HORMONAL REGULATION OF GENE EXPRESSION IN SERTOLI CELLS

FSH和雄激素作用于Sertoli细胞，调节其分泌 在控制精子发生过程中起作用的蛋白质。然而， 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。 支持细胞功能的调节。尽管总部基地在这方面的作用 精子发生和精子成熟尚不清楚，激素控制 ABP的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基因作为模型系统来确定 激素调节支持细胞分泌的机制 流程。 为了达到这些目标，我们在Lambda中准备了一个大鼠睾丸cdna文库 Gt11，并分离出含有与ABP互补的cdna的重组子。 MRNA.用ABP抗血清鉴定重组子及其同源性 经杂交抑制翻译和表位选择证实。这个 ABP分离株的全核苷酸序列揭示了该氨基酸 ABP前体及其信号肽的序列。其他研究 提示ABP的两个原核糖体均由一种mRNA物种编码， 只有一个基因。 利用ABP基因，我们提出了分离ABP基因、图谱和序列 内含子-外显子区域，鉴定转录起始点并定位 调控的DNA序列。我们将特别强调 检测该基因可能含有激素的5‘-上游区域 调控序列。ABP cDNAs也将作为探针用于研究 荷尔蒙调节基因表达。这些研究将包括在体内 去垂体大鼠和TFM大鼠的体外实验 分离的生精小管和培养的支持细胞的实验。 通过Northern印迹杂交和蛋白质印迹杂交分析ABP基因的表达水平 通过与RNA探针的溶液杂交进一步定量。我们会 也尝试通过杂交来测量ABP基因的转录速率 免疫沉淀法对ABP基因的筛选、鉴定及转化率。 这项工作将建立一套系统来研究FSH和雄激素的作用机制 使用在支持细胞中特异表达的基因的作用。

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.