ANDROGEN RECEPTOR EXPRESSION & ACTIVITY IN SERTOLI CELLS

雄激素受体表达

• 批准号: 6440545
• 负责人: TERRY R. BROWN
• 金额: $ 17.42万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6440545
• 关键词: DNA footprinting; Leydig cells; Sertoli cells; androgen receptor; follicle stimulating hormone; gel mobility shift assay; gene expression; genetic promoter element; genetic transcription; germ cells; in situ hybridization; laboratory rat; northern blottings; reporter genes; retinoids; spermatogenesis; testis; tissue /cell culture; transcription factor; transfection; western blottings; yeast two hybrid system

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.

正常的精子发生取决于足够的水平 肠内雄激素，主要是睾丸激素和雄激素 受体（AR）对于介导这些类固醇的作用至关重要。我们 提出细胞内水平和转录活性 AR可能在确定雄激素作用中起关键作用 Sertoli细胞，因此在精子发生中。在AIM 1中，我们将检查 假设Sertoli中AR基因表达的阶段特异性调节 细胞通过内源性的差分表达受到影响 与AR启动子结合并增强或压抑的转录因子 AR基因的转录。功能调节区域 将在转染的Sertoli细胞中确定大鼠AR启动子。这 推定转录因子表达的阶段特异性将是 通过DNA-蛋白质结合在凝胶移位和DNase足迹上确定 使用Sertoli细胞核提取物的测定 精神小管。 Norther和Western印迹将用于确定 转录因子的特定阶段特异性Sertoli细胞表达 与AR启动子结合。转录因子将过表达 瞬态转染测定法以确定其对AR基因的影响 启动子活动。我们将确定雄激素，FSH或维生素A是 影响培养的大鼠Sertoli细胞中的AR启动子活性。 AIM 2意志 检查AR能够募集内源性共同的假设 Sertoli细胞中的调节蛋白，这些蛋白质蛋白 相互作用激活或抑制基因的AR转录活性 精子发生过程中需要。 AR将在酵母中使用两种 混合蛋白质相互作用测定法以筛选大鼠睾丸cDNA文库和 确定与AR相互作用的共同调节蛋白的队列 激活或按下雄激素调节的转录。体内转染 和推定共同调节器的CDNA的表达将验证 特定蛋白质与AR相互作用以改变雄激素的反式激活 响应迅速的记者基因。直接蛋白质相互作用将通过 体外结合测定。编码推定相互作用蛋白的cDNA将会 用作北印迹上的探针来确定mRNA的表达 在各种组织和睾丸细胞中（Sertoli细胞，Leydig细胞，生殖 细胞）和原位杂交作为将表达相关的一种手段 与AR共同调节器的。我们的目标是了解AR的控制 Sertoli细胞中的表达和转录活性作为主要 精子发生期间调节雄激素作用的机制 循环。