The ovarian steroid hormones, estrogen and progesterone (P), have profound modulatory influences upon an extensive network of hypothalamic cells that ensures the release of pituitary gonadotropins which trigger ovulation and synchronous expression of reproductive behavioral receptivity in female rodents. Although we have found that the neurotransmitter dopamine (DA) exerts an influence on hypothalamic P receptors (PRs) in the absence of ligand, the underlying molecular and cellular mechanisms are defined only in part. The goal of this project is to characterize critical signaling molecules in the ventromedial nucleus (VMN) that regulate PR-dependent reproductive behavior. Relative to the female VMN and reproductive behavior, the specific aims are to: 1) identify the signaling mechanisms by which membrane GTP-coupled pathways modify the PR-mediated genomic pathway; 2) ascertain the membrane pathway for membrane anchored proteins by which cAMP/PKA signaling is augmented or """"""""reinforced""""""""; 3) determine the patterns of receptor-coactivator phosphorylation and histone posttranslational modifications that are inherent to the genomic and reinforcing pathways; and 4) identify potential target genes for PR- and DA-induced transcription in specific cells within the VMN that regulate reproductive behavior. In our studies, we will employ a combination of animal models (rats, PR knockout mice expressing a PRE-driven reporter protein), pharmacologic agents (antagonists, antisense oligonucleotides and small interfering RNAs), well-established procedures (membrane binding assays, blot analysis, in situ hybridization, immunohistochemistry) fluorescence, confocal/deconvolution microscopy, PR-dependent behavior and new technologies (real-time RT-PCR, ChIP assays for patterns of targeted histone modifications, phospho-specific antibodies, single cell expression profiling, microarrays, and laser capture of individual cells). By monitoring coordinate changes in PR-dependent behavior, intracellular signaling, molecular changes within the nucleus, and gene expression induced by P and DAergic stimulation of individual VMN cells that mediate this behavior, we should gain unique insight into the underlying cellular and molecular mechanisms associated with steroid receptor regulation in the female hypothalamus that results in PR-dependent behavior and ovulation. In addition, such analyses should contribute to the development of new therapeutic strategies for the regulation of reproduction.
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