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(申请人提供)：精原干细胞(SSC)，是一种既能自我更新又能分化的成体干细胞，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调节男性生育能力提供必要的机制洞察，并对未来动物和人类男性不育的治疗至关重要。在我们不断努力了解支配SSC生物学的调控机制的过程中，我们调查了microRNAs(MiRs)的潜在作用，miRs是一种抑制mRNA靶标翻译的小RNA分子。众所周知，基因在干细胞调控中具有重要作用；然而，对SSC的miR调控知之甚少。生殖细胞中miR加工蛋白DICER的取消极大地减少了精子的发生，表明miR对SSC的增殖和分化是必不可少的，但还没有研究证明特定的miRs的直接功能。使用高通量序列分析，我们产生了高度富含SSCs的小鼠生殖细胞特有的miR图谱，其中包括高水平的miR-21，并表明miR-21的表达有助于SSC的干性。此外，我们和其他人已经证明了胶质细胞系衍生神经营养因子(GDNF)信号在促进SSC自我更新和增殖方面的重要性。我们的假设是，miRs对SSC自我更新的贡献部分由GDNF控制，GDNF信号和miR靶基因抑制这两个过程协同发挥作用，作为调节SSC命运的更大调控网络的一部分。因此，在特定的目标1中，我们将进一步评估GDNF诱导的转录因子依赖的miR的表达及其对下游靶mRNAs在调节GDNF信号和SSC自我更新、分化和凋亡中的作用。SSCs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信号，我们推测miRs也调节生殖细胞的分化。因此，在特定的目标2中，我们将使用已知的生殖细胞分化诱导剂维甲酸来评估miRs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株系来跟踪和量化SSCs向分化精子的进展。有了这些资源，我们就可以研究miRs在分化过程中的功能重要性。我们预测，这些研究将改变这一领域，就像我们最初的精原移植研究改变了我们对SSC生物学的理解一样。 公共卫生相关性：精原干细胞是一种成体干细胞，它经历自我更新，并产生分化的子细胞，成为精子，这是精子发生的关键过程，也是男性生育的基础。约15%的夫妇患有不孕症，其中近50%与男性有关，其中很大一部分涉及精子生成缺陷。建议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。