Regulation of spermatogonial stem cell proliferation and differentiation by UTF1

描述(申请人提供)：男性不育症是一种流行的疾病(7%的男性是不育的)，在美国影响许多育龄夫妇的生活质量。大多数男性不育症是由于男性完全不能产生精子(无精子症)或由低精子量(少精子症)引起的，是特发性的。因此，研究发现男性不育的新原因对生殖医学具有非常重要的意义，并可能导致创新的诊断或治疗。精原干细胞(SSCs)是哺乳动物睾丸中的成体干细胞，维持精子发生，对男性生育能力至关重要，但控制其活性的机制仍不清楚。未分化胚胎细胞转录因子1(UTF1)是一种在睾丸未分化精原细胞(包括SSCs)中表达的转录因子，对多能干细胞的增殖和分化具有既定的作用。初步研究表明，在培养的SSC中，UTF1的表达对分化信号有反应，提示UTF1在SSC的分化中起着重要作用。因此，我推测UTF1促进SSC增殖，是SSC分化所必需的。我将用创新的方法在小鼠身上验证这一假说，以证明UTF1是否在SSC的增殖和分化中发挥作用。在具体目标1中，我将使用慢病毒传递的、可诱导的、过度表达和击倒的构建物来操纵培养的Thy1+精原细胞(包含SSCs)中的UTF1蛋白水平。我将使用Id4-GFP小鼠的Thy1+精原细胞培养来检测SSC的增殖，并使用三种不同的方法来评估SSC的增殖。这些研究将确定当UTF1水平改变时Id4-GFP+SSCs的增殖指数。AIM 1的结果将决定1)过量的UTF1是否促进SSC的增殖，2)UTF1缺乏是否抑制SSC的增殖。在特定的目标2中，我将确定在GFP+Thy1+精原细胞培养中UTF1过度表达或下调时SSC分化的程度。对于这些研究，我将使用含有可诱导过表达或敲除结构的培养物通过维甲酸处理来强制SSC分化，我将通过移植测量分化标记基因的表达和SSC数量来评估分化。AIM 2的结果将决定1)UTF1过多是否促进SSC分化，2)UTF1缺乏是否阻碍SSC分化。本申请中提议的研究将检验UTF1促进SSCs生物学活性的基本机制。总体而言，这些研究结果阐明了UTF1在精子发生中的作用，并将确定UTF1(以及其靶基因)是否可能在男性不育的发病机制中发挥作用。此外，拟议中的研究将为我的职业发展提供充足的机会，包括掌握干细胞、发育和分子生物学的尖端技术，为精子发生领域做出学术贡献，并帮助我发展我的研究领域。

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.