I-1: Effect of High Intratesticular Estrogen on

Background: The presence of estrogen receptor beta and aromatase in the germ cell has highlighted the physiological role of the traditionally female hormone, estrogen, in spermatogenesis. Estrogen receptor alpha knockouts and aromatase knockouts have further accentuated the role of estrogen in germ cell maturation. To delineate effects of high intratesticular estradiol in the seminiferous epithelium and the mechanisms involved. The study was based on the fact that administration of exogenous estradiol suppresses the hypothalamus pituitary gonadal axis (HPG) with a dose-dependant concomitant increase in intratesticular estrogen levels. Materials and Methods: Three doses of 17-β Estradiol, namely 20, 100 and 200μg/kg/day were administered subcutaneously to different batches of adult male rats for 10 days. The effect of the three doses on serum hormonal profile, intratesticular testosterone (T) and estradiol (E) levels and testicular morphology were studied. Further studies to delineate the mechanism causing spermiation failure was carried out with 100 μg/kg/day for 10 days and the effect on Sertoli cell cytoskeleton and testis specific adherens junction was done by immunofluorescence and confocal imaging Results: Twenty micrograms per kilograms per day of 17-β estradiol affected the hypothalamus–pituitary axis, reducing serum gonadotropins and intratesticular testosterone; however, 100 μg/kg/day of 17-β estradiol decreased serum FSH and intratesticular testosterone, increased intratesticular estradiol, but had no effect on serum LH. Interestingly, 200 μg/kg/day of 17-β estradiol decreased serum and intratesticular T without any effect on serum gonadotropins. This could be attributed to the positive feedback effect of estrogens on gonadotropins. In the testis, morphologically two visible effects were seen, namely ’spermiation failure’ in all three doses attributed to the suppression of T and FSH and a ’maintenance effect’ in the 100 μg/kg/day attributed to E and/or 10% of available intratesticular T. The direct effect of an increase in intratesticular estradiol levels was observed in terms of a decrease in apoptosis in germ cell. The study, therefore, suggests that 100 μg/kg/day of 17-β estradiol could be used to study the effects of high intratesticular estradiol with a concomitant decrease in intratesticular T and serum FSH levels on spermatogenesis. Hence further studies on mechanism causing spermiation failure were carried with this estradiol dose. Spermiation is the final phase of spermatogenesis leading to release of mature spermatids into the lumen of the seminiferous tubules. Morphologically, it involves a series of events, namely removal of excess spermatid cytoplasm, removal of ectoplasmic specialization, formation of tubulobulbar complex, and final disengagement of the spermatid from the Sertoli cell. Electron microscopic and confocal studies revealed an absence of tubulobulbar complex in step 19 spermatids after estradiol treatment, highlighting the significance of these structures in spermiation. It was further observed that treatment affected the Sertoli cell cytoskeleton and Arp2/3 complex that is critical for de novo polymerization of actin during tubulobulbar complex formation. In addition estradiol treatment also affected microtubule bundling and distribution of vimentin filaments in Stage VII-VIII of the seminiferous epithelium cycle suggesting an effect on Sertoli cell cytoskeleton. Conclusion: The present study reports the role of 17-β estradiol in inhibiting the formation of tubulobulbar complex, which could be one of the mechanism by which environmental estrogens influence male fertility.