The broad long-term goal of this proposal is to understand the essential role that vitamin A plays in male reproductive function, seeking to understand the necessary movements and metabolism of vitamin A in the testis and epididymis. The specific research proposed seeks to clarify the roles of several specific transport or binding proteins for vitamin A in these organs. Particular emphasis is given to two previously undescribed retinoic acid-binding proteins present in the epididymis of the rat. It is proposed to purify and characterize these proteins including determination of sequence, binding specificity, and endogenous ligand. The distribution and cellular location of the proteins will be determined by preparation of antiserum that can be used for sensitive radioimmunoassay and immunolocalization. Other species will be examined for the presence of these proteins., The function of the proteins will be examined by studying their ability to deliver retinoic acid to developing sperm. Preliminary work has shown that Sertoli cells in primary culture will internalize retinol from its complex with retinol-binding protein, the plasma transport protein for vitamin A, in a receptor-mediated fashion. This process will be further characterized. The metabolism of the internalized retinol will be determined, with particular emphasis on esterification and the possible involvement of a novel enzyme activity, lecithinretinol acyl transferase. The possible release by the Sertoli cell of the internalized vitamin A to the medium will be examined. The plasma membrane receptor for RBP will be studied in cell-free systems, following the transfer of retinol from RBP to CRBP. Purification of the RBP receptor will be attempted in order to produce antiserum usable for radioimmunoassay and immunolocalization. Finally, the cell-specific location of cellular retinol-binding protein and cellular retinoic acid-binding protein in human reproductive tissue will be determined. With antiserum to human CRBP, localization studies in human testis and epididymis will be continued. Such studies will be extended to ovary and uterus, two tissues with abundant CRBP. Human CRABP will be purified and antiserum to CRABP obtained that will be used for radioimmunoassay and immunolocalization studies similar to those for CRBP.
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