VITAMIN A AND REPRODUCTION

维生素A，视黄醇，是一种必需的营养物质，是重要的激素维甲酸(RA)的前体。在正常发育的动物中，对视黄醇合成RA的控制相对较少，而且RA的作用部位是推测的，而不是证明的。以前的工作已经确定雌激素是一种生理信号，它诱导大鼠子宫中RA的合成，并协调指导发情周期中子宫中存在的三种细胞维甲酸结合蛋白的细胞特异性表达。拟议的研究将：L)通过RA刺激间接识别受雌激素控制的子宫基因。差异显示或消减杂交技术，然后进行文库筛选，或将用于鉴定这些基因。在发情周期中，将跟踪候选基因，以确认它们的生理意义。2)用原位杂交和免疫组织化学方法证实雌激素刺激的RA反应基因在动情周期中的表达部位(S)。在表达细胞维甲酸结合蛋白的细胞中表达/不表达候选基因的证明将检验该蛋白阻断或增强细胞的RA反应性的相互竞争的假设。3)建立雌激素直接调节子宫细胞维甲酸结合蛋白(II)表达的机制。老鼠基因的启动子区域将被克隆、解剖，并在雌激素反应细胞系中使用CAT报告结构进行测试。4)说明雌激素在子宫中诱导RA合成的机制。具体地说，这种诱导是雌激素对原有酶的直接影响，是需要转录，还是间接的？为此，发展了一种新的能够检测少量RA的放射受体分析方法。总之，这里要完成的工作将使我们能够剖析雌激素刺激的RA信号合成的影响，这是正常生理过程的一部分。这将为维甲酸在未经处理的完整动物体内的作用提供重要信息。雌激素调节维甲酸的产生对于了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意义。

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.