VITAMIN A AND REPRODUCTION

维生素 A 与生殖

• 批准号: 2199433
• 负责人: DAVID E ONG
• 金额: $ 21.95万
• 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 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2199433
• 关键词: VITAMIN; REPRODUCTION

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.

睾丸和附睾含有许多生化成分， 在向靶细胞和靶细胞内递送类维生素A中起重要作用 用于它们随后的作用或代谢。其中包括细胞外 类维生素A转运蛋白、视黄醇结合蛋白（RBP）和管腔 附睾视黄酸结合蛋白（E-RABP）;细胞内 类维生素A结合蛋白，细胞视黄醇结合蛋白（CRABP）， 细胞视黄酸结合蛋白（CRABP）;其新成员 家族，睾丸脂质结合蛋白（TLBP）;代谢 类维生素A转化为活性和非活性形式或可能的新代谢物。 类维生素A在睾丸中的传递和代谢方式的研究 和附睾可以揭示生产的调节机制， 这可能是其他类维生素A作用位点的范例， 同时也有助于我们理解 维甲酸在维持男性生殖功能中的作用。三 研究领域涉及分离的细胞和从细胞中提取的提取物。 睾丸和附睾。电镜免疫定位 水平、质谱法结构测定和标准品 生物化学和分子生物学技术，包括定量 将使用RT-PCR。 I.睾丸中类维生素A运动/代谢的模型是， 通过肌样细胞和Sertoli细胞摄取然后释放 新合成的RBP到达晚期生殖细胞， 视黄醇转化为视黄酸可能发生在支持细胞中。这将是 通过测定RBP中视黄醇的摄取和代谢进行测试， 分离细胞以描绘视黄酸的合成位点 精子发生的某些步骤。CRABP的限制是 后期精原细胞的细胞质表明这些细胞是 保护免受视黄酸的作用。 细胞色素P-450代谢 将测定分离细胞的视黄酸-CRABP， 将研究CRABP对细胞质的限制以检验这一点。 在EM水平上定位RBP和CRABP将测试模型。 二.睾丸精子积累视黄酯， 在附睾运输过程中。一种假设是它们代谢了 视黄酸酯作为精子产生信号释放， 附睾细胞通过E-RABP转运。这将由以下人员进行测试 检查精子视黄酯的代谢;确定是否管腔 类视黄酸，特别是全反式或9-顺式视黄酸，显示出结合 对E-RABP，将影响特定基因的主要细胞的表达 已知受精子相关因子调节，特别是 对于那些在静纤毛中含有CRABP的主细胞。的 合成E-RABP的头主细胞的作用， 将对CRBP对这一过程的影响进行审查。 三.将表征仅限于晚期生殖细胞的TLBP。其 将测定内源性配体。重组蛋白和天然蛋白将 用于结合能力和特异性的研究。