Estrogen Receptor Beta Regulation of the GnRH Neuron

描述（由申请人提供）：雌激素向下丘脑中的GnRH神经元提供负反馈和正反馈，以调节发情周期并维持生育能力。生育能力下降和月经不调是由于类固醇激素平衡紊乱所致，如多囊卵巢综合征患者。了解雌激素反馈到下丘脑的机制，特别是介导GnRH表达负调控的特异性转录复合物，将为此类病例的治疗提供新的靶点。虽然调节正反馈的多神经元通路已经开始阐明（Herbison，2007），但GnRH神经元中的负调节机制和雌激素的直接作用仍然未知。本研究的目的是研究雌激素的调节作用，作为介导的雌激素受体β（ER β）在GnRH神经元，这是主要的亚型。目的1询问是否ER β是正常发育，发情周期和生育所需的GnRH神经元。我们将开发一种仅在GnRH神经元中缺乏ER β的靶向敲除小鼠。将产生纯合的“floxed”ER β小鼠，使得当与GnRH-Cre小鼠杂交时，外显子3将缺失。将靶向载体注射到ES细胞中，并将阳性克隆注射到囊胚中以产生嵌合动物。将采用连续交配方案评估GnRH神经元特异性ER β敲除对生育力的影响。将对这些小鼠进行详细的生化和组织学评估，以确定ER β在GnRH神经元中的体内功能。同时，目的2将使用永生化GnRH神经元细胞系来阐明雌激素介导的GnRH负转录调节的细胞机制。将跨越GnRH近端启动子的缺失构建体克隆到荧光素酶报告质粒中，并转染到GnRH神经元细胞系中，以鉴定负调控期间所需的DMA序列。染色质免疫沉淀（ChIP）将补充这些研究，以确定调节复合物的特异性结合位点，并检查雌激素受体功能的要求。将使用ChIP和电迁移率变化试验来鉴定调节复合物的组分。这些研究将进一步加深我们对雌激素在调节GnRH神经元中的中心作用以及随之而来的生理效应（如生育）的理解。对这些途径的深入了解最终将使人类疾病的干预更具针对性。

DESCRIPTION (provided by applicant): 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.