Estrogen Receptor Beta Regulation of the GnRH Neuron

描述（由申请人提供）：雌激素向下丘脑GnRH神经元提供负反馈和正反馈，调节发情周期，维持生育能力。生育能力下降和月经不规律是由于类固醇激素平衡紊乱造成的，多囊卵巢综合征患者就会出现这种情况。了解雌激素反馈下丘脑的机制，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被阐明（Herbison, 2007），但GnRH神经元的负调节机制和雌激素的直接作用尚不清楚。本研究的目的是研究雌激素在GnRH神经元中由雌激素受体β (erβ)介导的调节作用，其中erβ是主要亚型。目的1询问ERbeta是否在GnRH神经元中是正常发育、发情周期和生育所必需的。我们将开发一种只在GnRH神经元中缺乏erβ的靶向敲除小鼠。将产生纯合子“floxed”ERbeta小鼠，当与GnRH-Cre小鼠杂交时，外显子3将被删除。靶向载体被注射到胚胎干细胞中，阳性克隆被注射到囊胚中产生嵌合动物。这种gnrh神经元特异性ERbeta敲除对生育能力的影响将通过持续的交配协议进行评估。将对这些小鼠进行详细的生化和组织学评估，以确定ERbeta在GnRH神经元中的体内功能。同时，aim 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。跨越GnRH近端启动子的缺失构建体将被克隆到荧光素酶报告质粒中，并转染到GnRH神经元细胞系中，以鉴定负调控过程中所需的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定结合位点，并检查对雌激素受体功能的需求。ChIP和电迁移转移分析将用于鉴定调节复合物的成分。综上所述，这些研究将进一步加深我们对雌激素在调节GnRH神经元中的中枢作用以及由此产生的生育等生理效应的理解。对这些途径的复杂理解最终将使对人类疾病的干预更加有针对性。

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.