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，为什么精子发生需要睾酮的分子基础还没有完全被理解。这在一定程度上是因为到目前为止，很少有雄激素调节基因被明确识别出来。睾丸中大多数雄激素调节基因可能通过激活或抑制其他由雄激素直接调节的反式作用因子的转录来间接响应雄激素。最近，我们发现睾丸中一类雄激素调控的反式作用因子是microRNAs (miRNAs)，这是一种小的非编码RNA分子，通过结合mRNA中互补的顺式调控位点来抑制目标转录物的翻译。具体来说，我们已经表明，在缺乏雄激素的情况下，这些mirna中的许多被上调（表明它们的基因靶标将被下调）。我们的长期目标是了解体内功能和机制，通过这些雄激素响应mirna调节关键途径至关重要的男性生育能力。为了实现这一目标，我们产生了表达miR-471的转基因小鼠，该小鼠由我们之前广泛表征的Sertoli细胞特异性启动子Rhox5Pp驱动。我们对miR-471转基因小鼠的初步分析显示，miR-471特异性过表达导致生殖细胞凋亡急剧增加，多核巨细胞提示精母细胞减数分裂流产，减数分裂后生殖细胞分化受损和广泛的生殖细胞脱落，这一表型与造血屏障上的支持细胞-支持细胞/生殖细胞粘附受损有关，这是精子发生过程中雄激素依赖的事件。这些发现使我们假设，通过靶向特异性转录本，miR-471在支持细胞中的表达调节了生殖细胞发育和进展中至关重要的特异性雄激素依赖步骤。我们提出了两个具体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。我们将使用分子和生化方法具体评估支持细胞-支持细胞/生殖细胞粘附的完整性以及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Sertoli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能力重要的雄激素调节miRNA。在目标2中，我们将验证miR-471基因靶点并解决其在介导中的作用

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.