GNRH GONADOTROPIN TRANSCRIPTION AND STEROID FEEDBACK

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GnRH pulses and sex steroids act on the pituitary to regulate the synthesis and secretion of LH and FSH. In the past funding period, we cloned the rat alpha-subunit promoter, identified GnRH-responsive DNA elements and transcription factors for rat LHbeta and alpha-subunit promoters, and characterized intracellular signaling pathways mediating GnRH responses. The LHbeta promoter requires pulsatile GnRH stimulation in vivo, and has two cooperating complex DNA elements, a distal element with overlapping Sp1 and CArG box sites, and a proximal element with two bipartite sites for SF-1 and Egr-1. Both elements bind Sp1 family zinc finger proteins that could differentially affect transcription. Relative roles of these transcription factors in GnRH stimulation will be assessed by DNA-protein binding, chromatin immunoprecipitation (CHIP) and real-time RT-PCR, and transfection ot deletion/mutation luciferase constructs in LbetaT2 ceils. Preliminary data show that steroids both enhance (17beta- estradiol (E) or pM dihydrotestosterone, DHT) and suppress (nM DHT) the GnRH transcriptional response, without steroid receptor binding to DNA. Coactivator/integrator proteins SNURF and CBP will be tested in cotransfection and protein-protein binding studies for modulation of the GnRH response. Parallel biochemical and functional strategies will be used to investigate both stimulatory and suppressive effects of steroids in normal gonadotropes and LbetaT2 cells. These include tests of altered promoter occupancy by Sp1/Egr- 1 family members in ChIP assays, rescue of suppression by overexpression of transcription factors, and alteration of gene expression by focused microarray analysis and real-time RT-PCR. Promoter regions and transcription factors mediating steroid responses will be defined. Nuclear receptor requirements will be tested with steroid antagonists, and the Tfm androgen receptor mutant mouse. Non-genomic steroid effects on intracellular signaling pathways will be measured in the absence or presence of GnRH. These studies will further our understanding of GnRH regulation of the gonadotropins, and steroid modulation of this response. This research has important implications for understanding fertility disorders such as PCOS, in which elevated circulating androgens are accompanied by altered GnRH and LH secretion.