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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 男性因素不育症是全世界普遍关注的问题。大多数男性不育病例是特发性的。在男性中，原始生殖细胞(PGC)迁移、增殖并在生殖器脊上定植，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不完全清楚。该项目的长期目标是描述生殖细胞与睾丸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性理解与男性不育的临床状况有关，这种不育表现为仅支持细胞综合征，目前还没有治疗方法。为了开始探索男性生殖细胞的发育生物学和了解人类仅支持细胞综合征的病理生物学，我们描述了自然发生的纯合子隐性小鼠突变--闭锁。睾丸发育不全突变的组织学与仅支持细胞综合征患者非常相似，可见仅有支持细胞排列的小管，不含生殖细胞。提出了三个特定的目标来检验中心假设，即细胞自主缺陷导致在闭锁突变的睾丸中完全没有生殖细胞。在特定的目的1中，我们将利用生殖细胞特异性的Oct4启动子在闭锁突变背景下表达GFP转基因，并用共聚焦显微镜分析PGCs的迁移和增殖。在具体目标2中，我们将确定生殖细胞丢失是由于PGC细胞自主缺陷还是由于体细胞壁龛缺陷所致。在体外共培养试验中，我们将确定来自闭锁突变株的支持细胞是否与 野生型生殖细胞并启动黏附连接的形成。此外，标记了LacZ的供体雄性生殖细胞将被移植到突变的小管中，并监测精子生成。在具体目标3中，我们将利用从绿色荧光蛋白阳性的闭锁突变胚胎中分离的流式分选的PGCs，通过定位克隆或基于基因表达的策略来鉴定候选基因(S)。总而言之，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。这些研究将确定导致先天性闭锁突变小鼠生殖细胞缺失的基因(S)，并提供一个起点 进一步了解功能丧失和功能获得的遗传学方法，以了解生殖细胞的迁移和功能。最后，这项工作将建立一种遗传上容易处理的新小鼠模型，用于人类男性不育与支持细胞仅有的小管和生殖细胞再生障碍相关的疾病，从而影响男性生育能力恢复的临床方案。

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.