermatocyte was also reduced. The ratios of pachytene spermatocyte: resting spermatocyte and stage-16 spermatozoa: stage-7 spermatids were however not affected by IFN treatment. In another experiment IFN was administered (2 micrograms/day) subcutaneously to male mice for 30 days. No effect of drug treatment on body weight, organ weight, sperm motility and histology (including morphometry) of the testis was observed. Our data suggest that IFN action at testis may be associated with the antiproliferative effect of interferon.

Effect of active immunization to luteinizing-hormone-releasing hormone on the fertility and histoarchitecture of the reproductive organs of male rat.

The feasibility of using a vaccine against luteinizing-hormone-releasing factor for suppression of pituitary and gonadal functions has been indicated for some time. Antibody production against this low-molecular-weight, naturally occurring decapeptide, however, requires to be coupled to a carrier protein to enhance its immunogenicity. LHRH was coupled to diphtheria toxoid (DT). Adult male Sprague-Dawley rats with a mean basal body weight of 200 g were immunized with anti-LHRH-DT (20 micrograms/injection/rat) at four-week intervals. An equal number of unexposed animals served as controls. Six animals were killed every two weeks up the end of the week 43. The vaccination schedule did not have any effect on the gain in body weight, nor was any adverse effect of vaccination observed in the course of the investigations. The pituitary, prostate, epididymis, testes, seminal vesicles, adrenal and thyroid were excised for determination of organ weight and histological examination. The adrenal, pituitary and thyroid showed no remarkable weight changes during the observation period, whereas the weights of the reproductive organs demonstrated significant reductions compared to those of the control group. The histopathology revealed marked to significant changes in the gonads and the accessory sex organs including the prostate. A progressive phase of regeneration of spermatogenesis was evident 98 days after vaccination. Total recovery of spermatogenesis was observed 300 days after vaccination. The mating studies showed the return of fertility 300 days after vaccination. The litters borne were normal. Prostate showed recovery after 154 days of vaccination. Our observations lend strong support to the hypothesis that anti-LHRH vaccine can be effectively used on the management of prostate carcinoma. If the vaccination is given together with a suitable dose of long-acting androgen, contained in an adequate delivery system, the regimen may be used for the regulation of male fertility.

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.

65Zn incorporation in the male reproductive organs following gossypol treatment.

Male hamsters and rats were administered gossypol 10 mg/kg/day for 45 and 56 days respectively. Twenty four hours before the last dose, animals were administered 65Zn (specific activity 0.258 uci/mg) subcutaneously. A marked decrease in 65Zn incorporation was observed in testis, epididymis, seminal vesicles and prostate following drug administration. A significant increase in 65Zn uptake was however observed in vas deferens in both rat and hamster following drug administration. Our results suggest that whatever the mechanism of gossypol action on testis-epididymis complex may be, the marked decrease in 65Zn uptake by testis--epididymis complex following gossypol treatment may be related to the antispermatogenic effect of gossypol.

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.

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.

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 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.

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.