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）：睾丸激素对于精子发生绝对必要，并通过雄激素受体（AR）靶向哺乳动物睾丸中的Sertoli细胞，以产生生殖细胞发育和生存所需的因素。我们现在有证据表明，睾丸激素刺激ERK MAP激酶的磷酸化（激活）和一级大鼠Sertoli细胞中CREB转录因子的替代性（<5分钟）机制。这种新颖的睾丸激素作用机制将影响Sertoli细胞中的大量过程，因为MAP激酶和CREB是控制众多信号通路的焦点。此外，该提案解决了对睾丸激素调节精子发生的机制的长期差距，因为已经确定了较少的AR调节基因。我们将测试总体假设，即睾丸激素与AR的结合激活SRC激酶和/或Ca2+介导的信号传导级联反应，从而导致MAP激酶和CREB的磷酸化和激活。 目的1是检验AR需要介导MAP激酶和CREB快速磷酸化的假设。雄激素诱导的ERK和CREB磷酸化将在正常和AR缺乏的大鼠原发性Sertoli细胞中测定。将确定雄激素信号所需的AR结构域。将测试与Sertoli细胞质膜相关的AR种群的假设。 AIM 2是测试雄激素通过SRC激酶调节的途径和/或通过CA2+介导的途径激活MAP激酶和CREB的假设。将确定雄激素诱导的SRC激酶活性，并在添加基于药理和基因的SRC活性抑制剂后测定ERK和CREB枯萎病的雄激素激活。 Ca2+通道和Ca2+动作的特定阻滞剂将用于检验以下假设：Ca2+介导的信号通路传播雄激素信号。 AIM 3是测试雄激素激活CREB和MAP激酶调节基因表达的假设。 MAP激酶和CREB介导的基因表达的雄激素调节将在瞬时转染测定中使用报告基因质粒进行测试。将使用RNase保护测定法评估对特定内源性Sertoli细胞靶基因的调节。从这项研究产生的信息将直接适用于分化睾丸激素支持精子发生并维持男性生育能力的机制。