OVARIAN LDL RECEPTOR AND STAR GENE REGULATION

卵巢 LDL 受体和星基因调控

• 批准号: 6182360
• 负责人: HOLLY ANNE LAVOIE
• 金额: $ 12.97万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-21 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6182360
• 关键词: DNA binding protein; DNA footprinting; RNase protection assay; animal tissue; cell differentiation; corpus luteum; female; follicle stimulating hormone; gel mobility shift assay; gene induction /repression; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; gonadotropins; graafian follicles; granulosa cell; hormone regulation /control mechanism; insulinlike growth factor; low density lipoprotein receptor; luteinizing hormone; site directed mutagenesis; steroid biosynthesis; tissue /cell culture; transcription factor

Normal fertility in the female requires the development of one or more follicles to the pre-ovulatory stage, and formation and maintenance of a functional corpus luteum. During these transitions the follicle acquires the ability to synthesize estradiol and progesterone and transforms following ovulation into a predominantly progesterone-secreting structure. Understanding how multiple genes are turned on by the gonadotropins during ovarian cell differentiation will help us understand why some follicles are selected to ovulate and others undergo atresia. The ability to synthesize steroid hormones is a marker of differentiation in granulosa cells and is under the control of pituitary gonadotropins and locally produced ovarian factors, including FSH, LH, and Insulin-like Growth Factor I (IGF-I). IGF-I augments gonadotropin action at several levels of the steroidogenic pathway, including cholesterol uptake, transport, and utilization by the cytochrome P450 cholesterol-side chain cleavage complex in ovarian granulosa cells. Little is known about how gonadotropins and IGF-I act in concert to control the transcription of genes regulating steroidogenic capacity. This proposal will investigate how gonadotropins and IGF-1 interact to regulate two genes critical to steroidogenesis, namely the low density lipoprotein (LDL) receptor and Steroidogenic Acute Regulatory protein (StAR). (Years 1-2): The LDL receptor and StAR promoters will be mapped for putative transcription factors responding to gonadotropins and IGF-I. Deletional fragments of each gene promoter linked to a luciferase reporter gene will be transfected into FSH and LH responsive primary cultures of ovarian cells to identify hormone-responsive enhancer regions of each promoter and repressor regions. Dnase footprinting studies will also be performed to delineate responsive regions. (Years 2-4): Mutations of potential cis-acting elements will be made to determine the relative contributions of each site to hormone mediated transactivation of each gene promoter. (Years 2-5): Granulosa cell nuclear extracts from hormone-treated cells will be subjected to gel shift analyses with hormone responsive regions of each promoter. Trans-acting proteins necessary for transcription of these genes induced by gonadotropins and/or IGF-I will be identified. (Years 2-5: Parallel studies with dated ovarian follicle nuclear proteins will help identify which factors are regulated and expressed concurrently with StAR and LDL receptor mRNA in vivo. These studies will promote our understanding of how multiple genes are coordinated by different trophic factors in the ovary during follicular differentiation and add to our understanding of mechanisms of infertility.

女性的正常生育需要一个或多个卵泡发育到排卵前阶段，以及功能黄体的形成和维持。在这些转变过程中，卵泡获得了合成雌二醇和孕酮的能力，并在排卵后转变为以孕酮为主的分泌结构。了解促性腺激素如何在卵巢细胞分化过程中激活多个基因，将有助于我们理解为什么一些卵泡被选择排卵，而另一些卵泡则经历闭锁。合成类固醇激素的能力是颗粒细胞分化的标志，受垂体促性腺激素和局部产生的卵巢因子控制，包括FSH、LH和胰岛素样生长因子I(IGF-I)。IGF-I在类固醇合成途径的多个水平上增强促性腺激素的作用，包括卵巢颗粒细胞中胆固醇的摄取、运输和细胞色素P450胆固醇侧链裂解复合体的利用。关于促性腺激素和IGF-I如何协同作用来控制调节类固醇生成能力的基因的转录，人们知之甚少。这项建议将研究促性腺激素和IGF-1如何相互作用来调节两个对类固醇合成至关重要的基因，即低密度脂蛋白(LDL)受体和类固醇生成急性调节蛋白(STAR)。(1-2岁)：低密度脂蛋白受体和STAR启动子将被映射为对促性腺激素和IGF-I反应的假定转录因子。每个基因启动子的缺失片段与荧光素酶报告基因相连，将其导入卵巢细胞对FSH和LH反应的原代培养中，以确定每个启动子和抑制区的激素反应增强区。还将进行DNA酶足迹研究，以描绘响应区。(2-4年)：将对潜在的顺式作用元件进行突变，以确定每个位置对激素介导的每个基因启动子反式激活的相对贡献。(2-5年)：从激素处理的细胞中提取的颗粒细胞核提取物将进行凝胶位移分析，以确定每个启动子的激素响应区。将鉴定由促性腺激素和/或IGF-I诱导的这些基因转录所需的反式作用蛋白。(第2-5年：对过期的卵泡核蛋白进行的平行研究将有助于确定哪些因子在体内与STAR和低密度脂蛋白受体mRNA同时受到调控和表达。这些研究将促进我们对卵泡分化过程中卵巢中不同营养因子如何协调多个基因的理解，并增加我们对不孕症机制的理解。