Regulation of Spermatogenesis by X-linked miRNAs

描述（由申请人提供）：在精子发生过程中，减数分裂性染色体失活（MSCI）的特征是在粗线期中后期精子细胞中X染色体和Y染色体上的基因转录沉默。MSCI被认为是由于未配对DNA的减数分裂沉默，因为在第一次减数分裂前期，X和Y染色体在很大程度上保持未配对。因此，与女性胚胎或体细胞中的x染色体失活不同（据报道有25-30%的x连锁结构基因可以逃脱失活），在原代精母细胞中，没有关于基因可以逃脱MSCI沉默效应的报道。然而，我们最近发现许多X-linked microrna （mirna）在粗线精母细胞中被转录和加工。这种前所未有的MSCI逃逸表明，这些mirna在精子发生的这一阶段参与了一个或多个关键功能。我们的初步发现证实了这一点，即嵌合小鼠携带高百分比的细胞，携带一个主要的x连锁miRNA基因簇的敲除，在精子发生的减数分裂阶段表现为生育缺陷。这一点很重要，因为尽管最近的报道描述了在精子发生过程中大量的mirna和其他小的非编码rna的表达，但基本上对这些rna的功能一无所知。在本应用中，我们首先提出研究这些x连锁miRNA基因逃避MSCI的分子机制（Aim 1）。然后，我们提议测试关于这些mirna功能的两种假设（这两种假设并不相互排斥），包括它们作为常染色体mrna的转录后调节剂的作用，这些mrna在减数分裂期间合成，但直到减数分裂后才翻译（Aim 2），以及它们在调节MSCI本身过程中的作用（Aim 3）。这项研究非常新颖，因为它旨在揭示一种前所未有的逃避MSCI的机制，并确定在精子发生过程中经历这种逃避的X-linked mirna的实际功能。

DESCRIPTION (provided by applicant): Meiotic sex chromosome inactivation (MSCI) during spermatogenesis is characterized by transcriptional silencing of genes on both the X and Y chromosomes in mid to late pachytene spermatocytes. MSCI is believed to result from meiotic silencing of unpaired DNA because the X and Y chromosomes remain largely unpaired throughout first meiotic prophase. Thus, unlike X-chromosome inactivation in female embryonic or somatic cells, where 25-30% of X-linked structural genes have been reported to escape inactivation, there have been no previous reports of genes that escape the silencing effects of MSCI in primary spermatocytes. However, we recently discovered that many X-linked microRNAs (miRNAs) are transcribed and processed in pachytene spermatocytes. This unprecedented escape from MSCI suggests that these miRNAs participate in one or more critical functions at this stage of spermatogenesis. This is corroborated by our preliminary finding that chimeric mice carrying a high percentage of cells bearing a knockout of a major cluster of X-linked miRNA genes display a fertility defect manifest as a block during the meiotic phase of spermatogenesis. This is significant because despite recent reports describing expression of an abundance of miRNAs and other small, non-coding RNAs during spermatogenesis, essentially nothing is known about the function of any of these. In this application, we first propose to investigate the molecular mechanism by which these X-linked miRNA genes escape MSCI (Aim 1). We then propose to test two hypotheses (which are not mutually exclusive) regarding function of these miRNAs, including their role as post- transcriptional regulators of autosomal mRNAs that are synthesized during meiosis but not translated until the postmeiotic period (Aim 2), and their role in regulating the process of MSCI itself (Aim 3). This study is highly novel in that it is designed to reveal an unprecedented mechanism of escape from MSCI, and to identify actual functions of those X-linked miRNAs that undergo this escape during spermatogenesis. PUBLIC HEALTH RELEVANCE: Understanding the molecular and genetic mechanisms by which sperm are produced is critically important for diagnosis and treatment of male infertility, as well as for the development of non-hormonal male-specific contraceptives. Data from the present study will contribute insight into the function of X-linked miRNAs in the control of sperm production and male fertility.