NONCLASSICAL ESTROGEN RECEPTOR ALPHA ACTION IN THE OVARY

雌激素在女性生殖中起着至关重要的作用。在卵巢内，雌激素与卵泡刺激素和多种旁分泌因子，如IGF-1和激活素一起作用，诱导卵巢卵泡成熟。雌激素的作用是通过核雌激素受体(ER)α和β来实现的，它们通过改变基因表达和改变信号通路来发挥作用。ERα主要在卵泡膜和间质细胞中表达，而ERβ主要在颗粒细胞中表达。雌激素受体通过两个基本途径调节基因转录。 不同的机制：(1)通过经典的雌激素反应元件(ERE)介导的途径；(2)通过非经典的或连接的途径，涉及ER与靶基因上存在的其他转录因子的相互作用。我们已经建立了一种新的敲入小鼠模型，在该模型中，ERpha DNA结合域的两个氨基酸被突变为Ala(ER[AA])，从而选择性地消除了经典的通路信号。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，表现出更多的卵泡闭锁，并有更多富含脂质的间质 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介导的途径选择性地丢失基因表达。这一独特的遗传模型将与ER基因敲除小鼠(ER-/-)杂交，以研究ERα经典和非经典途径在卵巢功能中的不同作用。目的1研究ERα基因突变对卵泡发育和排卵的基因剂量效应，包括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的调控卵巢功能的基因分析来 描述受经典和非经典途径调控的基因。目的3将结合体外转基因研究和对不同ER基因类型细胞中基因调控的分析，研究ER AA突变体改变细胞功能的机制。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促性腺激素对卵巢的刺激。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。

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.