Estrogen provides both negative and positive feedback to GnRH neurons in the hypothalamus to regulate the estrous cycle and maintain fertility. Reduced fertility and irregular menses result from perturbations in the balance of steroid hormones, as happens in patients with polycystic ovarian syndrome. Understanding the mechanisms of estrogen feedback to the hypothalamus, in particular the specific transcription complexes mediating negative regulation of GnRH expression will provide new targets for developing therapies in such cases. While multi-neuron pathways regulating positive feedback have begun to be elucidated (Herbison, 2007), the mechanisms of negative regulation and the direct actions estrogen in GnRH neurons are still unknown. The goal of this research is to study the regulatory effects of estrogen, as mediated by estrogen receptor beta (ERbeta) in GnRH neurons, where this is the predominant isoform. Aim 1 asks whether ERbeta is required in the GnRH neuron for normal development, estrous cycling and fertility. We will develop a targeted knock out mouse that lacks ERbeta only in GnRH neurons. A homozygous "floxed" ERbeta mouse will be generated such that, when crossed with the GnRH-Cre mouse, exon 3 will be deleted. The targeting vector has been injected into ES cells and positive clones injected into blastocysts to generate chimeric animals. The effects of this GnRH-neuron specific ERbeta knock-out on fertility will be assessed with continuous mating protocols. Detailed biochemical and histologic assessments of these mice will be undertaken to identify the in vivo functions of ERbeta in GnRH neurons. Concurrently, aim 2 will use immortalized GnRH neuronal cell lines to elucidate the cellular mechanisms of estrogen mediated negative transcriptional regulation of GnRH. Deletion constructs spanning the GnRH proximal promoter will be cloned into luciferase reporter plasmids and transfected into GnRH neuronal cell lines to identify DMA sequences required during negative regulation. Chromatin immunoprecipitation (ChIP) will complement these studies to identify the specific binding sites of regulatory complexes and examine the requirement for estrogen receptor functions. Both ChIP and an Electromobility Shift Assay will be used to identify the components of the regulatory complex. Together these studies will further our understanding of the central actions of estrogen in the regulation of GnRH neurons and the consequent physiological effects such as fertility. A sophisticated understanding of these pathways will ultimately enable more targeted interventions in human disorders.