Somatic control of germline differentiation in spermatogenesis.

精子发生中种系分化的体细胞控制。

• 批准号: 10741641
• 负责人: Erika A Bach
• 金额: $ 16.95万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-04 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10741641
• 关键词: Adhesions; Adult; Affinity Chromatography; Age; Alleles; Amalgam; Antibodies; Back; Binding; Biochemistry; Biological Assay; Blood - brain barrier anatomy; Blood-Testis Barrier; Cell Aggregation; Cell Culture Techniques; Cell Differentiation process; Cell Separation; Cell-Cell Adhesion; Cells; Cultured Cells; Cyst; Defect; Development; Dextrans; Drosophila genus; Dyes; Elderly; Embryo; Encapsulated; Epithelium; Exclusion; Experimental Designs; Extracellular Domain; Fostering; Gametogenesis; Genetic; Germ Cells; Human; Immunofluorescence Immunologic; Immunoglobulin Domain; In Vitro; Infertility; Integral Membrane Protein; Interphase Cell; Length; Ligands; Maintenance; Male Infertility; Maps; Mass Spectrum Analysis; Mediating; Membrane; Modeling; Neuroglia; Neurons; Order Coleoptera; Organism; Permeability; Phenotype; Process; Production; Protein Secretion; Proteins; RNA Interference; RNA interference screen; Research; Septate; Signal Transduction; Site-Directed Mutagenesis; Somatic Cell; Spermatocytes; Spermatogenesis; Spermatogonia; Structure; Supporting Cell; Surveys; System; Techniques; Tertiary Protein Structure; Testing; Testis; Tight Junctions; Tissues; Transcript; Variant; Work; age related; cell envelope; cell growth; daughter cell; design; dimer; fertility preservation; fly; functional disability; germline stem cells; gliotactin; in vitro Assay; in vivo; insight; knowledge base; male; mutant; neuronal cell body; novel therapeutics; offspring; overexpression; protein degradation; receptor; reconstitution; recruit; sertoli cell; sperm cell; stem cells; transcriptomics

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.

项目摘要 本应用的广泛，长期目标是表征在 成人精子发生。该提案将利用生物化学，培养细胞和 成人的免疫荧光，遗传学，RNA干扰，靶向蛋白质降解和救援测定 果蝇睾丸确定分泌，保守的，免疫球蛋白（Ig）结构蛋白如何保持 成人测试的体细胞支撑细胞中的血尾屏障（BTB）。我们将利用强大的遗传学 可在果蝇中获得，以及明确鉴定生态细菌干细胞（GSC）的能力 果蝇睾丸中的精子和体支持细胞。该提议得到未出版的支持 结果表明（1）分泌蛋白在睾丸的体细胞中表达，是必需的 精子分化和BTB结构蛋白的稳健表达； （2）唯一已知的受体 因为分泌的蛋白是一种神经元粘附蛋白，在成人睾丸中未表达或不需要 表明涉及另一个受体； （3）表达了另一个具有高同源性的受体 在成人睾丸及其耗竭的体细胞中，表现类似于分泌的损失 蛋白质; （4）在发育过程中维持血脑屏障需要的其他受体需要 建议配置的屏障功能。目的1以使用体外评估（细胞细胞聚集，亲和力）为中心 纯化，然后进行质谱，结构功能分析），以确定分泌的配体如何 受体在培养的细胞和在体内分析中的就业中相互作用（分裂GFP重建和降解-FPP- 依赖性蛋白质与遗传“添加”降解）以测试成年人中这些相互作用是否发生 睾丸。目标2的重点是确定BTB的渗透性屏障功能是否受到损害 在缺乏配体或接收器的测试中。这些实验旨在揭示机械的见解 BTB结构域维持在成人中。该提案中的研究将增加有关的知识基础 在成人阶段维持精子发生的信号，并将促进新的研究途径 与年龄相关的男性不育症的机制和治疗方法。