Sperm maturation requires interaction of sperm with specific secretory products of the epididymal epithelium. Secretion of many of these products is dependent upon the stimulation of the epithelium by androgen, presumably locally produced dihydrotestasterone. However, testoserone is metabolized to dihydrotestosterone in epithelial cells proximal in the epididymis to most sites where epithelial products important in sperm maturation are secreted. Thus, elimination of the local supply of dihydrotestosterone would stop sperm maturation. Testosterone and androgen binding protein are available to the epididymal epithelium in rete testis fluid which carries sperm into the epididymis. Receptor-mediated endocytosis of androgen binding protein occurs and it is postulated that this occurs in cells containing 5 alpha-reductase. Furthermore, it is postulated that androgen binding protein serves as a carrier to transport dihydrotestosterone from the principal cells where it is produced to secretory cells located distally int he duct where dihydrotestosterone, still bound to androgen binding proteins, is taken into cells (dependent upon this androgen) by receptor- mediated endocytosis. If this is correct, blockage of this process would provide an approach for male contraception. Procedures are established for culture of ovine principal cells; fractionation of ovine rete testis fluid to obtain androgen binding protein, semi-purified alpha 2-macroglobulin, albumin, transferrin, and other proteins from rete testis fluid; quantification of steroids and androgen binding protein; and to study endocytosis with gold-labeled proteins. Endocytosis will be studied by subjective and quantitative electron microscopy and by immunocytochemistry. The hypothesis will be tested in vivo using micropuncture techniques to sample fluid form 9 regions of the ram epididymis to allow analyses of testosterone, dihydrotestosterone and androgen binding protein concentrations. Cultured principal cells (an in vitro system) will be used to determine the effects of steroid and location within the epididymis on the endocytosis of androgen binding protein, as compared to other proteins present in rete testis fluid or secreted by the epididymal epithelium. Dihydrotestosterone production will be measured. Corroboration will be provided by in vivo studies following uptake by the epididymal epithelium of exogenously labeled proteins reinjected into the rete testis.
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