REGULATION OF REPRODUCTION

繁殖调节

• 批准号: 3329580
• 负责人: SYNTHIA H MELLON
• 金额: $ 23.06万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3329580
• 关键词: DNA binding protein; DNA footprinting; adrenal ferredoxin; adrenal glands; adrenocorticotropic hormone; alternatives to animals in research; cell type; chorionic gonadotropin; cyclic AMP; gel mobility shift assay; gene expression; genetic regulation; hormone regulation /control mechanism; lyase; molecular cloning; oxygenases; phospholipase C; polymerase chain reaction; site directed mutagenesis; southern blotting; steroid hormone biosynthesis; testis; tissue /cell culture; transcription factor; transfection

The long term objective of this project is a thorough understanding of the roles of steroid hormones in mammalian reproduction. To begin these studies we will use laboratory rodents and rodent cell systems derived from reproductive tissues. In general, reproductive and other tissues (ovary, testis, placenta, adrenal) chronically regulate steroid hormone synthesis by regulating the abundance of the mRNAs encoding the enzymes that synthesize steroids. It is still unclear how this occurs; the abundances of these mRNAs may be regulated both transcriptionally and post-transcriptionally. Furthermore, although the same small set of enzymes mediate steroidogenesis in different tissues, the genes encoding these enzymes are regulated differently in different tissues, and differently in the same tissue among different mammals. Our work will focus initially on P450c 17. This one enzyme has two distinct activities, 17alpha hydroxylase and 17,20 lyase, but is encoded by only one gene. In the absence of any P45Ocl7 activity, mineralocorticoids are synthesized. In the presence of only the hydroxylase activity, glucocorticoids are synthesized, and in the presence of both hydroxylase and lyase activities, sex steroids are synthesized. P45Ocl7 thus is a key branch in the synthesis of all steroid hormones. We are just beginning to understand what signals on the genes for some steroidogenic enzymes are necessary for regulation by some factors, but little is known about the regulatory sequences or factors necessary for tissue-specific regulation of these genes. The physiologic regulation of steroidogenesis in the rat is well characterized. We have already cloned rat P45Ocl7 cDNA, and now wish to clone the rat P450c 17 gene and study its regulation. By stably transfecting it into cultured adrenal and Leydig cell lines, we can study how this gene is regulated in a tissue-specific manner by factors influencing both transcriptional and post-transcriptional events. In addition, by transiently transfecting adrenal, Leydig and non-endocrine fibroblast cell lines, with putative regulatory sequences of the rat P450c 17 gene, we can determine how this gene is regulated by stimulatory/repressive- and tissue-specific factors. We will identify the DNA sequences necessary for hormonal stimulation/repression and tissue-specific expression of this gene, and will begin to characterize the nuclear proteins necessary for this regulation. Successful completion of these studies will give us a better understanding of how adrenals and testes, using the same genes, synthesize different classes of steroid hormones.

该项目的长期目标是彻底了解 类固醇激素在哺乳动物生殖中的作用。 开始这些 研究我们将使用实验室啮齿动物和啮齿动物细胞系统衍生 从生殖组织。 一般来说，生殖组织和其他组织 （卵巢、睾丸、胎盘、肾上腺）长期调节类固醇激素 通过调节编码酶的mRNA的丰度来合成 合成类固醇。 目前还不清楚这是如何发生的; 这些mRNA的丰度可以在转录和转录水平上调节， 转录后的 此外，虽然同样的一小部分 在不同的组织中，酶介导类固醇的生成， 这些酶在不同的组织中受到不同的调节， 在不同的哺乳动物的同一组织中是不同的。 我们的工作将 首先关注P450c 17。 这种酶有两种不同的 活性，17 α羟化酶和17，20裂解酶，但仅由 一个基因。 在没有任何P45Ocl7活性的情况下，盐皮质激素是 合成了 在仅存在羟化酶活性的情况下， 糖皮质激素的合成，并在存在羟化酶， 和裂解酶活性，合成性类固醇。 P45OC17是一种 所有类固醇激素合成的关键分支。 我们只是 开始了解一些类固醇生成基因上的信号 酶是某些因子调节所必需的，但目前知之甚少 关于组织特异性的调节序列或因子， 这些基因的调控。 类固醇生成的生理调节 在大鼠中的作用是很好的。 我们已经克隆了大鼠P45Ocl7 目的：克隆大鼠P450c17基因，并研究其表达调控机制。 调控 通过稳定地将其接种到培养的肾上腺和睾丸间质细胞中， 细胞系，我们可以研究这个基因是如何在组织特异性 影响转录和 转录后事件。 此外，通过瞬时检测， 肾上腺、Leydig和非内分泌成纤维细胞系， 大鼠P450c 17基因的调控序列，我们可以确定这是如何发生的。 基因受刺激/抑制因子和组织特异性因子调节。 我们将鉴定出荷尔蒙分泌所必需的DNA序列， 该基因的刺激/阻遏和组织特异性表达，和 我们将开始研究这一过程所必需的核蛋白 调控 成功完成这些研究将使我们更好地 了解肾上腺和睾丸如何使用相同的基因， 合成不同种类的类固醇激素。