VITAMIN A AND REPRODUCTION

维生素 A 与生殖

• 批准号: 6636836
• 负责人: DAVID E ONG
• 金额: $ 23.78万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-30 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636836
• 关键词: chemical binding; chloramphenicol acetyltransferase; differential display technique; estrogens; estrus; female; gene expression; gene induction /repression; genetic promoter element; hormone regulation /control mechanism; in situ hybridization; laboratory rat; molecular cloning; reporter genes; retinoate; retinoid binding proteins; subtraction hybridization; tissue /cell culture; uterus; vitamin biosynthesis; vitamin metabolism

Vitamin A, retinol, is an essential nutrient that serves as precursor to the important hormone, retinoic acid (RA). Relatively little is known of the control of synthesis of RA from retinol in the normal, fully-developed animal and sites of action of RA are inferred, rather than demonstrated. Previous work has identified estrogen as a physiological signal which induces the synthesis of RA in the rat uterus and that coordinately directs cell-specific expression of the three cellular retinoid-binding proteins present in the uterus during the estrous cycle. Proposed studies will: l) Identify uterine genes that are under estrogen control indirectly, via RA stimulation. The techniques of differential display or subtractive hybridization followed by library screening or will be used to identify these genes. Candidate genes will be followed during the estrous cycle to confirm their physiological significance. 2) Demonstrate the site(s) of expression of the estrogen-stimulated RA responsive genes by in situ hybridization and immunolocalization during the estrous cycle. Demonstration of expression/non-expression of candidate genes in cells expressing cellular retinoic acid-binding protein will test the competing hypotheses that this protein either blocks or enhances the RA responsiveness of cells. 3) Establish the mechanism by which estrogen directly regulates cellular retinoic-acid binding protein (II) expression in the uterus. The promoter region of the rat gene will be cloned, dissected and tested using CAT reporter constructs in an estrogen responsive cell line. 4) Demonstrate the mechanism by which estrogen induces RA synthesis in the uterus. Specifically, is this induction a direct effect of estrogen on pre-existing enzymes, does it require transcription, or is it indirect? A novel radioreceptor assay capable of detecting small amounts of RA has been developed for this aim. In summary, the work to be accomplished here will allow dissection of the effects of the demonstrated estrogen-stimulated synthesis of RA signal that is part of a normal physiological process. This will provide important information on retinoic acid action in the unmanipulated, intact animal. Regulation of retinoic acid production by estrogen has direct importance for understanding/treating conditions such as endometriosis, breast cancer, and cancers of the female reproductive system.

维生素A，视黄醇，是一种必需的营养物质，是重要激素视黄酸（RA）的前体。相对而言，我们对正常发育的动物视黄醇合成RA的控制知之甚少，RA的作用部位是推断的，而不是证明的。先前的工作已经确定雌激素是一种生理信号，它诱导大鼠子宫内RA的合成，并协调指导在发情周期中子宫内存在的三种细胞类维甲酸结合蛋白的细胞特异性表达。拟开展的研究将：1)通过类风湿性关节炎刺激，确定受雌激素间接控制的子宫基因。鉴别这些基因的技术将采用差示或减法杂交，然后进行文库筛选。将在发情周期对候选基因进行跟踪，以确认其生理意义。2)通过原位杂交和免疫定位证实雌激素刺激的RA应答基因在发情周期中的表达位点。在表达细胞维甲酸结合蛋白的细胞中表达或不表达候选基因将验证该蛋白阻断或增强细胞RA反应性的竞争性假设。3)建立雌激素直接调控子宫细胞维甲酸结合蛋白（retinoic-acid binding protein， II）表达的机制。大鼠基因的启动子区域将被克隆、解剖，并在雌激素应答细胞系中使用CAT报告结构进行测试。4)阐明雌激素诱导子宫内RA合成的机制。具体来说，这种诱导是雌激素对已有酶的直接作用，需要转录，还是间接作用？一种新型的能够检测少量类风湿性关节炎的放射受体测定方法已经为此目的而开发。总之，这里要完成的工作将允许解剖已证实的雌激素刺激的RA信号合成的影响，这是正常生理过程的一部分。这将为维甲酸在未处理的完整动物中的作用提供重要信息。雌激素对维甲酸产生的调节对于理解和治疗子宫内膜异位症、乳腺癌和女性生殖系统癌症等疾病具有直接的重要性。