microRNA regulation of spermatogonial stem cell self-renewal and differentiation

描述（申请人提供）：精原干细胞（SSC）是一种能够自我更新和分化的成体干细胞，因此在精子发生中起着关键作用。了解支配SSC自我更新和分化的过程将为男性生育力的调节提供必要的机械见解，并且对于动物和人类的男性不育症的未来治疗至关重要。在我们正在进行的努力，以了解管理SSC生物学的调控机制，我们研究了微RNA（miR）的潜在作用，小RNA分子，抑制mRNA的目标翻译。众所周知，基因在调节干细胞中具有重要作用;然而，关于SSC的miR调节知之甚少。生殖细胞中miR加工蛋白Dicer的去除大大减少了精子发生，表明miR对于SSC的增殖和分化是必不可少的，但是还没有研究证明特异性miR的直接功能。使用高通量序列分析，我们生成了高度富集SSC的鼠生殖细胞特有的miR谱，其中包括高水平的miR-21，并已表明miR-21表达有助于SSC干性。此外，我们和其他人已经证明了胶质细胞源性神经营养因子（GDNF）信号在促进SSC自我更新和增殖的重要性。我们的假设是miR对SSC自我更新的贡献部分由GDNF控制，并且GDNF信号传导和miR靶基因抑制的两个过程作为介导SSC命运的更大调控网络的一部分协同起作用。因此，在具体目标1中，我们将进一步评估GDNF诱导的转录因子依赖性miR表达的作用及其对下游靶mRNA在调节GDNF信号传导和SSC自我更新、分化和凋亡中的影响。精原细胞和精子的多阶段分化过程需要大量的细胞外和细胞内的信号线索，我们假设miR也调节生殖细胞的分化。因此，在特定目标2中，我们将使用视黄酸（一种已知的生殖细胞分化诱导剂）来评估miR在早期精子发生过程中的表达和功能。此外，我们将利用专门为这些研究开发的创新转基因小鼠品系来跟踪和定量精原干细胞向分化精子的进展。有了这些资源，我们可以研究miR在分化过程中的功能重要性。我们预测，这些研究将以与我们最初对精原细胞移植的研究改变了我们对SSC生物学的理解相同的方式改变该领域。 公共卫生相关性：精原干细胞是一种成体干细胞，其经历自我更新并产生分化的子细胞，子细胞成为精子，这是精子发生所必需的过程，是男性生育力的基础。不孕症影响约15%的夫妇，其中近50%与男性有关，其中大部分涉及精子发生缺陷。这些研究对于了解精子发生的调节和男性不育的原因以及改善治疗方案至关重要。

DESCRIPTION (provided by applicant): The spermatogonial stem cell (SSC), is an adult stem cell capable of both self-renewal and differentiation, thereby playing a critical role in spermatogenesis. Understanding the processes that govern SSC self-renewal and differentiation will provide essential mechanistic insight into the regulation of male fertility and is critical for future therapeutic treatment of male infertility in both animals and humans. In our ongoing efforts to understand the regulatory mechanisms governing SSC biology, we investigated the potential role of micro-RNAs (miRs), small RNA molecules that inhibit the translation of mRNA targets. It is well established that genes have an important role in regulating stem cells; however, little is known about miR regulation of the SSC. Abrogation of the miR processing protein, Dicer, in germ cells greatly reduces spermatogenesis, suggesting miRs are essential for the proliferation and differentiation of the SSC, but there have been no studies to demonstrate a direct function of specific miRs. Using high-throughput sequence analysis, we generated a miR profile unique to murine germ cells highly enriched for SSCs, which includes high levels of miR-21, and have shown that miR-21 expression contributes to SSC stemness. Moreover, we and others have demonstrated the importance of glial cell line-derived neurotrophic factor (GDNF) signaling in promoting SSC self-renewal and proliferation. Our hypothesis is that the contribution of miRs to SSC self-renewal is in part controlled by GDNF, and that the two processes of GDNF signaling and miR-target gene repression function in concert as part of a larger regulatory network mediating the fate of the SSC. Therefore, in Specific Aim 1, we will further evaluate the role of GDNF-induced transcription factor-dependent miR expression and its consequent effects on downstream target mRNAs in regulating GDNF signaling and SSC self-renewal, differentiation, and apoptosis. The multistage differentiative process of SSCs into spermatogonia and spermatozoa requires a multitude of extra- and intracellular signaling cues and we hypothesize that miRs also regulate the differentiation of germ cells. Therefore, in Specific Aim 2, we will use retinoic acid, a known inducer of germ cell differentiation, to evaluate the expression and function of miRs during early-stage spermatogenesis. Moreover, we will utilize innovative transgenic mouse lines specifically developed for these studies to track and quantitate the progress of SSCs towards differentiated spermatozoa. With these resources we can investigate the functional importance of miRs in the process of differentiation. We predict that these studies will transform the field in much the same way that our original studies on spermatogonial transplantation transformed our understanding of SSC biology. PUBLIC HEALTH RELEVANCE: The spermatogonial stem cell is an adult stem cell that undergoes both self-renewal and produces differentiated daughter cells that become spermatozoa, a process that is essential to spermatogenesis and is the foundation of male fertility. Infertility affects ~15% of couples, with nearly 50% being male-related and much of this involves defective spermatogenesis. The studies proposed are essential to understanding the regulation of spermatogenesis and causes of male infertility as well as for improvement of therapeutic treatment options.