Regulation of insulins by the Rhox5 homeobox gene supports spermatogenesis

描述(申请人提供)：同源框基因编码转录因子，控制广泛的生物事件。我最近发现了一组同源框基因，它们是调节男性生殖发育和生理的很好的候选基因。许多这些生殖同源框(ROX)基因是雄激素调节的，并且大多数在支持精子发生的支持细胞(Sertoli Cell)中表达，这表明它们调节对生殖细胞发育至关重要的体细胞基因产物的表达。事实上，这些基因中的一种Rhox5(Pem)的定向缺失会导致男性不育，其特征是生殖细胞凋亡增加和精子运动异常。为了开始揭示Rhox5在Sertoli细胞中触发的分子事件，我使用微阵列分析来鉴定Rhox5调节的基因，其中许多编码在新陈代谢中起作用的蛋白质，包括胰岛素II(Ins2)、抵抗素和脂联素，以及核激素受体PPAR。由于胰岛素样因子最近被证明支持生殖细胞存活，我最初的研究重点是Rhox5对Ins2的调控。瞬时转染Rhox5表达载体可提高Ins2启动子驱动的荧光素酶报告基因的活性。这对我们的实验室来说是一个突破性的发现，因为还没有发现任何可能的Rhox5反应元件。Rhox5介导的Ins2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和从第12天纯化的支持细胞中，而不是来自其他组织的细胞系。这表明Rhox5介导的转录激活需要Sertoli特异的辅因子。在这项应用中，我建议描述Rhox5调节睾丸靶基因的机制。这些实验旨在确定与Rhox5共同作用的辅因子，以实现Rhox5调控基因的细胞类型特异性表达。由于Rhox5正向调节支持生殖细胞存活的代谢因子，阐明导致Rhox5基因缺失小鼠不育的转录控制机制为理解人类不育提供了有用的模型系统。在就诊于生育问题的患者中，75%的患者是由于精液质量差而导致的无法治愈的不育症。这主要是由于精子数量(少精子症)和精子活力缺陷(弱精子症)造成的。我提出的研究致力于了解Rhox5同源框转录因子控制支持男性生殖细胞发育的基因表达的机制。缺乏Rhox5基因的小鼠表现出精子数量、精子活力和繁殖效率的异常。因此，Rhox5缺失小鼠的特征也可以作为理解人类生育问题的模型系统。

DESCRIPTION (provided by applicant): 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 (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 been 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. 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.