The epididymis provides the proper physiological environment in which sperm maturation occurs, and therefore serves a vital role in the establishment and maintenance of male fertility. Knowledge of epididymal function will contribute to a greater understanding of the development of the fertilization capacity of spermatozoa, and will give insights into the factors affecting both animal and human fertility. The long term objectives of the proposed research are to characterize testicular factors involved in regulating epididymal biosynthesis and function, and to gain an understanding of the role that regulated epididymal biosynthetic products serve in the sperm maturation process. Testicular factors have been recognized as essential in maintaining normal epididymal function. Gonadal androgens are required for proper epididymal function, and androgen-dependent transcription of epididymis-specific genes has been demonstrated. Experimental results have suggested that additional, non-androgen testicular factors are also necessary for the maintenance of normal epididymal biosynthesis. The expression of the proenkephalin gene in rat epididymal epithelial cells has been shown to be highly regulated by a non-androgen testicular factor. A specific aim of this proposal is to isolate this non-androgen testicular factor regulating proenkephalin gene expression in the epididymis. A related specific aim is to isolate cDNA clones of other genes expressed in the epididymis under the control of non-androgen testicular factor(s). Methods for maintaining epididymal epithelial cells in primary culture will be established. By utilizing the regulated expression of the proenkephalin gene in cultured cells as an assay system, the testicular factor regulating expression of this gene will be characterized. This regulatory factor will then be isolated from testicular extracts. Previous results have suggested that synthesis of a number of proteins in the epididymis is regulated in a manner similar to proenkephalin. A subtractive hybridization cDNA cloning strategy will be employed to isolate clones of genes expressed in the epididymis under the control of non-androgen testicular factors. Sequence analysis of these regulated genes will serve to identify proteins of potential importance to epididymal function.