Studies on Mechanisms of Surface Modifications in Ram Spermatozoa During the Final Stages of Differentiation1

To elucidate mechanisms involved in surface changes of ram spermatozoa during their final stages of differentiation in the epididymis we have studied: 1) trypsin accessibility of intrinsic surface proteins of testicular spermatozoa; 2) trypsin accessibility of adsorbed rete testis fluid (RTF) and cauda epididymal fluid (CEF) components bound to testicular spermatozoa; and 3) the regional distribution of intrinsic surface proteins and adsorbed fluid components over testicular and ejaculated spermatozoa. Electrophoretic analysis of testicular spermatozoa incubated in radioiodinated RTF revealed adsorption of several fluid components of molecular weights ranging from 17,500 to 41,000 daltons. Mild trypsinization removed almost all adsorbed 125I-labeled proteins, but after prolonged incubation traces of label remained inaccessible to trypsin. Incubation of testicular spermatozoa in 125I-labeled CEF resulted in selective and time-dependent adsorption. A 24,000 dalton component was predominant among the adsorbed proteins, and between 21% and 30% of this fluid component was inaccessible to trypsin. Because internalization of intrinsic surface components might be associated with the striking transformation of the radioiodination pattern of maturing ram spermatozoa, radioiodinated testicular spermatozoa were also subjected to trypsinization. The results suggest that some of the surface radioactivity becomes inaccessible to trypsin and is slowly degraded by endogenous protease to yield new radioactive components protected from exogenous proteolytic enzymes. To determine the distribution of intrinsic surface proteins and adsorbed fluid components over the sperm cell body, surface-labeled spermatozoa or spermatozoa incubated in 125I-labeled fluids were fractionated after disruption by a brief ultrasonification. The subcellular fractions obtained were homogenous as judged by morphological criteria, but loss of outer membranes from the head and tail fractions complicated interpretation of the results. Although electrophoretic analysis of the subcellular fractions revealed striking differences in staining patterns, there was little evidence for regional specialization of adsorbed or intrinsic surface components in both testicular and ejaculated spermatozoa. The significance of this fluid adsorption is not clearly resolved but the results suggest that internalization and degradation within the membrane of maturing spermatozoa may be important mechanisms of sperm differentiation in the epididymis.