PROJECT 1 - Clinical and basic studies in Male Reproduction

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Spermatogenesis in mammals is dependent on androgens produced by testicular Leydig cells. Genetic and pharmacological experiments suggest that androgen levels, working through Sertoli cells, support progression through spermatogenesis. This proposal addresses how androgen receptor function in Sertoli cells supports spermatogenesis. We have identified the Claudin 3 (Cldn3) gene as an androgen responsive gene in Sertoli cells. We have shown that CLDN3, a four-pass transmembrane protein, is localized to Sertoli cell tight junctions during the stages of highest androgen receptor expression. Sertoli cell tight junctions create one the most impermeable barriers in mammals. Even a modest disruption of this barrier results in infertility, while at the same time spermatocytes are constantly crossing the tight junction barrier into the adluminal compartment of the testis. Together with peritubular myoid cells and regulatory T cells, the Sertoli cell tight junctions collectively form the blood testis barrier (BTB). In mice with conditional ablation of Ar in Sertoli cells, CldnS is down-regulated, the permeability of the BTB to small molecules is increased, and the immune privilege provided to germ cells is compromised. This proposal addresses the hypothesis that androgens support spermatogenesis by creating a microenvironment permissive for spermatogenesis. The Sertoli cell tight junctions regulate this microenvironment. In aim 1 we characterize a Sertoli cell conditional knockout of Cldn3 and determine if the permeability of the BTB is relaxed and the immune privilege is compromised. Functional characterization of the Sertoli cell tight junctions will be assessed in primary culture by measuring transepithelial resistance. In aim 2 we test the importance of the stage-specific expression of CldnS by driving its expression throughout the cycle of the seminiferous epithelium using an inducible transgene. Characterization will include light and electron microscopy and functional assays that probe the integrity of the BTB. These studies will lead to a better understanding of the function of testosterone for normal spermatogenesis and fertility, and to the molecular mechanism of hormone-based contraceptives for men.