Estrogen Receptor Beta Regulation of the GnRH Neuron

说明(申请人提供)：雌激素为下丘脑中的促性腺激素释放激素神经元提供负反馈和正反馈，以调节发情周期和维持生育能力。生育力下降和月经不调是类固醇激素平衡失调的结果，就像多囊卵巢综合征患者的情况一样。了解雌激素反馈到下丘脑的机制，特别是介导GnRH表达负调控的特定转录复合体，将为开发此类病例的治疗方法提供新的靶点。虽然调节正反馈的多个神经元通路已经开始阐明(Herbison，2007)，但GnRH神经元中负调节的机制和雌激素的直接作用仍不清楚。这项研究的目的是研究雌激素在促性腺激素释放激素神经元中的调节作用，这种调节作用是由雌激素受体β(ERbeta)介导的。目的1探讨促性腺激素释放激素神经元的正常发育、动情周期和生育是否需要ERbeta。我们将开发一种仅在GnRH神经元中缺乏ERbeta的靶向基因敲除小鼠。当与GnRH-CRE小鼠杂交时，将产生一个纯合子的“有花蕾的”ERbeta小鼠，外显子3将被删除。靶向载体已经注射到ES细胞中，并将阳性克隆注射到囊胚中以产生嵌合动物。这种GnRH神经元特异性ERbeta基因敲除对生育力的影响将通过连续交配方案进行评估。将对这些小鼠进行详细的生化和组织学评估，以确定GnRH神经元中ERbeta的体内功能。同时，Aim 2将利用永生化的GnRH神经元细胞系来阐明雌激素介导的GnRH负转录调控的细胞机制。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载体中，并导入GnRH神经元细胞系，以确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，以确定调节复合体的特定结合部位，并检查对雌激素受体功能的要求。芯片和电子迁移率漂移分析都将用于识别监管复合体的组件。总之，这些研究将进一步加深我们对雌激素在调节GnRH神经元以及由此产生的生理效应(如生育)中的中枢作用的理解。对这些途径的深入了解最终将使我们能够对人类疾病进行更有针对性的干预。

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.