Regulation of insulins by the Rhox5 homeobox gene supports spermatogenesis

项目摘要 同源异型盒基因编码转录因子，这些转录因子控制多种生物学行为。 事件我最近发现了一组同源异型盒基因， 男性生殖发育和生理。许多这些生殖同源框上的 X染色体（Rhox）基因受雄激素调节，大多数在支持细胞中表达 (the支持精子发生的滋养细胞），这表明它们调节 体细胞基因产物对生殖细胞发育至关重要。事实上，有针对性地删除一个 这些基因中，Rhox 5（Pem）导致男性不育，其特征是生殖细胞数量增加， 细胞凋亡和异常精子活力。为了开始揭示由 Rhox 5在Sertoli细胞中的表达，我使用微阵列分析来鉴定Rhox 5调控的基因， 其编码在代谢中起作用的蛋白质，包括胰岛素II（Ins 2）、β-淀粉样蛋白， 脂联素和核激素受体PPAR。因为胰岛素样因子 最近被证明支持生殖细胞存活，我把我的初步研究集中在Rhox 5上 Ins 2的规则。Rhox 5表达质粒的瞬时转染增加了活性 Ins 2启动子驱动的荧光素酶报告基因构建体。这是一个突破性的发现， 我们的实验室，因为没有假定Rhox 5反应元件曾经发现。Rhox 5介导 Ins 2启动子的激活是支持细胞特异性的，因为它发生在支持细胞系中， 从第12天睾丸纯化的支持细胞，但不是来自其他组织的细胞系。这 表明支持细胞特异性辅因子是Rhox 5介导的转录所必需的。 activation.在本申请中，我建议描述Rhox 5 调节睾丸中的靶基因。这些实验被设计来鉴定辅因子， 与Rhox 5一起工作以实现Rhox 5调节基因的细胞类型特异性表达。因为 Rhox 5正调控支持生殖细胞存活的代谢因子，阐明了生殖细胞的存活机制。 导致Rhox 5缺失小鼠生育力低下的转录控制机制提供了一种有用的 了解人类生育力低下的模型系统。无法治疗的低生育力造成的贫穷 精液质量占咨询生育问题患者的75% 75%的患者因精液质量差而导致无法治疗的生育能力低下 咨询生育问题这主要是由于精子数量的缺陷 （少精子症）和精子活力（弱精子症）。我所提出的研究旨在 了解Rhox 5同源框转录因子控制细胞凋亡的机制。 支持男性生殖细胞发育的基因表达。缺乏Rhox 5基因的小鼠 精子数量、精子活力和繁殖效率出现异常。因此 Rhox 5缺失小鼠的表征也可用作理解 人类生育问题。

Project Summary Homeobox genes encode transcription factors that control a wide variety of biological events. I recently identified a cluster of homeobox genes that are good candidates to regulate male reproductive development and physiology. Many of these reproductive homeobox on the X chromosome (Rhox) genes are androgen regulated and most are expressed in Sertoli cells (the nurse cells that support spermatogenesis), suggesting that they regulate the expression of somatic-cell gene products crucial for germ-cell development. Indeed, targeted deletion of one of these genes, Rhox5 (Pem), results in male subfertility, marked by increased germ-cell apoptosis and abnormal sperm motility. To begin to uncover the molecular events triggered by Rhox5 in Sertoli cells, I used microarray analysis to identify Rhox5-regulated genes, many of which encode proteins that have roles in metabolism, including insulin II (Ins2), resistin, and adiponectin, and the nuclear hormone receptor PPAR. Because insulin-like factors have recently been shown to support germ cell survival, I focused my initial studies on Rhox5 regulation of Ins2. Transient transfection of Rhox5 expression plasmids increased the activity of Ins2 promoter-driven luciferase reporter constructs. This was a breakthrough discovery for our laboratory, as no putative Rhox5-response elements had ever discovered. Rhox5-mediated activation of the Ins2 promoter was Sertoli-cell specific, as it occurred in Sertoli cell lines and Sertoli cells purified from day 12 testes, but not cell lines derived from other tissues. This suggests that a Sertoli-specific cofactor is required for Rhox5-mediated transcriptional activation. In this application, I propose to characterize the mechanism by which Rhox5 regulates target genes in the testes. The experiments are designed to identify cofactors that work with Rhox5 to achieve cell-type specific expression of Rhox5-regulated genes. Because Rhox5 positively regulates metabolic factors that support germ cell survival, elucidation of the transcription control mechanism that leads to subfertility in Rhox5-null mice provides a useful model system for understanding human subfertility. Untreatable subfertility caused by poor semen quality accounts for 75% of patients consulting for fertility problems Project Narrative Untreatable subfertility caused by poor semen quality accounts for 75% of patients consulting for fertility problems. This is due mainly to defects in sperm numbers (oligozoospermia) and sperm motility (asthenozoospermia). My proposed research strives to understand the mechanism by which the Rhox5 homeobox transcription factor controls the expression of genes that support male germ cell development. Mice which lack the Rhox5 gene exhibit abnormalities in sperm number, sperm motility, and breeding efficiency. Thus, the characterization of Rhox5-null mice may also be useful as a model system for understanding human fertility problems.