HORMONAL REGULATION OF GENE EXPRESSION IN SERTOLI CELLS

支持细胞基因表达的激素调节

• 批准号: 3320823
• 负责人: DAVID R JOSEPH
• 金额: $ 11.42万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 1989-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3320823
• 关键词: Sertoli cells; androgen binding protein; androgens; complementary DNA; follicle stimulating hormone; gene expression; genetic library; genetic manipulation; genetic mapping; genetic promoter element; genetic transcription; hormone regulation /control mechanism; hypophysectomy; laboratory rat; messenger RNA; molecular cloning; nucleic acid hybridization; nucleic acid sequence; protein sequence; regulatory gene; spermatogenesis; tissue /cell culture

FSH and androgens act on Sertoli cells to regulate the secretion of proteins that function in the control of spermatogenesis. However, the mechanisms by which these hormones regulate Sertoli gene expression remain incompletely understood. One of the secretory proteins, androgen binding protein (ABP), has been used extensively as a marker for studies on the regulation of Sertoli cell function. Although the role of ABP in spermatogenesis and sperm maturation is not known, the hormonal control of ABP secretion closely parallels the control of spermatogenesis. Our goal is to develop the rat ABP gene as a model system to determine the mechanisms of hormone mediated regulation of Sertoli cell secretory processes. Toward these goals, we have prepared a rat testis cDNA library in Lambda gt11 and have isolated recombinants that contain cDNA complementary to ABP mRNA. Recombinants were identified with ABP antiserum and their identity was confirmed by hybrid-arrested translation and epitope selection. The total nucleotide sequence of ABP cDNA isolates has revealed the amino acid sequence of the ABP precursor and its signal peptide. Other studies suggest that both protomers of ABP are coded for by one mRNA species and one gene. With the ABP cDNA we propose to isolate the gene for ABP, map and sequence intron-exon regions, identify the transcription initiation site and locate regulatory DNA sequences. A particular emphasis will be placed on examination of the 5'-upstream region of the gene which may contain hormone regulatory sequences. ABP cDNA will also be used as a probe to study hormone regulated gene expression. These studies will include in vivo experiments with hypophysectomized rats and Tfm rats as well as in vitro experiments with isolated seminiferous tubules and cultured Sertoli Cells. The levels of ABP mRNA will be analyzed by northern blot hybridization and further quantitated by solution hybridization with RNA probes. We will also attempt to measure transcription rates of the ABP gene by hybrid selection assay and translation rates of ABP mRNA by immune precipitation. This work will develop a system to study the mechanisms of FSH and androgen action using a gene expressed specifically in Sertoli Cells.

FSH和雄激素作用于支持细胞，调节 控制精子发生的蛋白质。 但 这些激素调节Sertoli基因表达的机制仍然存在 不完全理解。 一种分泌蛋白，雄激素结合 蛋白（ABP），已被广泛用作研究的标志物， 支持细胞功能的调节。 虽然ABP在 精子发生和精子成熟是未知的，激素的控制， ABP分泌与精子发生的控制密切相关。 我们的目标 是开发大鼠ABP基因作为模型系统，以确定 激素介导的支持细胞分泌调节机制 流程. 为了实现这些目标，我们制备了大鼠睾丸cDNA文库， gt 11，并分离了含有与ABP互补的cDNA的重组体 mRNA。 用ABP抗血清鉴定澄清剂， 通过杂交停滞翻译和表位选择得到证实。 的 ABP cDNA分离物的总核苷酸序列揭示了氨基酸 ABP前体及其信号肽的序列。 其他研究 表明ABP两种原聚体由一种mRNA种类编码， 一个基因。 利用ABP cDNA，我们建议分离ABP基因，作图和测序 内含子-外显子区域，确定转录起始位点并定位 调节DNA序列。 将特别强调 检查可能含有激素的基因的5 '上游区域 调节序列 ABP cDNA也将被用作研究的探针 激素调节基因表达。 这些研究将包括体内 垂体切除大鼠和Tfm大鼠的实验以及体外实验 用分离的生精小管和培养的支持细胞进行实验。 将通过北方印迹杂交分析ABP mRNA水平， 通过与RNA探针的溶液杂交进一步定量。 我们将 我还试图通过杂交来测量ABP基因的转录率， 通过免疫沉淀法测定ABP mRNA的选择测定和翻译速率。 本工作将建立一个研究FSH和雄激素作用机制的系统 使用在支持细胞中特异性表达的基因的作用。