COORDINATION OF FOLLICULOGENESIS AND OOGENESIS

卵泡发生和卵子发生的协调

• 批准号: 6851662
• 负责人: DAVID F ALBERTINI
• 金额: $ 26.46万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-19 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6851662
• 关键词: basement membrane; cadherins; cell cell interaction; cell differentiation; cell growth regulation; cell proliferation; cellular polarity; confocal scanning microscopy; digital imaging; flow cytometry; follicle stimulating hormone; gap junctions; granulosa cell; hormone regulation /control mechanism; immunocytochemistry; in situ hybridization; integrins; laboratory mouse; membrane channels; mesenchyme; oogenesis; phenotype; protein localization; protein quantitation /detection; tissue /cell culture; transforming growth factors

DESCRIPTION (provided by applicant): Folliculogenesis is a dynamic and progressive process that begins with the initiation of development in primordial follicles and culminates in ovulation of healthy oocytes from follicles that transform into corpora lutea. Information exchanged between the oocyte (OO) and its surrounding somatic follicle cells coordinates folliculogenesis with the process of oogenesis. The central hypothesis being tested in this proposal is that GDF-9 and FSH exert antagonistic influences on an epithelial to mesenchymal transition (EMT) in granulosa cells (GCs). Three specific aims will be undertaken:(1) To study the regulation of follicle development by GDF-9 in vivo and in vitro. The endogenous distribution of GDF-9 will be determined in normal developing follicles and the hypothesis that GDF-9 modulates granulosa cell proliferation and differentiation will be tested by (1) culturing null phenotype follicles with recombinant GDF-9 or (2) coculturing null phenotype follicles with recombinant GDF-9 impregnated beads, wild-type (WT) follicles, or denuded oocytes. The spatiotemporal influence of GDF-9 on GC polarity, cell cycle state and differentiation will be determined at mRNA and protein levels with respect to maintenance of an epithelial phenotype.(2) To characterize the phenotypic response of granulosa cells to FSH that leads to antrum formation and the ontogeny of mural and cumulus lineages. The hypothesis that FSH modulates cell-cell and cellmatrix interactions and mesenchymal fate will be tested in GC from FSH-beta-deficient animals responding to FSH under in vivo or in vitro conditions, mRNA, protein expression and localization studies of cadherins, beta-catenin, connexins, and integrins will be determined with respect to oocyte, granulosa and basement membrane influences or granulosa cell phenotype.(3) To elucidate the importance of granulosa cell polarity and contact with the oocyte in establishing the epithelial-mesenchymal transition of granulosa. GCs isolated from WT pre-antral/antral or GDF-9, Cx37, or FSH-beta null animals will be analyzed in OO/GC co-culture or follicle culture to ascertain GC cell cycle status prior to or following ligand stimulation. These studies will define the basis for cell cycle regulation at critical stages of follicle development.This work will shed light on the paracrine and hormonal mechanisms deployed within ovarian follicles to coordinate the processes of oogenesis and folliculogenesis. This knowledge will be useful in designing new contraceptive strategies for timely interruption of oocyte development and should enhance the potential utility of in vitro follicle systems for human finical applications in ARTs.

描述（由申请人提供）：卵泡发生是一个动态且渐进的过程，从原始卵泡开始发育开始，最终从卵泡中排出健康卵母细胞，转化为黄体。卵母细胞 (OO) 与其周围体细胞卵泡细胞之间交换的信息协调卵泡发生和卵子发生过程。该提案中测试的中心假设是 GDF-9 和 FSH 对颗粒细胞 (GC) 的上皮间质转化 (EMT) 产生拮抗影响。将实现三个具体目标：(1)研究GDF-9在体内和体外对卵泡发育的调节作用。将确定正常发育卵泡中 GDF-9 的内源分布，并通过 (1) 与重组 GDF-9 培养无效表型卵泡或 (2) 共培养无效表型来测试 GDF-9 调节颗粒细胞增殖和分化的假设含有重组 GDF-9 浸渍珠的卵泡、野生型 (WT) 卵泡或裸露的卵母细胞。 GDF-9 对 GC 极性、细胞周期状态和分化的时空影响将在 mRNA 和蛋白质水平上确定，以维持上皮表型。(2) 表征颗粒细胞对导致胃窦的 FSH 的表型反应壁画和积云谱系的形成和个体发育。 FSH 调节细胞-细胞和细胞基质相互作用以及间充质命运的假设将在体内或体外条件下对 FSH 作出反应的 FSH-β 缺陷动物的 GC 中进行测试，以及钙粘蛋白、β-连环蛋白的 mRNA、蛋白质表达和定位研究、连接蛋白和整合素将根据卵母细胞、颗粒和基底膜影响或颗粒细胞表型来确定。(3) 阐明颗粒细胞极性以及在建立颗粒上皮-间质转化过程中与卵母细胞的接触。从 WT 前窦/窦或 GDF-9、Cx37 或 FSH-β 缺失动物中分离的 GC 将在 OO/GC 共培养或滤泡培养中进行分析，以确定配体刺激之前或之后的 GC 细胞周期状态。这些研究将确定卵泡发育关键阶段细胞周期调节的基础。这项工作将揭示卵巢卵泡内协调卵子发生和卵泡发生过程的旁分泌和激素机制。这些知识将有助于设计新的避孕策略，以及时中断卵母细胞的发育，并应增强体外卵泡系统在人类辅助生殖技术中的潜在效用。