Importance of Androgen regulated miRNAs in male fertility

描述（由申请人提供）：即使经过几十年的研究，精子发生需要睾酮的分子基础仍然没有完全理解。这部分是因为到目前为止，很少有雄激素调节基因被确定。睾丸中大多数雄激素调节基因可能通过激活或抑制由雄激素直接调节的其他反式作用因子的转录而间接应答雄激素。最近，我们已经表明，在睾丸中的此类雄激素调节的反式作用因子的一类是微小RNA（miRNA），小的非编码RNA分子，其通过结合到mRNA中的互补顺式调节位点来抑制靶转录物的翻译。具体地说，我们已经证明，在雄激素缺乏的情况下，这些miRNAs中的一些被上调（表明它们的基因靶点将被下调）。我们的长期目标是了解这些雄激素反应性miRNAs调节男性生育力所必需的关键途径的体内功能和机制。为了实现这一目标，我们产生了转基因小鼠表达miR-471驱动的塞尔托利细胞特异性启动子Rhox 5 Pp，我们以前已经广泛的特点。我们对miR-471转基因小鼠的初步分析显示，支持细胞特异性过表达miR-471导致生殖细胞凋亡显著增加，多核巨细胞表明精母细胞减数分裂失败，减数分裂后生殖细胞分化受损和广泛的生殖细胞脱落，这是一种与血液测试屏障处支持细胞-支持细胞/生殖细胞粘附受损相关的表型，这是精子发生过程中的雄激素依赖性事件。这些发现使我们假设，通过靶向特定的转录本，支持细胞中的miR-471表达调节生殖细胞发育和进展的关键的特定雄激素依赖性步骤。提出了两个具体的目标来验证我们的假设：在目标1中，我们将扩展我们的初步分析，并进一步表征miR-471转基因小鼠。我们将具体评估支持细胞-支持细胞/生殖细胞粘附的完整性，以及使用分子和生物化学方法的生殖细胞减数分裂阻滞。这项研究将是非常重要的，因为miR-471转基因小鼠代表了第一个研究支持细胞中特定miRNA功能的遗传模型，miR-471将是第一个被证明对男性生育力重要的雄激素调节的miRNA。在目标2中，我们将验证miR-471基因靶点并探讨它们在介导中的作用 使用体外和离体方法的miR-471的支持细胞特异性功能。这项建议所关注的雄激素调节的miRNA提供了开始解决这个问题的机会。这项工作将为清楚地了解雄激素调节男性生育力关键途径的分子机制提供重要线索。此外，这项研究计划可能会提供对miRNA在雄激素相关疾病（如前列腺癌和雄激素不足综合征）中的假定作用的见解。

DESCRIPTION (provided by applicant): Even after decades of research, the molecular basis for why spermatogenesis requires testosterone is not completely understood. This is in part because very few androgen-regulated genes have been definitively identified so far. It is likely that most androgen-regulated genes in the testis respond indirectly to androgen via activation or suppression of transcription by other trans-acting factors directly regulated by androgen. Recently, we have shown that one class of such androgen-regulated trans-acting factors in the testis are microRNAs (miRNAs), small non-coding RNA molecules that inhibit the translation of the target transcript by binding to complementary cis-regulatory sites in the mRNA. Specifically, we have shown that a number of these miRNAs are upregulated (suggesting their gene targets would be downregulated) in the absence of androgen. Our long term goal is to understand the in vivo function and mechanism by which these androgen-responsive miRNAs regulate critical pathways essential for male fertility. Toward this goal, we generated transgenic mice expressing miR-471 driven by the Sertoli cell-specific promoter Rhox5Pp that we have previously extensively characterized. Our preliminary analyses of miR-471 transgenic mice reveal that Sertoli cell-specific overexpression of miR-471 resulted in dramatically increased germ cell apoptosis, multinucleated giant cells suggesting abortive meiosis in spermatocytes, impaired postmeiotic germ cell differentiation and extensive germ cell sloughing, a phenotype that correlates with impaired Sertoli cell-Sertoli cell/germ cell adhesion at the blood-test barrier, which is an androgen-dependent event during spermatogenesis. These findings led us to hypothesize that by targeting specific transcripts, miR-471 expression in the Sertoli cells regulates specific androgen-dependent steps critical for germ cell development and progression. Two specific aims are proposed to test our hypothesis: In aim 1, we will extend our preliminary analysis and further characterize miR-471 transgenic mice. We will specifically assess the integrity of Sertoli cell-Sertoli cell/germ cell adhesion as well as germ cell meiotic arrest using molecular and biochemical approaches. This study will be highly significant as miR-471 transgenic mice represent the first genetic model to study the function of specific miRNAs in the Sertoli cells and miR-471 will be the first proven androgen-regulated miRNA important for male fertility. In aim 2, we will validate miR-471 gene targets and address their role in mediating Sertoli cell-specific function of miR-471 using in vitro and ex-vivo approaches. The androgen-regulated miRNA on which this proposal is focused provide an opportunity to begin to address this. The proposed work will provide important leads to clearly understand the molecular mechanism by which androgen regulates critical pathways essential for male fertility. Furthermore, this research initiative may provide insights into the putative role of miRNA in androgen related diseases such as prostate cancer and androgen insufficiency syndrome.