Effect of anti-estrogen and anti-progesterone on spermatogenesis, testosterone production and expression of steroidogenic enzyme genes in adult male rats.

The present study was planned to investigate the anti-spermatogenic and anti-steroidogenic effects of Clomiphene Citrate (CC) an anti-estrogen and Mifepristone (MT) an anti-progesterone in the testis of male rats. Following the oral administration of 1.0 mg and 5.0 mg/kg b.w/day of each for the duration of 30 and 60 days, quantitation of spermatogenesis, RIA for serum and intra-testicular testosterone levels, western blotting and RT-PCR for expression of StAR, 3ß-HSD and P450arom enzymes in the testis was done. Clomiphene Citrate at 5.0 mg/kg b.w/day for 60 days significantly reduced testosterone (T) levels however the effect was not significant with the lower doses. Reproductive parameters in animals treated by Mifepristone remained mostly unaffected, however, a significant decline in testosterone levels and altered expression of selected genes was observed in 5.0 mg for the 30d treatment group. Clomiphene Citrate at higher doses affected the weights of the testis and secondary sex organs. Seminiferous tubules revealed hypo-spermatogenesis with a significant decrease in the number of maturing germ cells and a reduction in tubular diameter. Attenuation in serum testosterone was associated with the downregulation of expression in StAR, 3ß-HSD, and P450arom mRNA and protein levels in the testis even after 30 d of CC administration. The results indicate that the anti-estrogen (Clomiphene Citrate) but not anti-progesterone (Mifepristone) induces hypo-spermatogenesis in rats which are associated with a downregulation of expression of two of the steroidogenic enzymes, 3ß-HSD and P450arom mRNA and StAR protein.

Complete sperm suppression in rats with dienogest plus testosterone undecanoate is facilitated through apoptosis in testicular cells.

Complete suppression of the production of sperm in rats with dienogest (DNG, 40 mg/kg body weight [bw]) plus testosterone undecanoate (TU, 25 mg/kg bw), every 45 days, was found to be associated with a significant increase in germ cell apoptosis. Caspase 3 activity and expression in testis were simultaneously upregulated. Rise in the activities of caspase 8 and 9 was associated with overexpression of upstream marker proteins from extrinsic (Fas [Fatty acid synthase], FasL [Fatty acid synthase ligand], and caspase 8) and intrinsic (Bax [Bcl2-associated-x protein], Bcl2 [B-cell lymphoma 2], and caspase 9) pathways of apoptosis. Apart from the germ cells, interstitial cell apoptosis was also observed along with a decline in the number of functional Leydig cells. It is therefore concluded that complete suppression of the production of sperm with DNG + TU is facilitated mainly through the removal of precursor germ cells through apoptosis. The process is largely modulated by upregulation of upstream and downstream marker proteins from intrinsic as well as extrinsic pathway of metazoan apoptosis.

Complete sperm suppression induced by dienogest plus testosterone undecanoate is associated with down-regulation in the expression of upstream steroidogenic enzyme genes in rat testis.

BACKGROUND: We had shown that dienogest (DNG) + testosterone undecanoate (TU) induced complete sperm suppression in rats when administered together every 45 days. On the other hand, individual drugs given alone in a similar fashion failed to achieve the same result. STUDY DESIGN: The present study was therefore undertaken to determine the reason for such a differential sperm suppression and to correlate it with the expression of steroidogenic enzyme genes in the rat testis. RESULTS: Administration of DNG (40 mg/kg body weight [bw]) + TU (25 mg/kg bw) every 45 days for a duration of 90 days induced spermatogenic arrest, leading to a significant reduction in testicular weight and number of precursor germ cells. Flow cytometric analysis further confirmed the same result, leading to a significant shift in the distribution of haploid cells. Measurement of testosterone (serum and intratesticular) was significantly low. Complete sperm suppression coincided with significant down-regulation in the expression of upstream steroidogenic enzyme genes represented serially by cytochrome P450 side-chain cleavage, P450 17α-hydroxylase, 3ß-hydroxysteroid dehydrogenase and steroidogenic acute regulatory protein (StAR) in the testis. On the other hand, rats administered with either DNG or TU alone demonstrated incomplete sperm suppression in which the expression of all the above genes remained characteristically nonuniform. CONCLUSION: Taken together, the above findings corroborate the fact that regulation of expression of three of the upstream steroidogenic enzymes genes and the StAR protein in rat testis is crucial in leading to complete sperm suppression as observed with DNG+TU treatment.

Extended intervention time and evaluation of sperm suppression by dienogest plus testosterone undecanoate in male rat.

BACKGROUND: The potential of using dienogest [DNG, 40 mg/kg body weight (bw)] plus testosterone undecanoate (TU, 25 mg/kg bw) in rats for development of a once-a-month male hormonal contraceptive has been reported earlier in our laboratories. STUDY DESIGN: In the present study, we report a separate efficacy evaluation of the same combination, DNG (40 mg/kg bw) and TU (25 mg/kg bw) in which interval of drug administration has been extended further to 45 and 60 days instead of every 30 days. RESULTS: Complete sperm suppression was observed in rats sacrificed either 60 or 90 days after DNG+TU administration, for two injections at 45-day interval. The neutral α-glucosidase activity in these treated rats remained in the normal range. Germ cell loss due to apoptosis was frequently observed both after 60 or 90 days of combination treatment. Significant decline in serum gonadotropin and testosterone, both serum and intratesticular levels, were observed in the treated rats. Following stoppage of treatment (given at 45-day interval) after two (0 and 45 days) or three injections (0, 45 and 90 days), complete restoration of spermatogenesis was observed by 120 and 165 days, respectively. The sperm suppression, however, could not be sustained when the period of combined drug administration was extended from every 45 to 60 days. CONCLUSIONS: Dienogest plus testosterone undecanoate in the above doses retained contraceptive effectiveness when administered every 45 days but not 60 days. The spermatogenic arrest was completely reversible once drug treatment is stopped. The dose and the frequency of intervention can be extrapolated in future clinical trials.