Estrogen Receptor Beta Regulation of the GnRH Neuron

GnRH 神经元的雌激素受体 Beta 调节

• 批准号: 7541559
• 负责人: Jennifer Sophie Mammen
• 金额: $ 5.33万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-08 至 2010-09-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7541559
• 关键词: Animals; Binding; Binding Sites; Biochemical; Biological Assay; Breeding; Cell Line; Chromatin; Complement; Complex; Cultured Cells; DNA Sequence; Development; Disease; Disruption; Electrophoretic Mobility Shift Assay; Equilibrium; Estrogen Receptor beta; Estrogen Receptors; Estrogens; Estrous Cycle; Exons; Feedback; Fertility; Gene Expression; Genes; Genetic Transcription; Goals; Gonadotropin Hormone Releasing Hormone; Histocytochemistry; Histologic; Human; Hypothalamic structure; In Situ Hybridization; In Vitro; Intervention; Investigation; Knock-out; Knockout Mice; Luciferases; Mediating; Menstrual fluid; Menstruation; Mus; Neurons; Ovarian; Partner in relationship; Pathway interactions; Patients; Periodicity; Phenotype; Physiological; Plasmids; Precipitation; Promoter Regions; Protein Isoforms; Protocols documentation; Regulation; Relative (related person); Reporter; Research; Role; Series; Signal Transduction; Syndrome; Testing; Transcriptional Regulation; Transgenic Mice; blastocyst; chromatin immunoprecipitation; embryonic stem cell; in vivo; mutant; promoter; receptor function; recombinase; reproductive; research study; steroid hormone; vector

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.

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，以调节动情周期并维持生育能力。类固醇激素平衡的紊乱会导致生育能力下降和月经不规律，就像多囊卵巢综合征患者所发生的情况一样。了解雌激素反馈下丘脑的机制，特别是介导 GnRH 表达负调节的特定转录复合物，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神经元通路已开始被阐明（Herbison，2007），但负调节机制和雌激素在 GnRH 神经元中的直接作用仍然未知。本研究的目的是研究 GnRH 神经元中雌激素受体 β (ERbeta) 介导的雌激素调节作用，ERβ 是 GnRH 神经元中的主要亚型。目标 1 询问 GnRH 神经元的正常发育、动情周期和生育能力是否需要 ERbeta。我们将开发一种仅在 GnRH 神经元中缺乏 ERbeta 的靶向敲除小鼠。将产生纯合“floxed”ERbeta 小鼠，当与 GnRH-Cre 小鼠杂交时，外显子 3 将被删除。将靶向载体注射到ES细胞中，并将阳性克隆注射到囊胚中，产生嵌合动物。这种 GnRH 神经元特异性 ERbeta 敲除对生育力的影响将通过连续交配方案进行评估。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，目标 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，以识别负调节过程中所需的 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异性结合位点并检查雌激素受体功能的要求。 ChIP 和电动移动分析都将用于识别调节复合物的组成部分。这些研究将进一步加深我们对雌激素在 GnRH 神经元调节中的核心作用以及随之而来的生理效应（如生育能力）的理解。对这些途径的深入了解最终将能够对人类疾病进行更有针对性的干预。