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/TFG a-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 proposes a phased development plan where didactic training will be completed during the first three years along with performance 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 TGFa/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.
