NONCLASSICAL ESTROGEN RECEPTOR ALPHA ACTION IN THE OVARY

卵巢中非经典雌激素受体 α 的作用

• 批准号: 6849152
• 负责人: James Larry JAMESON
• 金额: $ 25万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6849152
• 关键词: biological signal transduction; cAMP response element binding protein; cyclic AMP; estrogen receptors; estrus; follicle stimulating hormone; gel mobility shift assay; gene expression; gene targeting; genetic promoter element; genetic regulatory element; genetic transcription; genetically modified animals; granulosa cell; hormone regulation /control mechanism; in situ hybridization; inhibin; laboratory mouse; ovary; protein protein interaction; site directed mutagenesis; transcription factor; western blottings

Estrogen plays a critical role in female reproduction. Within the ovary, estrogen acts together with FSH and a variety of paracrine factors, such as IGF-1 and activin, to induce ovarian follicle maturation. Estrogen action is mediated through nuclear estrogen receptors (ER) alpha and beta, which act by altering gene expression and by modifying signaling pathways. ERalpha is expressed primarily in ovarian theca and stroma cells whereas ERbeta is predominant in granulosa cells. The estrogen receptor modulates gene transcription by two fundamentally different mechanisms: (1) via a classical estrogen response element (ERE)-mediated pathway and (2) via a non-classical or tethered pathway that involves ER interactions with other transcription factors present on target genes. We have created a novel knock-in mouse model in which two amino acids in the DNA-binding domain of ERalpha were mutated to Ala (ER[AA]), thereby selectively eliminating classical pathway signaling. Heterozygous females containing one mutant ER allele and one wild-type ER allele (ER AA/WT) are infertile-- they are anovulatory, exhibit increased follicular atresia, and have increased numbers of lipid-laden stromal cells. We hypothesize that the phenotypic features of this mouse model reflect the selective loss of gene expression via the classical ERE-mediated pathway. This unique genetic model will be crossed to ER knockout mice (ER -/-) to examine the distinct roles of the ERalpha classical and nonclassical pathways in ovarian function. Aim 1 will examine gene dosage effects of the ER AA mutation on follicular development and ovulation in the following six ERalpha genotypes: ER WT/WT, ER AA/WT, ER AA/AA, ER WT/-, ER AA/-, and ER -/-. Aim 2 will use microarrays in combination with analyses of genes known to regulate ovarian function to characterize genes regulated by the classical and nonclassical pathways. Aim 3 will examine the mechanism by which the ER AA mutant alters cellular function by using a combination of in vitro transfection studies and analyses of gene regulation in cells derived from the various ER genotypes. Aim 4 will examine how the ER AA mutation alters ER-mediated neuroendocrine signaling and gonadotropin stimulation of the ovary. Taken together, these proposed studies will use the ER AA genetic model to gain insight into how estrogen acts by classical and nonclassical pathways to regulate ovarian function.

雌激素在女性生殖中起着关键作用。在卵巢内，雌激素与FSH和各种旁分泌因子（如IGF-1和激活素）共同作用，诱导卵泡成熟。雌激素作用通过核雌激素受体（ER）α和β介导，其通过改变基因表达和通过修饰信号通路起作用。ER α主要表达于卵泡膜和基质细胞，而ER β主要表达于颗粒细胞。雌激素受体从两个方面调节基因转录 不同的机制：（1）通过经典的雌激素反应元件（ERE）介导的途径和（2）通过涉及ER与靶基因上存在的其他转录因子相互作用的非经典或栓系途径。我们已经创建了一种新的敲入小鼠模型，其中ER α的DNA结合结构域中的两个氨基酸突变为Ala（ER[AA]），从而选择性地消除经典途径信号传导。含有一个突变型ER等位基因和一个野生型ER等位基因（ER AA/WT）的杂合子女性是不育的--她们无排卵，表现出卵泡闭锁增加，并且具有增加的脂质负载基质细胞数量。 细胞我们假设，这种小鼠模型的表型特征反映了通过经典的ERE介导的途径选择性丧失基因表达。将这种独特的遗传模型与ER敲除小鼠（ER -/-）杂交，以检查ER α经典和非经典途径在卵巢功能中的不同作用。目的1将研究ER AA突变对以下6种ER α基因型卵泡发育和排卵的基因剂量效应：ER WT/WT、ER AA/WT、ER AA/AA、ER WT/-、ER AA/-和ER -/-。 Aim 2将使用微阵列结合已知调节卵巢功能的基因分析， 表征由经典和非经典途径调控的基因。目的3将研究ER AA突变体改变细胞功能的机制，通过使用体外转染研究和来自各种ER基因型的细胞中的基因调控分析的组合。目的4将研究ER AA突变如何改变ER介导的神经内分泌信号和卵巢促性腺激素刺激。综上所述，这些拟议的研究将使用ER AA遗传模型来深入了解雌激素如何通过经典和非经典途径调节卵巢功能。