Nongenomic Androgen Signaling in Sertoli Cells

支持细胞中的非基因组雄激素信号传导

• 批准号: 7049420
• 负责人: WILLIAM H WALKER
• 金额: $ 22.84万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-09 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7049420
• 关键词: SDS polyacrylamide gel electrophoresis; Sertoli cells; androgen receptor; androgens; biological signal transduction; cAMP response element binding protein; confocal scanning microscopy; fertility; gene expression; genetic transcription; hormone regulation /control mechanism; immunoprecipitation; laboratory rat; mitogen activated protein kinase; phosphorylation; polymerase chain reaction; spermatogenesis; testosterone; thin layer chromatography; tissue /cell culture; transcription factor; western blottings

DESCRIPTION (provided by applicant): Testosterone is absolutely essential for spermatogenesis and targets Sertoli cells in the mammalian testis through the androgen receptor (AR) to produce factors required for germ cell development and survival. We now have evidence for an alternative, rapid (< 5 min) mechanism by which testosterone stimulates the phosphorylation (activation) of the Erk MAP kinase and the CREB transcription factor in primary rat Sertoli cells. This novel mechanism of testosterone action will impact a broad number of processes in Sertoli cells because MAP kinase and CREB are focal points for the control of numerous signaling pathways. Furthermore, this proposal addresses a longstanding gap in the understanding of the mechanisms by which testosterone regulates spermatogenesis as few AR-regulated genes have been identified. We will test the overall hypothesis that testosterone binding to AR activates a Src kinase and/or Ca2+-mediated signaling cascade resulting in the phosphorylation and activation of MAP kinase and CREB. Aim 1 is to test the hypothesis that AR is required to mediate the rapid phosphorylation of MAP kinase and CREB. Androgen-induced ERK and CREB phosphorylation will be assayed in normal and AR deficient rat primary Sertoli cells. AR domains required for androgen signaling will be identified. The hypothesis that populations of AR are associated with the Sertoli cell plasma membrane will be tested. Aim 2 is to test the hypothesis that androgen activates MAP kinase and CREB by a Src kinase-regulated pathway and/or via a Ca2+-mediated pathway. Androgen-induced Src kinase activity will be determined and androgen-activation of Erk and CREB wilt be assayed after addition of pharmacological and gene-based inhibitors of Src activity. Specific blockers of Ca2+ channels and Ca2+ actions will be used to test the hypothesis that Ca2+-mediated signaling pathways propagate androgen signals. Aim 3 is to test the hypothesis that androgen activates CREB and MAP kinase-regulated gene expression. Androgen regulation of MAP kinase and CREB-mediated gene expression will be tested using reporter plasmids in transient transfection assays. The regulation of specific endogenous Sertoli cell target genes via CREB will be assessed using RNAse protection assays. The information generated from this study will be directly applicable to divining the mechanisms by which testosterone supports spermatogenesis and maintains male fertility.

描述（由申请人提供）：睾酮对于精子发生绝对重要，并通过雄激素受体（AR）作用于哺乳动物睾丸中的支持细胞，产生生殖细胞发育和存活所需的因子。我们现在有证据表明，睾酮可通过另一种快速（< 5 分钟）机制刺激原代大鼠支持细胞中 Erk MAP 激酶和 CREB ​​转录因子的磷酸化（激活）。这种睾酮作用的新机制将影响支持细胞中的许多过程，因为 MAP 激酶和 CREB ​​是控制众多信号通路的焦点。此外，该提案解决了对睾酮调节精子发生机制的理解中长期存在的空白，因为目前尚未发现 AR 调节基因。我们将测试总体假设，即睾酮与 AR 结合会激活 Src 激酶和/或 Ca2+ 介导的信号级联，从而导致 MAP 激酶和 CREB ​​的磷酸化和激活。 目标 1 是检验 AR 是介导 MAP 激酶和 CREB ​​快速磷酸化所必需的假设。雄激素诱导的 ERK 和 CREB ​​磷酸化将在正常和 AR 缺陷的大鼠原代支持细胞中进行测定。雄激素信号传导所需的 AR 域将被确定。 AR 群体与支持细胞质膜相关的假设将得到检验。目标 2 是检验雄激素通过 Src 激酶调节途径和/或 Ca2+ 介导途径激活 MAP 激酶和 CREB ​​的假设。在添加Src活性的药理学和基于基因的抑制剂后，将测定雄激素诱导的Src激酶活性，并测定Erk和CREB的雄激素激活。 Ca2+ 通道和 Ca2+ 作用的特异性阻断剂将用于测试 Ca2+ 介导的信号通路传播雄激素信号的假设。目标 3 是检验雄激素激活 CREB ​​和 MAP 激酶调节基因表达的假设。雄激素对 MAP 激酶的调节和 CREB ​​介导的基因表达将在瞬时转染测定中使用报告质粒进行测试。将使用 RNAse 保护测定来评估通过 CREB ​​对特定内源支持细胞靶基因的调节。这项研究产生的信息将直接应用于推测睾酮支持精子发生和维持男性生育能力的机制。