Regulation of Leydig cell function by small noncoding RNAs

描述（由申请人提供）：间质细胞是睾丸的间质细胞，是男性睾酮(T)产生的主要来源。正常的睾酮分泌不仅对正常的精子产生和男性生育能力至关重要，而且对正常的性行为和男性特有的生理机能也至关重要。间质细胞发育和功能的分子机制在很大程度上仍不清楚。我们最近在小鼠中证明，当我们通过灭活两种关键的RNase III酶（Dicer或Drosha）来抑制microRNA （miRNA）的产生时，间质细胞不能正常工作。这两种酶是miRNA生物合成所必需的。在缺乏mirna的情况下，成年小鼠的间质细胞过量产生睾酮，下丘脑-垂体-睾丸负反馈系统出现故障，导致T水平持续升高，雌二醇（E2）水平下降，促性腺激素（FSH和LH）水平轻微升高。这种异常的激素谱伴随着精子缺陷，其特征是精子头尾分离，精子头缺乏运动性和变形，导致雄性突变小鼠完全不育。我们的初步数据表明，正常的miRNA产生对于正常的间质细胞功能（即正常的T和E2产生以及正常的下丘脑-垂体-睾丸（HPT）轴反馈）、正常的精子产生和男性生育能力至关重要。因此，我们提出这项初步研究来回答关于间质细胞mirna的两个基本问题：1)间质细胞中通常表达多少mirna ？为了回答这个问题，我们将使用miRNA深度测序（miRNA- seq）来定义对照和缺乏miRNA的间质细胞中的miRNA转录组。2) mirna的缺失如何影响间质细胞的mRNA转录组和蛋白质组？为了回答这些问题，我们将使用基于mRNA- seq和itraq的质谱（MS）来定义成人对照和mirna缺陷的间质细胞的mRNA转录组和蛋白质组。这项研究的数据将使我们能够建立导致类固醇生成异常的失调基因与缺乏关键的间质细胞miRNAs（本研究的终点）之间的相关性，这将为进一步研究miRNAs控制间质细胞功能的具体机制奠定基础（未来R01提案的起点）。基于这一初步研究的未来研究可能会为内分泌失调导致的男性不育带来新的诊断和治疗方法，mirna可能代表一类新的男性避孕药。

DESCRIPTION (provided by applicant): Leydig cells are interstitial cells of the testis and represent the major source of testosterone (T) production in men. Normal T production is critical not only for normal sperm production and male fertility, but also for normal sexual behavior and male-specific physiology in general. The molecular mechanism underlying the development and function of Leydig cells remains largely undefined. We recently demonstrated in mice that when we inhibited the microRNA (miRNA) production by inactivating either of the two key RNase III enzymes, Dicer or Drosha, which are known to be essential for miRNA biosynthesis, Leydig cells could not function normally. In the absence of miRNAs, Leydig cells in adult mice overproduced testosterone, and the hypothalamus-pituitary-testis negative feedback system malfunctioned, leading to persistently elevated levels of T, decreased levels of estradiol (E2), and slightly increased levels of both gonadotropins (FSH and LH). This abnormal hormonal profile is accompanied by sperm defects characterized by separation of sperm heads from tails, lack of motility and deformation of sperm heads, which led to complete infertility in male mutant mice. Our preliminary data suggest that normal miRNA production is essential for normal Leydig cell functions [i.e. normal T and E2 production and normal hypothalamus-pituitary- testis (HPT) axis feedback], normal sperm production and male fertility. We, therefore, propose this pilot study to answer two fundamental questions concerning Leydig cell miRNAs: 1) How many miRNAs are normally expressed in Leydig cells? To answer this question, we will define the miRNA transcriptome in control and miRNA-deficient Leydig cells using miRNA deep sequencing (miRNA-Seq). 2) How does depletion of miRNAs affect mRNA transcriptome and proteome in Leydig cells? To answer these questions, we will define the mRNA transcriptome and proteome in adult control and miRNA-deficient Leydig cells using mRNA-Seq and iTRAQ-based mass spectrometry (MS). Data from this study will allow us to establish correlations between dysregulated genes leading to abnormal steroidogenesis and the absence of critical Leydig cell miRNAs (end-point of this study), which will serve as the basis for a further study on the specific mechanisms underlying the control of Leydig cell functions by miRNAs (starting point of the future R01 proposal). Future investigation based upon this pilot study may lead to novel diagnostics and treatments for male infertility due to endocrine disorders, and miRNAs may represent a novel class of male contraceptive agents.