This proposal addressed the autocrine and paracrine function of growth factors in the development of the porcine ovarian follicle. Two classes of growth factors will emphasized: the insulin-like growth factors (IGFs) and epidermal growth factor-like activity (EGF-1a), since we have found such factors in the porcine ovarian follicle and they exert profound effects on the replication and differentiation of cells from such follicles. We propose, first, to develop a better appreciation of the biochemical nature of these growth factors through chromatography, electrophoresis, and cDNA hybridization studies. These studies will determine if IGF-I and IGF-II are both secreted in the ovarian follicle and if the EGF-like peptides are more similar to EGF or its homologue transforming growth factor-alpha (TGF-alpha). In the course of these studies, antibodies and cDNA probes for the two IGFs and ovarian EGF-1a will be defined and validated. Next, the manner in which the secretion of these peptides is interfaced with gonadotropins and gonadal steroids, and the role of such factors in ovarian follicular growth and development will be elucidated. This goal will be approached by parallel in vitro and in vivo studies. In vitro, the secretion of these growth factors by cultured granulosa and theca cells and its hormonal regulation will be established. Autocrine or paracrine effects of the growth factors or follicular cell replication and steroidogenesis will be examined with blocking antibodies. Using flow cytometry, we will correlate the replicative activity of cultured cells, and the presence of IGFs, EGF-like activity, and steroidogenic enzymes with appropriate immunofluorescent probes. To investigate the physiological role of such growth factors in vivo, follicles from cycling and gonadotropin-treated pigs will be analyzed for intrafollicular content of growth factors, ovarian steroids, and granulosa cell proliferative activity (by flow cytometric analysis). Supplement the data from dissected follicles, these ovaries will be analyzed chemically to localize growth factors and the side chain cleavage enzyme in the several ovarian components; later in the course of the proposal, hybridization histochemistry with cDNA probes for this enzyme and the IGFs and EGF-like peptides will be employed. Collectively, these several approaches should allow a more complete understanding of the nature of ovarian growth factors and their physiological role.
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