PROJECT 1 - Clinical and basic studies in Male Reproduction

哺乳动物的精子发生依赖于睾丸间质细胞产生的雄激素。遗传和 药理学实验表明，雄激素水平，通过支持细胞，支持 通过精子发生进行。这项建议涉及雄激素受体如何在支持 细胞支持精子发生。我们已经确定Claudin 3（Cldn3）基因作为雄激素反应性调节因子。 Sertoli细胞中的基因。我们已经表明，CLDN3，一个四次跨膜蛋白，定位于支持细胞 在雄激素受体表达最高阶段的细胞紧密连接。支持细胞紧密连接 创造了哺乳动物中最不透气的屏障之一。即使是对这一屏障的适度破坏， 不育，而同时精母细胞不断地穿过紧密连接屏障进入 睾丸的近腔室。支持细胞与肾小管周围肌样细胞和调节性T细胞一起， 细胞紧密连接共同形成血睾屏障（BTB）。在条件性消融Ar的小鼠中， 在支持细胞中，CldnS下调，BTB对小分子的渗透性增加， 提供给生殖细胞的免疫豁免受到损害。这一建议涉及的假设是， 雄激素通过创造允许精子发生的微环境来支持精子发生。的 支持细胞紧密连接调节这种微环境。在目的1中，我们描述了支持细胞条件 敲除Cldn3并确定BTB的渗透性是否松弛以及免疫豁免是否被抑制。 暴露了支持细胞紧密连接的功能表征将在原代培养中进行评估。 通过测量跨上皮电阻进行培养。在目标2中，我们测试了特定阶段的重要性 通过在生精上皮的整个周期中使用免疫抑制剂驱动CldnS的表达， 可诱导转基因表征将包括光学和电子显微镜以及功能测定， 探测BTB的完整性这些研究将有助于更好地了解 睾酮对正常精子发生和生育能力的影响，并以睾酮的分子机制为基础 男性避孕药

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.