Polycystic ovarian disease (PCOD) is a common gynecologic problem in which patients suffer from hyperandrogenism, hirsutism and infertility. The role of peptide growth factors as autocrine or paracrine contributors to this disease is unclear. E. Anderson (see collaboration letter) and others have developed a rat model for polycystic ovary in which the in vitro and in vivo effects of hyperandrogenism on granulosa cells can be tested experimentally. The goal of this proposal is to study, by using the rat model, the potential roles of transforming growth factor-alpha (TGF-a), epidermal growth factor (EGF) (the biochemical and functional analog of TGF-a), and their common receptor, the EGF receptor, in the pathophysiology of PCOD.
Specific aims are: I. To study TGF-a and granulosa function in cells subjected to hyperandrogenic environments. Preliminary results indicate that normal granulosa cells contain TGF-a mRNA and respond to exogenously added TGF-a. These experiments are planned: (1) In vitro, rat granulosa cells cultured with androgens and TGF-a or EGF will be studied for changes in TGF-a mRNA production, in estrogen and progesterone production, in levels of mRNA of the enzymes of steroidogenesis, in the kinetics of DNA synthesis and in cellular morphology. (2) In vivo, the change in TGF-a gene expression during polycystic ovary formation in rats injected with dehydroepiandrosterone will be analyzed by in situ hybridization and immunohistochemistry. II. To understand TGF-a signal transduction in hyperandrogenic granulosa cells. Normal granulosa cells contain receptors for TGF-a. The effects of hyperandrogenism on TGF-a signal transduction are unknown. These experiments are planned: (1) Analysis of changes in EGF receptors in hyperandrogenism in terms of change in receptor binding affinity, in level of EGF receptor mRNA production, in cellular localization and in subcellular (vesicular/cytoskeletal) localization. (2) Study of blockage of TGF-a signal transduction in hyperandrogenism. TGF-a signal transduction will be blocked with anti-TGF-a antibodies and with genistein (a tyrosine kinase inhibitor) and the cells will be analyzed for these changes: TGF-a mRNA expression; c-fos and c-myc proto-oncogene mRNA expression; steroid production; and morphology. III. To investigate the regulation of TGF-a gene expression in hyperandrogenic granulosa cells. TGF-a gene expression is inducible with FSH and with estradiol. The rat TGF-a gene (6 exons, 85 kilobases in length) has been mapped and the promoter region has recently been sequenced. It is planned that the FSH and estradiol responsive sites of the TGF-a gene be identified and elucidated. After that, the regulation of TGF-a gene expression in hyperandrogenism will be compared to normal.
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