VITAMIN A AND REPRODUCTION

睾丸和附睾含有许多生化元素， 在将维甲酸运送到靶细胞和在靶细胞内发挥重要作用 为他们随后的行动或新陈代谢。这些细胞包括细胞外 视黄醇转运蛋白、视黄醇结合蛋白和管腔蛋白 附睾维A酸结合蛋白；细胞内 视黄醇结合蛋白、细胞视黄醇结合蛋白、 细胞维甲酸结合蛋白(CRABP)；新成员 睾丸脂结合蛋白家族(TLBP)；代谢酶 维甲酸转变为活性和非活性形式或可能的新代谢物。 维甲酸在睾丸中的递送方式和代谢研究 附睾可能揭示了生殖和生殖的调节机制 可能是视黄醇作用的其他部位的范例。 身体，以及有助于我们理解基本的作用 维甲酸在维持男性生殖功能方面发挥作用。三位一体 研究领域涉及分离的细胞和从植物中提取的 睾丸和附睾部。免疫组织化学在电子显微镜上的定位 水平、质谱学结构测定和标准 生化和分子生物学技术，包括定量 将使用RT-PCR法。 I.睾丸中的视黄醇运动/代谢模型是视黄醇 通过摄取通过肌样细胞和支持细胞，然后释放 关于新合成的RBP到达晚期生殖细胞及其代谢的研究 视黄醇向视黄酸的转化可能发生在支持细胞中。这将是 通过测定RBP对视黄醇的摄取和代谢来测试 分离的细胞来描绘维甲酸的合成部位 精子发生的某些步骤所必需的。CRABP的一个限制是 晚期精原细胞中的细胞质表明这些细胞是 保护不受维甲酸的作用。细胞色素P-450代谢 将为分离的细胞测定维甲酸-CRABP的含量 将研究CRABP对细胞质的限制以测试这一点。 RBP和CRABP在EM层面的本地化将检验该模型。 二、睾丸精子积累耗尽的视黄酸酯 在附睾期。一种假设是，它们会代谢出 酯到维甲酸被释放为精子产生的信号 附睾细胞，通过E-RABP运输。这将通过以下方式进行测试 检查精子视黄酸酯的代谢；确定管腔是否 维甲酸，特别是全反式或9-顺式维甲酸，显示与 对于E-RABP，会影响主细胞特定基因的表达 已知受精子相关因子(S)调节，尤其是 对于那些显示在其立体纤毛中含有CRABP的主细胞。这个 合成E-RABP的头状主细胞的作用 将考察CRBP对这一过程的影响。 TLBP，仅限于晚期生殖细胞，将被描述。它的 内源性配体将被确定。重组蛋白和天然蛋白将 用于结合能力和特异性的研究。

The testis and epididymis contain many of the biochemical elements that play important roles in delivering retinoids to and within target cells for their subsequent action or metabolism. These include the extracellular retinoid transport proteins, retinol-binding protein (RBP) and lumenal epididymal retinoic acid-binding protein (E-RABP); the intracellular retinoid-binding proteins, cellular retinol-binding protein (CRABP), cellular retinoic acid-binding protein (CRABP); a novel member of the same family, testis lipid-binding protein (TLBP); the enzymes that metabolize retinoids to active and inactive forms or possible novel metabolites. Studies of the mode of delivery and metabolism of retinoids in the testis and epididymis may reveal mechanisms of regulation of production and action that may be paradigms for other sites of retinoid action in the body, as well as to contribute to our understanding of the essential roles that retinoids play in maintaining male reproductive function. The three areas of investigation involve both isolated cells and extracts from the testis and epididymis. Immunolocalization at the electron microscopic level, structural determinations by mass spectrometry, and standard biochemical and molecular biological techniques, including quantitative RT-PCR will be used. I. A model for retinoid movement/metabolism in the testis is that retinol passes through both the myoid and Sertoli cell by uptake and then release on newly synthesized RBP to reach the late germ cells and that metabolism of retinol to retinoic acid probably occurs in Sertoli cells. This will be tested by determining the uptake and metabolism of retinol from RBP by isolated cells to delineate sites of synthesis of the retinoic acid required for certain steps of spermatogenesis. A restriction of CRABP to the cytoplasm in later stage spermatogonia suggests that these cells are protected from the action of retinoic acid. Cytochrome P-450 metabolism of retinoic acid-CRABP will be determined for isolated cells and the restriction of CRABP to the cytoplasm will be studied to test this. Localization of RBP and CRABP at the EM level will test the model. II. Testicular spermatozoa accumulate retinyl ester which is depleted during epididymal transit. One hypothesis is that they metabolize the ester to retinoic acid to be released as a spermatozoa-generated signal to the epididymal cells, transported via E-RABP. This will be tested by examining spermatozoan metabolism of retinyl ester; determining if lumenal retinoids, particularly all-trans- or 9-cis-retinoic acid, shown to bind to E-RABP, will affect the expression by principal cells of specific genes known to be regulated by spermatozoa associated factor(s), particularly for those principal cells shown to contain CRABP in their stereocilia. The role of the caput principal cells that synthesize E-RABP and are rich in CRBP to this process will be examined. III. TLBP, restricted to late germ cells, will be characterized. Its endogenous ligand will be determined. Recombinant and native protein will be used for studies of binding ability and specificity.