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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Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
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Reproductive Endocrinology Study Section (REN)
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Pennsylvania State University
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Ongeri, Elimelda Moige; Verderame, Michael F; Hammond, James M (2007) The TATA binding protein associated factor 4b (TAF4b) mediates FSH stimulation of the IGFBP-3 promoter in cultured porcine ovarian granulosa cells. Mol Cell Endocrinol 278:29-35
Cunningham, Melissa A; Zhu, Qin; Hammond, James M (2004) FoxO1a can alter cell cycle progression by regulating the nuclear localization of p27kip in granulosa cells. Mol Endocrinol 18:1756-67
Cunningham, Melissa A; Zhu, Qin; Unterman, Terry G et al. (2003) Follicle-stimulating hormone promotes nuclear exclusion of the forkhead transcription factor FoxO1a via phosphatidylinositol 3-kinase in porcine granulosa cells. Endocrinology 144:5585-94
Zaczek, Denise; Hammond, James; Suen, Lii et al. (2002) Impact of growth hormone resistance on female reproductive function: new insights from growth hormone receptor knockout mice. Biol Reprod 67:1115-24
Wandji, S A; Gadsby, J E; Barber, J A et al. (2000) Messenger ribonucleic acids for MAC25 and connective tissue growth factor (CTGF) are inversely regulated during folliculogenesis and early luteogenesis. Endocrinology 141:2648-57
Wandji, S A; Gadsby, J E; Simmen, F A et al. (2000) Porcine ovarian cells express messenger ribonucleic acids for the acid-labile subunit and insulin-like growth factor binding protein-3 during follicular and luteal phases of the estrous cycle. Endocrinology 141:2638-47
Wandji, S A; Wood, T L; Crawford, J et al. (1998) Expression of mouse ovarian insulin growth factor system components during follicular development and atresia. Endocrinology 139:5205-14
Samaras, S E; Canning, S F; Barber, J A et al. (1996) Regulation of insulin-like growth factor I biosynthesis in porcine granulosa cells. Endocrinology 137:4657-64
Gadsby, J E; Lovdal, J A; Samaras, S et al. (1996) Expression of the messenger ribonucleic acids for insulin-like growth factor-I and insulin-like growth factor binding proteins in porcine corpora lutea. Biol Reprod 54:339-46
Grimes, R W; Manni, A; Hammond, J M (1996) Postsynthetic regulation of insulin-like growth factor-binding protein-3 by MCF-7 human breast cancer cells in culture. Breast Cancer Res Treat 39:187-96

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