ANDROGEN RECEPTOR EXPRESSION & ACTIVITY IN SERTOLI CELLS

正常的精子发生依赖于足够的 睾丸内雄激素，主要是睾酮和雄激素 受体(AR)对这些类固醇的作用起着至关重要的调节作用。我们 提出了细胞内水平和转录活性 AR可能在决定雄激素的作用中发挥关键作用 Sertoli细胞，因此在精子发生中。在目标1中，我们将研究 支持细胞AR基因表达的阶段特异性调控假说 细胞受内源蛋白差异表达的影响 与AR启动子结合并增强或抑制的转录因子 AR基因的转录。《条例》内的职能监管区域 大鼠AR启动子将在转染的Sertoli细胞中确定。这个 推测转录因子表达的阶段特异性将是 由凝胶位移和DNA酶足迹上的DNA-蛋白质结合决定 阶段同步化Sertoli细胞核提取液的检测 生精小管。北方和西方的印迹将被用来确定 转录因子在支持细胞中的阶段特异性表达 结合AR启动子。转录因子将在 瞬时转染法检测其对AR基因的影响 启动子活性。我们将确定雄激素、卵泡刺激素或维生素A 影响培养的大鼠支持细胞AR启动子活性。目标2将 检验AR能够招募内源性共刺激因子的假设 支持细胞中的调节蛋白，以及这些蛋白-蛋白质 相互作用激活或抑制基因的AR转录活性 在精子发生过程中是必需的。AR将用于酵母两种- 杂交蛋白相互作用实验筛选大鼠睾丸cDNA文库和 确定一组与AR相互作用的共调控蛋白以 激活或按下雄激素调节的转录。体内转染法 而可能的协同调节因子的cDNA表达将验证 特定的蛋白质与AR相互作用，改变雄激素的反式激活- 反应灵敏的记者基因。直接的蛋白质相互作用将通过以下方式验证 体外结合试验。编码可能相互作用的蛋白质的cDNA将 作为Northern blotts的探针用于确定mRNAs的表达 在各种组织和睾丸细胞(支持细胞、间质细胞、生殖细胞 细胞)，并通过原位杂交作为关联表达的手段 与AR的联合监管机构。我们的目标是了解AR的控制 以支持细胞为原代细胞的表达和转录活性 雄激素作用在生精过程中的调控机制 周而复始。

Normal spermatogenesis is dependent upon adequate levels of intratesticular androgens, primarily testosterone, and the androgen receptor (AR) is essential for mediating the actions of these steroids. We propose that the intracellular level and the transcriptional activity of the AR are likely to play key roles in determining androgen action in Sertoli cells and hence, in spermatogenesis. In aim 1, we will examine the hypothesis that stage-specific regulation of AR gene expression in Sertoli cells is affected via the differential expression of endogenous transcription factors that bind to the AR promoter and enhance or repress transcription of the AR gene. The functional regulatory regions within the rat AR promoter will be determined in transfected Sertoli cells. The stage-specificity of expression of putative transcription factors will be determined by DNA-protein binding on gel shift and DNase footprinting assays using Sertoli cell nuclear extracts from stage-synchronized seminiferous tubules. Norther and Western Blots will be used to determine the stage-specific Sertoli cell expression of transcription factors that bind to the AR promoter. Transcription factors will be overexpressed in transient transfection assays to determine their effects on AR gene promoter activity. We will determine whether androgens, FSH, or vitamin A affect AR promoter activity in cultured rat Sertoli cells. Aim 2 will examine the hypothesis that the AR is able to recruit endogenous co- regulatory proteins in Sertoli cells, and that these protein-protein interactions activate or repress AR transcriptional activity of genes required during spermatogenesis. The AR will be used in the yeast two- hybrid protein interaction assays to screen a rat testis cDNA library and to identify a cohort of co-regulatory proteins that interact with AR to activate or press androgen-regulated transcription. In vivo transfection and expression of cDNAs for putative co-regulators will verify that specific proteins interact with AR to alter transactivation of androgen- responsive reporter genes. Direct protein interactions will be verified by in vitro binding assays. cDNAs encoding putative interacting proteins will be used as probes on Northern blots to determine the expression of mRNAs in various tissues and testicular cells (Sertoli cells, Leydig cells, germ cells) and by in situ hybridization as a means to correlate the expression of co-regulators with AR. Our goal is to understand the control of AR expression and transcriptional activity in Sertoli cells as a primary mechanism by which androgen action is regulated during the spermatogenic cycle.