microRNA regulation of spermatogonial stem cell self-renewal and differentiation

microRNA对精原干细胞自我更新和分化的调控

基本信息

批准号：
8214793
负责人：
Ralph Lawrence Brinster
金额：
$ 24万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-16 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8214793
关键词：
Affect Animals Apoptosis Biological Assay Cell Differentiation process Cell Transplants Cell physiology Cells Couples Cues Culture Techniques Evaluation Fertility Foundations Functional disorder Future Gene Targeting Genes Genetic Translation Genetic Variation Germ Cells Goals Hematopoiesis Human In Vitro Infertility Male Infertility Malignant Neoplasms Measures Mediating Medical Messenger RNA Methods MicroRNAs Mus Play Population Process Protamines Proteins Regulation Research Resources Role Sequence Analysis Signal Transduction Small RNA Spermatids Spermatogenesis Spermatogonia Staging Stem cells System Testis Therapeutic Transgenic Mice Transplantation Tretinoin adult stem cell daughter cell enhanced green fluorescent protein extracellular gene repression glial cell-line derived neurotrophic factor human DICER1 protein in vivo innovation insight male promoter reproductive function research study self-renewal sperm cell stem cell biology stem cell fate stemness transcription factor transgene expression

项目摘要

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.

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，从而在精子发生中发挥关键作用。了解控制 SSC 自我更新和分化的过程将为了解男性生育力的调节提供重要的机制见解，并且对于未来动物和人类男性不育症的治疗至关重要。在我们不断努力了解控制 SSC 生物学的调控机制的过程中，我们研究了 micro-RNA (miR) 的潜在作用，这种小 RNA 分子抑制 mRNA 靶标的翻译。众所周知，基因在调节干细胞中发挥着重要作用。然而，人们对 miR 对 SSC 的调节知之甚少。生殖细胞中 miR 加工蛋白 Dicer 的废除大大减少了精子发生，表明 miR 对于 SSC 的增殖和分化至关重要，但尚未有研究证明特定 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 的小鼠生殖细胞特有的 miR 谱，其中包括高水平的 miR-21，并表明 miR-21 表达有助于 SSC 干细胞性。此外，我们和其他人已经证明了胶质细胞系源性神经营养因子（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，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 靶基因抑制这两个过程作为介导 SSC 命运的更大调控网络的一部分协同发挥作用。。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传导，我们假设 miR 也调节生殖细胞的分化。因此，在具体目标 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品系来跟踪和定量 SSC 向分化精子的进展。借助这些资源，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改变这个领域，就像我们最初对精原细胞移植的研究改变了我们对 SSC 生物学的理解一样。公共健康相关性：精原干细胞是一种成体干细胞，可以进行自我更新并产生分化的子细胞，从而成为精子，这是精子发生所必需的过程，也是男性生育能力的基础。不孕症影响约 15% 的夫妇，其中近 50% 与男性有关，其中大部分与精子发生缺陷有关。所提出的研究对于了解精子发生的调节和男性不育的原因以及改进治疗方案至关重要。