Role of integrins and activin in granulosa cell growth

在卵泡发育的早期阶段，调节颗粒细胞生长的因素尚不清楚。 初步研究表明，细胞外基质（ECM）在早期卵泡的生长中起作用， 激活素-A，并提出了整合素和激活素/TFG 1 3信号传导途径之间的相互作用。的说明， 参与早期卵泡颗粒细胞生长的分子机制可能导致 预防卵巢早衰、治疗年龄相关性不孕、延迟绝经和预防 癌症药物导致的性腺损伤申请人着重于对早期阶段的监管机制， 卵泡生长，因为在生殖内分泌学的临床培训完成。经过一年的研究 在英国接受国际公认的生殖生物学家的培训后，候选人开始研究整合素 与本项目的建议导师合作，研究卵泡生长中的信号传导。申请人和 导师的机构是一个三个机构的联合体，在研究和顶级研究资源的世界公认。 导师是NIH资助的世界公认的整合素信号传导专家。申请人的短期目标是 加强细胞和分子生物学知识的基础，成为研究细胞信号传导的有效技术 并生成初步数据。候选人的长期目标是成为独立的临床科学家， 专注于将早期卵泡生长的基础研究转化为临床应用。为了实现这些目标，申请人 ）提出了一个分阶段的发展计划，教学培训将在头三年完成， - 具体目标的实验-1。第四年和第五年将重点进行第二年的实验 第三个目标。这一建议的具体目的是调查和描绘的相互作用的机制 在自发永生化（SIGC）和原代颗粒细胞中ECM和激活素A之间的平行关系，以及 确定是否可以通过阻断这种相互作用来调节颗粒细胞增殖。为了实现这些 为了达到特定目的，将在存在或不存在激活素的情况下在聚赖氨酸、纤连蛋白、层粘连蛋白或胶原蛋白上培养SIGC。 A.相互作用的机制将通过研究整合素和激活素受体的表达来研究， 以及关键整合素和TGFI 3/Activin信号分子的表达和磷酸化。 印迹、体内磷酸化、激酶测定和免疫荧光。转录因子如 c-jun和c-fos受整联蛋白信号转导的调节，将通过Western印迹和启动子测定进行研究。到 明确ECM和Activin-A之间的相互作用机制，显性阴性转染技术， 涉及激活素A和ECM之间相互作用的信号传导蛋白的突变体，以及激活素A和ECM之间相互作用的特异性抑制剂。 将使用信号分子。将在转染前和转染后测定细胞增殖和存活， 显性负突变体。

The factors that regulate granulosa cell growth during the early stages of follicular development are unknown. Preliminary work showed that extracellular matrix (ECM) plays a role in growth of early stage follicles in concert with Activin-A, and suggested an interaction between integrin- and Activin/TFGl3-signaling pathways. The elucidation of the molecular mechanisms involved in the growth of granulosa cells of early stage follicles may result in the development of therapeutic strategies that prevent premature ovarian failure, treat age-related infertility, delay menopause and prevent gonadal damage due to cancer drugs. The applicant focused on the mechanisms behind regulation of early stages of follicle growth since the completion of the clinical training in Reproductive Endocrinology. After a year of research training in the UK with an internationally recognized reproductive biologist, the candidate began studying integrin signaling in ovarian follicle growth in collaboration with the proposed mentor of this project. The applicant's and the mentor's institution is a tri-institutional conglamerate with worldwide recognition in research and top research resources. The mentor is an NIH-funded and world recognized expert in integrin-signaling. The applicant's short-term goals are to strengthen the fund of knowledge in cell and molecular biology, become efficient in techniques to study cell signaling and to generate preliminary data. The candidates' long-term goal is to become an independent clinician-scientist with focus on translating basic research on early follicle growth to clinical applications. To achieve these goals, the applicant )roposes a phased development plan where didactic training will be completed during the first three years along with _erformance of the experiments of specific aim-1. The fourth and fifth years will focus on the experiments of second and third aims. The specific aims of this proposal are to investigate and delineate the mechanisms of the interaction between the ECM and Activin-A in spontaneously immortalized (SIGC) and primary granulosa cells in parallel, and to determine whether granulosa cell proliferation can be modulated by blockage of this interaction. In pursuit of these specific aims, SIGC will be cultured on polylysine, fibronectin, laminin or collagen in the presence or absence of Activin- A. The mechanisms of interaction will be investigated by studying the expression of integrins and activin receptors as well as the expression and phosphorylation of the key integrin and TGFI3/Activin signaling molecules by Western blotting, in vivo phosphorylation, kinase assays, and immunofluorescence. Expression of transcription factors such as c-jun and c-fos, which are regulated by integrin signaling, will be studied by Western blotting and promoter assays. To pinpoint the mechanism of interaction between the ECM and Activin-A, transfection techniques with dominant-negative mutants of the signaling proteins implicated in the interaction between Activin-A and the ECM, and specific inhibitors of signaling molecules will be utilized. Cell proliferation and survival will be determined before and after transfection with the dominant-negative mutants.