ANDROGEN RECEPTOR EXPRESSION & ACTIVITY IN SERTOLI CELLS

正常的精子发生依赖于足够水平的 睾丸内雄激素，主要是睾酮， 受体（AR）对于介导这些类固醇的作用是必需的。我们 提出，细胞内水平和转录活性的 AR可能在决定雄激素作用中起关键作用， 支持细胞，因此，在精子发生。在目标1中，我们将检查 睾丸支持细胞AR基因表达阶段特异性调控假说 细胞是通过内源性的差异表达的影响， 与AR启动子结合并增强或抑制 AR基因的转录。内的功能调节区域 将在转染的Sertoli细胞中测定大鼠AR启动子。的 假定转录因子表达的阶段特异性将被 通过DNA-蛋白质结合凝胶位移和DNA酶足迹法测定 使用来自阶段同步的睾丸支持细胞核提取物的测定 生精小管将使用Northern和Western印迹法测定 阶段特异性Sertoli细胞转录因子表达， 与AR启动子结合。转录因子将过度表达， 瞬时转染试验，以确定它们对AR基因的影响 启动子活性我们将确定雄激素FSH或维生素A 影响培养的大鼠Sertoli细胞中AR启动子的活性。目标2将 检查AR能够招募内源性共- 支持细胞中的调节蛋白，这些蛋白-蛋白 相互作用激活或抑制基因的AR转录活性 在精子发生过程中需要。AR将用于酵母二- 杂交蛋白质相互作用试验筛选大鼠睾丸cDNA文库， 鉴定一组与AR相互作用的共调节蛋白， 激活或压制雄激素调节的转录。体内转染 并且推定的共调节子的cDNA的表达将证实， 特定蛋白质与AR相互作用以改变雄激素的反式激活， 应答报告基因。直接蛋白质相互作用将通过以下方法进行验证： 体外结合测定。编码推定的相互作用蛋白质的cDNA将 用作北方印迹的探针，以确定mRNA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）中， 细胞）和通过原位杂交作为关联表达的手段， 与AR的协同调节。我们的目标是了解AR的控制 支持细胞中的表达和转录活性作为主要的 在精子发生过程中调节雄激素作用的机制 周期

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.