We have recently shown that the GT1-3 and GT1-7 cell lines, derived from gonadotropin-releasing hormone (GnRH) neurons of the rat hypothalamus, express immunoreactive glucocorticoid receptors (GRs). The GRs within these cell lines are functional and can activate transcription from exogenously introduced glucocorticoid responsive promoters, decrease expression of the endogenous GnRH gene, and repress transcription from transfected GnRH promoters. These results support the hypothesis that glucocorticoid effects on the reproductive axis could be mediated by the direct action of GRs contained within GnRH neurons. In this proposal we plan to investigate the molecular mechanisms of glucocorticoid repression of GnRH neurons. In this proposal we plan to investigate the molecular mechanisms of glucocorticoid repression of GnRH gene expression. One of our specific aims is to identify sequences within the mouse GnRH promoter that are responsible for glucocorticoid mediated repression. Various deletion and point mutants of the mouse GnRH promoter will be tested for glucocorticoid repression upon transient transfection into the GT1-7 cell line. These analyses should identify the minimal promoter segment required for glucocorticoid repression and reveal whether this negative glucocorticoid response element (nGRE) also possesses any other overlapping transcriptional regulatory elements. We also plan to determine whether repression of mouse GnRH promoter activity involves direct DNA binding of the GR and/or other GT1-7 cell nuclear factors. Segments of the mouse GnRH promoter, including the putative nGRE, will be tested for their ability to be bound in vitro by purified GR, or nuclear factors extracted from isolated GT1-7 nuclei. The results of these studies should reveal whether glucocorticoid repression of GnRH transcription is associated with alterations of promoter bound transcription factors, perhaps including the GR. Finally, we plan to examine the effect of glucocorticoids on GnRH secretion and gene expression in vivo. GT1-7 cells will be attached to Cytodex beads and perifused to determine whether glucocorticoids alter GnRH secretion. Effects of glucocorticoids on GnRH mRNA expression in vivo will be assessed by a sensitive solution hybridization/RNase protection assay and by in situ hybridization using two rat models, adult males and immature females treated with estradiol to induce gonadotorpin surges.
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