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

• 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
• 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

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（由申请人提供）：精子干细胞（SSC）是一个能够自我更新和分化的成年干细胞，因此在精子发生中起着关键作用。了解管理SSC自我更新和分化的过程将为男性生育能力的调节提供基本的机械洞察力，对于将来对动物和人类男性不孕症的治疗治疗至关重要。在我们为了解有关SSC生物学的调节机制的持续努力中，我们研究了微RNA（miRS）的潜在作用，即抑制mRNA靶标转换的小RNA分子。众所周知，基因在调节干细胞中具有重要作用。但是，关于SSC的miR调节知之甚少。生殖细胞中miR加工蛋白DICER的废除大大降低了精子发生，这表明MIR对于SSC的增殖和分化至关重要，但没有研究证明特定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的鼠类生殖细胞独有的miR谱，其中包括高水平的miR-21，并表明miR-21表达有助于SSC茎。此外，我们和其他人证明了神经胶质细胞系衍生的神经营养因子（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，miR对SSC自我更新的贡献部分由GDNF控制，而GDNF信号传导和miR-target基因抑制功能的两个过程是作为较大的调节网络的一部分，介导了SSC的命运。因此，在特定的目标1中，我们将进一步评估GD​​NF诱导的转录因子依赖性miR表达及其对下游靶标mRNA的作用在调节GDNF信号传导和SSC自我更新，分化和凋亡方面的作用。 SSC的多阶段分化过程中的精子和精子需要多种细胞内信号线索，我们假设MIR还调节生殖细胞的分化。因此，在特定的目标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系来跟踪和量化SSC向分化精子的进展。通过这些资源，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对精子移植的原始研究相同的方式改变领域，从而改变了我们对SSC生物学的理解。 公共卫生相关性：精子干细胞是一种成年干细胞，既经历自我更新，又产生分化的子细胞，这些细胞成为精子，这对于精子发生至关重要，是男性生育能力的基础。不育会影响约15％的夫妇，近50％与男性有关，其中大部分涉及精子发生不良。提出的研究对于理解精子发生和男性不育症的原因以及改善治疗治疗方案至关重要。