Post-testicular developmental changes in the ram sperm cell surface and their relationship to luminal fluid proteins of the reproductive tract.

Abstract

Surface proteins of ram spermatozoa were studied at three stages of post-testicular development: immediately after transport out of the testis; after epididymal transit; and after exposure to accessory sex gland secretions. Testicular and cauda epididymal spermatozoa, together with the fluids which normally surround them, were collected from conscious rams through catheters inserted into the rete testis and vas deferens, respectively. Ejaculated spermatozoa were obtained by electrical stimulation. The washed, viable spermatozoa were radioiodinated by the surface selective hactoperoxidase-glucose oxidase technique. Total sodium dodecyl sulfate-soluble cellular proteins were fractionated by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeled surface components were identified and distinguished from unlabeled cellular proteins by autoradiography of dried gel slices. The electrophoretic analysis reveals that the predominant radioiodinated surface components of testicular spermatozoa are seven polypeptides that migrate with apparent molecular weights of 78,000–132,000. Spermatozoa collected from the cauda epididymidis lack the major components characteristic of testicular spermatozoa, but have other unique surface features; these include two large polypeptides (molecular weights, 71,000 and 116,000 daltons) and several smaller components. Most prominent among the latter is a diffuse band of apparent molecular weight 24,000. These striking changes in accessible surface proteins take place during epididymal transit without alterations in the electrophoretic pattern of total cellular proteins. Further surface renovation after spermatozoal maturation is minimal, as shown by the similarity in the radioiodination pattern of ejaculated and cauda epididymal spermatozoa. Since extracellular fluids may play a role in surface modifications of spermatozoa in the epididymis, testicular spermatozoa were incubated in the presence of radioiodinated proteins of rete testis and cauda epididymal fluid. Uptake of labeled protein from rete testis fluid is nonspecific, but adsorption of cauda epididymal fluid proteins by testicular spermatozoa is highly selective. The major adsorbed component migrates with an apparent molecular weight of 24,000 and appears to correspond to the major surface component detectable by radioiodination of cauda epididymal spermatozoa. The anomalous migration of this protein on two-dimensional gel suggests that it is highly glycosylated. These results provide direct biochemical evidence that spermatozoa undergo extensive surface modifications during passage through the epididymis where they become almost totally dependent on exogenous substrates for survival and develop the capacity for coordinated movement. Adsorption of extracellular proteins from the luminal fluids of the testis and epididymis may contribute to these critical functions of the fully differentiated cell.