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.

雌激素在女性繁殖中起着至关重要的作用。在卵巢中，雌激素与FSH和多种旁分泌因子（例如IGF-1和激活素）一起作用，以诱导卵巢卵泡成熟。雌激素作用是通过核雌激素受体（ER）α和β介导的，它们通过改变基因表达和修改信号通路来起作用。 Eralpha主要在卵巢塞子中表达，基质细胞和基质细胞表示，而Erbeta则主要在颗粒细胞中。雌激素受体通过两个根本上调节基因转录 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）通过非经典或束缚的途径，该途径涉及与目标基因上存在的其他转录因子的ER相互作用。我们创建了一种新型的敲入小鼠模型，其中将Eralpha的DNA结合结构域中的两个氨基酸突变为ALA（ER [AA]），从而选择性地消除了经典途径信号传导。杂合的雌性含有一个突变的ER等位基因和一个野生型ER等位基因（ER AA/WT）是不育的 - 它们是无卵形的，表现出卵泡闭锁的增加，并且脂质含量增加的基质数量增加 细胞。我们假设该小鼠模型的表型特征反映了通过经典ERE介导的途径选择性基因表达的选择性丧失。这种独特的遗传模型将越过ER敲除小鼠（ER - / - ），以检查Eralpha经典和非经典途径在卵巢功能中的不同作用。 AIM 1将检查ER AA突变对卵泡发育和排卵的基因剂量效应，在以下六种Eralpha基因型中：ER WT/WT，ER AA/WT，ER AA/WT，ER AA/AA/AA，ER WT/ - ，ER AA/ - 和ER - / - 。 AIM 2将使用微阵列与已知调节卵巢功能的基因的分析结合使用 表征由经典和非经典途径调节的基因。 AIM 3将检查ER AA突变体通过使用体外转染研究和对源自各种ER基因型的细胞中基因调节的分析来改变细胞功能的机制。 AIM 4将检查ER AA突变如何改变ER介导的神经内分泌信号传导和卵巢刺激的卵巢刺激。综上所述，这些提出的研究将使用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来调节卵巢功能。