Testicular spermatozoa are immotile and infertile. It is during transit through the epididymis that they develop the capacity for motility and the ability to fertilize ova. An understanding of the role of the epididymis in mediating sperm maturation is of great importance in male fertility and infertility. The specific aim of this proposal is to develop a system whereby the events governing sperm maturation within the epididymis can be studied in vitro. Epididymal epithelial cells isolated from different regions of the epididymis will be grown as polarized monolayers on filters impregnated with defined extracellular matrix components in novel cell culture chambers. Cells growing in the chambers will be characterized morphologically by electron microscopy and the ability of monolayers to exclude tracer compounds and lgG. Protein and glycoprotein secretion by the epithelial cells will be monitored by incorporating radiolabeled amino acids and sugars into proteins and detecting these labeled compounds by SDS polyacrylamide gel electrophoresis and fluorography. The effects of androgen deprivation, anti-androgens, protein and glycoprotein synthesis inhibitors on the synthesis and secretion of labeled proteins will also be tested. The role of testicular derived proteins, for example androgen binding protein, in protein synthesis will be investigated simply by incubating cells in medium depleted of these proteins. Confluent monolayers of cells in the chambers will then be exposed to culture medium at their basal surface and to rete testis fluid with or without teticular or epididymal spermatozoa at their apical surface. Spermatozoa in the apical chamber will be maintained in contact with the epithelial cells for varying times and their maturation assessed by estimates of cytoplasmic droplet migration, changes in surface properties, motility, and the ability of spermatozoa to bind eggs. The effects of various components in the basal medium, for example, serum and androgen, on these parameters of sperm maturation will also be investigated. Similarly, the effects of components in the rete testis fluid at the apical surface, for example, androgen binding protein will be tested. Using this system, it should be possible to determine whether testicular spermatozoa require passage through particular regions of the epididymis before they mature and also, what positive contributions the epididymal cells themselves make to this process.