OVARIAN LDL RECEPTOR AND STAR GENE REGULATION

卵巢 LDL 受体和星基因调控

• 批准号: 2908599
• 负责人: HOLLY ANNE LAVOIE
• 金额: $ 12.6万
• 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/2908599
• 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.

女性的正常生育力需要一个或多个卵泡发育到排卵前阶段，以及功能性黄体的形成和维持。在这些转变过程中，卵泡获得合成雌二醇和孕酮的能力，并在排卵后转变为主要分泌孕酮的结构。了解在卵巢细胞分化过程中促性腺激素是如何开启多个基因的，将有助于我们理解为什么一些卵泡被选择排卵，而另一些卵泡则发生闭锁。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（包括FSH、LH和胰岛素样生长因子I（IGF-I））的控制。IGF-I在类固醇生成途径的几个水平上增强促性腺激素的作用，包括卵巢颗粒细胞中细胞色素P450胆固醇侧链裂解复合物对胆固醇的摄取、转运和利用。关于促性腺激素和IGF-I如何协同作用以控制调节类固醇生成能力的基因的转录，我们知之甚少。这项提案将研究促性腺激素和IGF-1如何相互作用，以调节两个关键的类固醇生成基因，即低密度脂蛋白（LDL）受体和类固醇生成急性调节蛋白（星星）。（1-2年级）：LDL受体和星星启动子将被定位为对促性腺激素和IGF-I有反应的假定转录因子。将与荧光素酶报告基因连接的每个基因启动子的缺失片段转染到卵巢细胞的FSH和LH应答原代培养物中，以鉴定每个启动子和阻遏物区域的FSH应答增强子区域。还将进行DNA酶足迹研究以描绘反应区域。（2-4年级）：将进行潜在顺式作用元件的突变，以确定每个位点对每个基因启动子的激素介导的反式激活的相对贡献。（2-5年级）：将对来自经酶处理的细胞的颗粒细胞核提取物与每个启动子的激素响应区进行凝胶位移分析。将鉴定促性腺激素和/或IGF-I诱导的这些基因转录所必需的反式作用蛋白。（2-5年级：与卵巢卵泡核蛋白的平行研究将有助于确定哪些因子在体内与星星和LDL受体mRNA同时调节和表达。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，并增加我们对不孕机制的理解。