Regulation of spermatogonial stem cell proliferation and differentiation by UTF1

UTF1对精原干细胞增殖和分化的调控

• 批准号: 8873983
• 负责人: KAZADI NADINE MUTOJI
• 金额: $ 6.22万
• 依托单位: UNIVERSITY OF TEXAS SAN ANTONIO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8873983
• 关键词: Address; Affect; Age; Apoptosis; Biological; Biological Assay; Biology; Bromodeoxyuridine; Cell Count; Cell Death; Cell Differentiation process; Cell Proliferation; Couples; Defect; Developmental Biology; Diagnostic; Differentiation Antigens; Disease; Ensure; Equilibrium; Etiology; Fellowship; Fertility; Gene Targeting; Genes; Gentian Violet; Infertility; Lead; Learning; Male Infertility; Measures; Messenger RNA; Methods; Molecular; Molecular Biology; Mus; Oligospermia; Pathogenesis; Play; Pluripotent Stem Cells; Postdoctoral Fellow; Process; Production; Proteins; Quality of life; Regulation; Reporting; Reproductive Medicine; Research; Role; Signal Transduction; Spermatogenesis; Spermatogonia; Staining method; Stains; Stem cells; Techniques; Testing; Testis; Therapeutic; Training; Transcriptional Regulation; Transplantation; Tretinoin; Undifferentiated; United States; Universities; Washington; adult stem cell; blastomere structure; career development; indexing; innovation; knock-down; male; man; meetings; men; novel; public health relevance; reproductive; research study; self-renewal; sperm cell; stem cell biology; stem cell differentiation; stem cell fate; stem cell population; targeted treatment; transcription factor

DESCRIPTION (provided by applicant): Male infertility is a prevalent disease (7% of men are infertile) that affects the quality-of-life of many reproductive-age couples in the US. Most cases of male infertility which result from a man's complete inability to produce sperm (azoospermia) or arise from low sperm production (oligospermia) are idiopathic. Therefore, research to uncover novel causes of male infertility are highly significant to reproductive medicine and may lead to innovative diagnostics or therapeutics. Spermatogonial stem cells (SSCs) are adult stem cells in mammalian testes that maintain spermatogenesis and are essential for male fertility, yet the mechanisms that control their activity remain elusive. Undifferentiated embryonic cell transcription factor 1 (UTF1) is a transcription factor expressed in undifferentiated spermatogonia of the testis (including SSCs) which has an established role in proliferation and differentiation of pluripotent stem cells. Preliminary studies demonstrated that UTF1 expression responds to differentiation signals in cultured SSCs, suggesting it plays a prominent role in SSC differentiation. Thus, I hypothesized that UTF1 promotes SSC proliferation and is required for SSC differentiation. I will test this hypothesis using innovative methods in mice to demonstrate whether UTF1 plays a role in SSC proliferation and differentiation. In Specific Aim 1, I will manipulate UTF1 protein levels in cultured Thy1+ spermatogonia (which contain SSCs) using lentiviral-delivered, inducible, over-expression and knockdown constructs. I will examine SSC proliferation using Thy1+ spermatogonial cultures from Id4-GFP mice, which express GFP specifically in SSCs, and evaluate proliferation using three separate assays. These studies will determine the proliferative index among Id4-GFP+ SSCs when UTF1 levels are altered. The results of Aim 1 will determine whether 1) excess UTF1 promotes SSC proliferation and 2) UTF1 deficiency blocks SSC proliferation. In Specific Aim 2, I will determine the degree of SSC differentiation when UTF1 is over-expressed or knocked-down in GFP+ Thy1+ spermatogonial cultures. For these studies, I will force SSC differentiation by retinoic acid treatment using cultures harboring inducible over-expression or knockdown constructs and I will assess differentiation by measuring expression of differentiation marker genes and SSC numbers by transplantation. The results of Aim 2 will determine whether 1) excess UTF1 promotes SSC differentiation and 2) UTF1 deficiency blocks SSC differentiation. The studies proposed in this application will examine the fundamental mechanisms through which UTF1 contributes to the biological activity of SSCs. Overall, the results of these studies clarify the role of UTF1 in spermatogenesis and will determine whether UTF1 (and, by extension, its target genes) may play a role in the pathogenesis of male infertility. Moreover, the proposed studies will provide ample opportunities for my career development, including mastery of cutting-edge techniques in stem cell, developmental, and molecular biology, scholarly contributions to the field of spermatogenesis, and help me to develop my research niche.

描述（由申请人提供）：男性不育症是一种普遍的疾病（男性的7％），影响了美国许多生殖年龄夫妇的生活质量。大多数男性不育症的病例是由男人完全无法产生精子（Azoospermia）或由低精子产生（寡植物）引起的。因此，揭示新的不育症的新原因的研究对于生殖医学非常重要，可能导致创新的诊断或治疗疗法。精子干细胞（SSC）是哺乳动物睾丸中的成年干细胞，可维持精子发生并且对于男性生育力至关重要，但是控制其活性的机制仍然难以捉摸。未分化的胚胎细胞转录因子1（UTF1）是在未分化的睾丸精子（包括SSC）中表达的转录因子，该因子在多能干细胞的增殖和分化中具有确定的作用。初步研究表明，UTF1表达对培养的SSC中的分化信号做出响应，这表明它在SSC分化中起着重要的作用。因此，我假设UTF1促进了SSC的增殖，这是SSC分化所必需的。我将使用小鼠中的创新方法检验该假设，以证明UTF1是否在SSC增殖和分化中起作用。在特定的目标1中，我将使用慢病毒降低，可诱导的，过表达和敲低构建体中培养的Thy1+精子（包含SSC）的UTF1蛋白水平。我将使用来自ID4-GFP小鼠的THY1+精子培养物检查SSC增殖，这些培养物专门在SSC中表达GFP，并使用三个单独的测定法评估增殖。当UTF1水平改变时，这些研究将确定ID4-GFP+ SSC之间的增殖指数。 AIM 1的结果将确定1）多余的UTF1是否促进了SSC增殖，而2）UTF1缺乏阻滞SSC增殖。在特定目标2中，我将确定当UTF1在GFP+ THY1+精子培养物中过度表达或击倒时，SSC分化的程度。对于这些研究，我将使用具有诱导过表达或敲低构建体的培养物通过视黄酸处理来迫使SSC分化，并通过测量通过移植来测量分化标记基因和SSC数的表达来评估分化。 AIM 2的结果将确定1）多余的UTF1是否促进SSC分化和2）UTF1缺乏症阻断SSC分化。本应用程序中提出的研究将研究UTF1对SSC生物活性有助于的基本机制。总体而言，这些研究的结果阐明了UTF1在精子发生中的作用，并将确定UTF1（并且通过扩展其靶基因）是否可能在男性不育的发病机理中发挥作用。此外，拟议的研究将为我的职业发展提供足够的机会，包括掌握干细胞，发育和分子生物学的尖端技术，对精子发生领域的学术贡献，并帮助我发展研究莱基。