Somatic control of germline differentiation in spermatogenesis.

项目摘要 这个应用程序的广泛的长期目标是描述体细胞-生殖系在 成体精子发生。该提案将利用生物化学、在培养细胞中进行定点突变和 成人的免疫荧光、遗传学、RNA干扰、靶向蛋白降解和救援分析 以确定分泌的、保守的免疫球蛋白(Ig)结构域蛋白如何维持 成人睾丸支持细胞的血-睾丸屏障(BTB)。我们将利用强大的遗传学 在果蝇中可用，以及明确识别利基、生殖系干细胞(GSCs)的能力， 果蝇睾丸的精原细胞和体细胞支持细胞。这项提议得到了未出版的 结果表明：(1)分泌的蛋白质在睾丸的体细胞中表达，是 精原细胞分化和BTB结构域蛋白的强健表达；(2)唯一已知的受体 因为分泌的蛋白是一种神经元黏附蛋白，在成人睾丸中不表达或不需要， 表明另一个受体参与；(3)另一个与已知受体同源性很高的受体被表达 在成人睾丸的体细胞膜中，它的耗竭导致类似于分泌的表型的丧失 蛋白质；(4)在发育过程中，这个另一个受体是维持血脑屏障所必需的， 暗示了一种守恒的屏障功能。目标1集中于使用体外分析(细胞-细胞聚集、亲和力 纯化后进行质谱学、结构-功能分析)以确定分泌的配体是如何 和受体在培养的细胞中相互作用，并在体内使用检测(Split-GFP重组和去GradFP- 依赖于蛋白质降解与基因的“加回”)，以测试这些相互作用是否发生在成人 睾丸。目标2集中于确定BTB的渗透屏障功能是否受到损害 在缺乏配体或受体的睾丸中。这些实验旨在揭示机械的洞察力如何 BTB区域在成人中保持不变。这项建议的研究将增加关于以下方面的知识库 在成体阶段维持精子发生的信号，并将培育研究的新途径 年龄相关性男性不育的机制和治疗。

Project Summary The broad, long-term objectives of this application are to characterize soma-germline interactions during adult spermatogenesis. The proposal will utilize biochemistry, site-directed mutagenesis in cultured cells and immunofluorescence, genetics, RNA interference, targeted protein degradation and rescue assays in the adult Drosophila testis to determine how a secreted, conserved, immunoglobulin (Ig) domain protein maintains the blood-testis barrier (BTB) in somatic support cells of adult testes. We will capitalize upon the powerful genetics available in Drosophila, as well as the ability to unequivocally identify the niche, germline stem cells (GSCs), spermatogonia and somatic support cells in the Drosophila testis. This proposal is supported by unpublished results demonstrating that (1) the secreted protein is expressed in somatic cells of the testis and is required for spermatogonial differentiation and for robust expression of a BTB domain protein; (2) the only known receptor for the secreted protein is a neuronal adhesion protein that is not expressed or required in the adult testis, indicating that another receptor is involved; (3) another receptor with high homology to known one is expressed in somatic membranes in the adult testis and its depletion leads to phenotypes similar to loss of the secreted protein; (4) this other receptor is required for maintenance of the blood-brain-barrier during development, suggesting a conserved barrier function. Aim 1 centers on using in vitro assays (cell-cell aggregation, affinity purification followed by mass spectrometry, structure-function analysis) to determine how the secreted ligand and receptor interact in cultured cells and on employing in vivo assays (split-GFP reconstitution and deGradFP- dependent protein degradation with genetic “add back”) to test whether these interactions occur in the adult testis. The Aim 2 is focused on determining whether the permeability barrier function of the BTB is compromised in testes lacking the ligand or receptor. These experiments are designed to reveal mechanistic insights into how the BTB domain is maintained in adults. The studies in this proposal will increase the knowledge base about signals that maintain spermatogenesis during adult stages and will foster new avenues of research into mechanisms and treatments for age-related male infertility.