microRNA regulation of spermatogonial stem cell self-renewal and differentiation

描述（由申请人提供）：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。了解控制SSC自我更新和分化的过程将为男性生育能力的调节提供必要的机制见解，对未来动物和人类男性不育的治疗至关重要。在我们持续努力了解SSC生物学调控机制的过程中，我们研究了微RNA （miRs）的潜在作用，即抑制mRNA靶标翻译的小RNA分子。众所周知，基因在调节干细胞中起着重要作用；然而，miR对SSC的调控知之甚少。生殖细胞中miR加工蛋白Dicer的缺失大大减少了精子的发生，这表明miR对SSC的增殖和分化至关重要，但目前还没有研究证明特异性miR的直接功能。利用高通量序列分析，我们生成了高度富集SSC的小鼠生殖细胞所特有的miR谱，其中包括高水平的miR-21，并表明miR-21的表达有助于SSC的干细胞性。此外，我们和其他人已经证明了神经胶质细胞系来源的神经营养因子（GDNF）信号在促进SSC自我更新和增殖中的重要性。我们的假设是，miRs对SSC自我更新的贡献部分由GDNF控制，GDNF信号传导和mir -靶基因抑制这两个过程作为一个更大的调节网络的一部分协同起作用，介导SSC的命运。因此，在Specific Aim 1中，我们将进一步评估GDNF诱导的转录因子依赖性miR表达及其对下游靶mrna的影响在调节GDNF信号传导和SSC自我更新、分化和凋亡中的作用。ssc向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信号提示，我们假设miRs也调节生殖细胞的分化。因此，在Specific Aim 2中，我们将使用维甲酸（一种已知的生殖细胞分化诱导剂）来评估早期精子发生过程中mir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系来跟踪和量化ssc向分化精子的进展。有了这些资源，我们可以研究miRs在分化过程中的功能重要性。我们预测，这些研究将改变这个领域，就像我们对精子移植的原始研究改变了我们对SSC生物学的理解一样。

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.