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 本申请的广泛、长期目标是表征在细胞周期中的体细胞-生殖系相互作用。 成年精子发生该提案将利用生物化学、培养细胞中的定点突变， 免疫荧光、遗传学、RNA干扰、靶向蛋白降解和成人中的拯救测定 果蝇睾丸，以确定分泌的，保守的，免疫球蛋白（IG）结构域蛋白如何维持 血睾屏障（BTB）在成年睾丸的体细胞支持细胞。我们将利用强大的基因 在果蝇中可用，以及明确识别小生境，生殖系干细胞（GSC）， 果蝇睾丸中的精原细胞和体细胞支持细胞。这一建议得到了未发表的支持。 结果表明（1）分泌蛋白在睾丸的体细胞中表达，并且是 精原细胞分化和BTB结构域蛋白的稳健表达;（2）唯一已知的受体 因为分泌的蛋白质是在成年睾丸中不表达或不需要的神经元粘附蛋白， 表明另一种受体参与;（3）另一种与已知受体具有高度同源性的受体表达 在体细胞膜在成年睾丸和它的消耗导致表型类似的损失分泌的 蛋白质;（4）这种其他受体是发育期间维持血脑屏障所必需的， 这表明了保守的屏障功能。目标1集中于使用体外分析（细胞-细胞聚集，亲和力 纯化，然后质谱分析，结构-功能分析）以确定分泌的配体如何 和受体在培养细胞中的相互作用，并采用体内测定（分裂-GFP重建和deGradFP- 依赖性蛋白质降解与遗传"加回"），以测试这些相互作用是否发生在成人中 睾丸目标2的重点是确定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见解， BTB结构域在成人中维持。本提案中的研究将增加以下方面的知识基础 在成年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。