KANSAS U COBRE: GERM CELL DEVELOPMENT IN THE ATRICHOSIS MUTANT MOUSE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 男性因素不育是全世界关注的一个重大问题。大多数男性不育病例是特发性的。在男性中，原始生殖细胞（PGCs）迁移、增殖并定殖于生殖嵴，最终形成睾丸索，在那里它们与支持细胞建立联系。调节原始生殖细胞迁移和增殖的关键因素尚未完全了解。本项目的长期目标是阐明睾丸中生殖细胞与支持细胞相互作用的机制。对生殖细胞如何发育和功能的机械理解与男性不育的临床状况有关，这些临床状况表现为目前没有治疗方法的仅支持细胞综合征。为了开始探索雄性生殖细胞的发育生物学和了解人类支持细胞综合征的病理生物学，我们对自然发生的纯合子隐性小鼠突变体-毛发萎缩进行了表征。无毛突变型睾丸组织学与仅支持细胞综合征患者非常相似，表现为小管内衬仅支持细胞，不含生殖细胞。提出了三个具体的目的，以测试中心的假设，即细胞自主缺陷导致完全缺乏生殖细胞的无毛突变睾丸。在特定目标1中，我们将在无毛突变背景中使用生殖细胞特异性Oct 4启动子表达GFP转基因，并通过共聚焦显微镜分析PGC的迁移和增殖。在具体目标2中，我们将确定生殖细胞损失是由于PGC细胞自主缺陷还是由于有缺陷的体细胞小生境隔室。在体外共培养试验中，我们将确定来自无毛突变体的支持细胞是否与 野生型生殖细胞并启动粘附连接的形成。此外，lacZ标记的供体雄性生殖细胞将被移植到突变体小管中，并监测精子发生。在具体目标3中，我们将通过定位克隆或基于基因表达的策略，使用从GFP阳性无毛突变体胚胎中分离的流式分选的PGC，鉴定无毛位点的候选基因。总的来说，这些研究将使我们能够阐明生殖细胞的发育，功能和命运的机制，并对它们如何影响睾丸中的体细胞提供新的见解。这些研究将确定基因负责缺乏生殖细胞的无毛突变小鼠，并提供一个起点 进一步的功能丧失和功能获得的遗传方法，以了解生殖细胞的迁移和功能。最后，这项工作将建立atrichosis突变体作为一种遗传上易于处理的新的小鼠模型，用于人类男性不育症与支持细胞仅小管和生殖细胞发育不全相关的疾病，从而影响男性生育力恢复的临床方案。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Male factor infertility is a significant concern throughout the world. The majority of the male infertility cases are idiopathic. In the male, primordial germ cells (PGCs) migrate, proliferate, and colonize the genital ridges to ultimately form testicular cords, where they establish contacts with the Sertoli cells. Key factors that regulate primordial germ cell migration and proliferation are not completely understood. The long-term goal of this project is to delineate the mechanisms of germ cell interactions with Sertoli cells in the testis. A mechanistic understanding of how germ cells develop and function is relevant to clinical conditions of male infertility that manifest as Sertoli cell-only syndrome for which there is currently no treatment. To begin to explore the developmental biology of the male germ cells and to understand the pathobiology of the human Sertoli cell-only syndrome, we have characterized atrichosis, the naturally occurring homozygous recessive mouse mutant. The atrichosis mutant testis histology closely resembles that of Sertoli cell-only syndrome patients and demonstrates tubules lined with only Sertoli cells and contains no germ cells. Three Specific Aims are proposed to test the central hypothesis that a cell autonomous defect leads to complete absence of germ cells in the atrichosis mutant testis. In Specific Aim 1, we will express a GFP transgene using a germ cell specific, Oct4 promoter in the atrichosis mutant background, and analyze the migration and proliferation of PGCs by confocal microscopy. In Specific Aim 2, we will determine whether the germ cell loss is due to a PGC cell autonomous defect or due to a defective somatic cell niche compartment. In a co-culture in vitro assay, we will determine whether the Sertoli cells from atrichosis mutants establish functional contacts with wild type germ cells and initiate the formation of adherens junctions. Additionally, lacZ-tagged donor male germ cells will be transplanted into the mutant tubules and spermatogeneis monitred. In Specific Aim 3, we will identify the candidate gene(s) at the atrichosis locus by positional cloning or a gene expression-based strategy using flow-sorted PGCs isolated from the GFP-positive atrichosis mutant embryos. Collectively, these studies will allow us to elucidate the mechanisms of development, function and fate of the germ cells and give new insights into how they influence somatic cells in the testis. These studies will identify the gene(s) responsible for the absence of germ cells in the atrichosis mutant mouse and provide a starting point for further loss-of-function and gain-of-function genetic approaches to understand germ cell migration and function. Finally, this work will establish atrichosis mutant as a genetically tractable new mouse model for human male infertility conditions associated with Sertoli cell-only tubules and germ cell aplasia, thus impacting clinical protocols of male fertility restoration.