Role of integrins and activin in granulosa cell growth

超出提供的空间。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)与激活素-A(Activin-A)共同参与早期卵泡的生长，提示整合素-和激活素/TFGl3-信号通路之间存在相互作用。早期卵泡颗粒细胞生长的分子机制的阐明可能导致开发预防卵巢早衰、治疗年龄相关性不孕症、推迟绝经和预防抗癌药物引起的性腺损伤的治疗策略。申请人侧重于自生殖内分泌学临床培训结束以来对卵泡生长早期阶段的调节机制。在英国与一位国际公认的生殖生物学家进行了一年的研究培训后，这位候选人开始与该项目的拟议导师合作，研究整合素信号在卵巢卵泡生长中的作用。申请者和导师所在的机构是一个在研究和顶级研究资源方面获得全球认可的三所院校的联合体。这位导师是美国国立卫生研究院资助的整合素信号方面的世界公认的专家。申请者的短期目标是加强细胞和分子生物学知识的基金，在研究细胞信号和产生初步数据的技术方面变得高效。候选人的长期目标是成为一名独立的临床科学家，专注于将早期毛囊生长的基础研究转化为临床应用。为了实现这些目标，申请者)提出了一个分阶段发展计划，在该计划中，教学培训将在头三年内完成，同时完成特定目标-1的实验。第四年和第五年将集中进行第二和第三个目标的实验。该方案的具体目的是研究和描述自发永生化颗粒细胞(SIGC)和原代颗粒细胞中ECM和激活素A相互作用的机制，并确定阻断这种相互作用是否可以调节颗粒细胞的增殖。为了实现这些特定的目标，SIGC将在有或没有激活素-A的情况下，在聚赖氨酸、纤维连接蛋白、层粘连蛋白或胶原上进行培养。通过Western blotting、体内磷酸化、激酶分析和免疫荧光等方法研究整合素和激活素受体的表达以及关键的整合素和TGFI3/激活素信号分子的表达和磷酸化来探讨相互作用的机制。C-jun和c-fos等受整合素信号调控的转录因子的表达将通过Western blotting和启动子分析进行研究。为了弄清细胞外基质和激活素-A之间的相互作用机制，将利用与激活素-A和细胞外基质相互作用有关的信号蛋白显性-负性突变体以及信号分子的特异性抑制剂的转基因技术。用显性-阴性突变体转染前后将确定细胞的增殖和存活情况。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE ========================================Section End===========================================