Questions concerning the regulation of folliculogenesis are clinically important because alterations in the process can lead to pathophysiology and infertility. Recently, a novel TGF-Beta family member, GDF-9 is ovary specific, being localized exclusively to oocytes. The importance of GDF-9 deficient mice is arrested and the females are infertile. Accordingly, GDF-9 secreted by the egg is obligatory for folliculogenesis and fertility. The clinical relevance of this new concept is demonstrated by the presence of GDF- 9 mRNA in the human oocyte. At present, virtually nothing is known about the mechanisms controlling GDF-9 expression, and nothing is known about the target cells for GDF-9 and the biological process that GDF-9 regulates. The purpose of this grant is to fill these fundamental gaps in our knowledge of GDF-9 in rat and human ovaries.
Three specific aims are proposed:
AIM1. Determine the cellular localization and changing pattern of expression of GDF-9 mRNA and protein by in situ hybridization and immunocytochemiestry, respectively, in human (normal and abnormal) and rat ovaries at various reproductive states and during aging. The latter could have implications for aged-related infertility. Novel preliminary data show a dramatic decrease in GDF-9 mRNA during atresia.
AIM 2. Test the hypothesis that atretogenic endocrine and autocrine/paracrine ligands negatively regulate GDF-9 expression and promote atresia. Using the immature rat, we will investigate the effects of known atretogenic hormones (testosterone, GnRH, low dose hCG, estrogen withdrawal, prolactin, growth hormone) on oocyte GDF-9 levels, and correlate the effects with changes in granulosa cell mitosis and apoptosis. Novel preliminary data indicate that prolactin and growth hormone cause dramatic decreases in GDF-9 mRNA which are accompanied by the cessation of granulosa mitosis.
Aim 3 Using recombinant human GDF-9, we will 1) determine the target cells for GDF-9 in rat and human ovaries by in situ autoradiography; 2) characterize the binding constants of the GDF-9 receptor by Scatchard analysis; and 3) elucidate the biological consequences of GDF-9 action in granulosa cells cultured in serum free medium. Novel preliminary data indicate theat rhGDF-9 acts on granulosa cells to stimulate mitosis. We anticipate that the results from our experiments will also suggest new targets and avenues for managing fertility and infertility.
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