Clinically, gonadotropin-releasing hormone (GnRH) agonists are frequently used to down-regulate hypothalamic GnRH and produce """"""""chemical castration."""""""" More recently, GnRH antagonists, which block the GnRH receptor, have been used for pituitary suppression in ovulation induction protocols. Pharmacologic doses of GnRH analog, used during fertility treatment, may disrupt the physiologic intra-ovarian GnRH regulatory system and directly modulate ovarian function. Because the physiologic role of ovarian GnRH is not completely understood, it is not clear whether direct ovarian effects of GnRH analogs are beneficial or detrimental. A more comprehensive understanding of the mechanisms regulating GnRH expression in the ovary may provide insights into its physiologic role. The overall objective of this proposal is to elucidate the molecular mechanisms that regulate GnRH gene expression in the ovary. Due to low levels of expression, the transcriptional regulation of ovarian GnRH has been extremely difficult to study. Using transgenic mice, generated with fragments of the mouse GnRH (mGnRH) gene promoter fused to the luciferase (LUC) reporter gene, mGnRH promoter activity was detected in the hypothalamus and ovary. The studies outlined in my proposal will use the mGnRH-LUC mice to isolate cis-regulatory elements in the mGnRH gene that regulate ovarian GnRH in vivo. These DNA elements will be characterized in vitro to identify factors that mediate expression and repression of mGnRH in the ovary.
My first aim will test the hypothesis that binding of ovarian proteins to specific sequences in the mGnRH gene promoter is critical for ovarian GnRH expression. DNA footprint analysis and gel mobility shift assays will be used to identify proteins in the ovarian granulosa cells that mediate mGnRH expression in the ovary. In my second aim, I will use in vitro and in vivo methods to test the hypothesis that estrogen exerts repressive effects on ovarian GnRH gene expression via specific DNA sequences in the mGnRH gene promoter. The mGnRH-LUC mice that I constructed provided a unique and powerful tool for investigating ovarian GnRH expression in vivo. Several factors, which might bind regulatory regions in the GnRH gene, appear to be temporally expressed during the estrus cycle, providing a compelling argument for my hypothesis that ovarian GnRH expression requires specific interactions of ovarian proteins with regulatory elements in the mGnRH gene. My proposed studies, by identifying regulators of ovarian GnRH expression, may reveal mechanisms by which stimulatory and inhibitory intracellular signals are integrated to regulate ovarian GnRH gene expression. Ultimately, elucidating the molecular mechanisms by which ovarian GnRH is regulated should provide further insights into the role of GnRH in ovarian function and will optimize the therapeutic use of GnRH analogs for maximal clinical benefit. Gonadotropin-releasing hormone (GnRH) is secreted by the hypothalamus and is the primary regulator of reproductive function. GnRH analogs are routinely used to suppress the hypothalamus during infertility treatment, but it is not known whether direct ovarian effects contribute to clinical efficacy. Elucidating the molecular mechanisms by which ovarian GnRH is regulated should lead to improved therapeutic regimens optimizing the differential use of GnRH agonists and antagonists. ? ? ? ?
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