As a control, an equal volume of fluid from the cauda containing both the sperm and the droplets was centrifuged and washed only with PBS.
The PBS- and percoll-washed cauda sperm, the cytoplasmic droplets, the caudal fluid and the vesicles pellet were run on SDS-PAGE (Fig 7A). Almost no difference was observed in the protein pattern between the two caudal sperm preparations (first and second lanes) and the sperm extracts from zones 2 and 6 (not shown).
The cytoplasmic droplet protein pattern was very different from those of the sperm extracts, the caudal fluid, and the vesicles. We have previously shown that acrosin could be released in the fluid by dying sperm, and thus, if some of the vesicles were derived from the acrosomal membranes of these damaged sperm, some of the acrosin should remain associated with it. With our ram anti-acrosin antibody, three different ac-rosin forms were observed in the sperm extracts between 43 and 50 kDa and a 43-kDa band was slightly visible in the caudal fluid (Fig. 7B). No reactive bands were found in cytoplasmic droplets or in the vesicle preparation, suggesting that the vesicles did not derive from the degraded acrosomal sperm membranes and indicating also that the cytoplasmic droplet preparation was not contaminated with sperm.
FIG. 7. Origin of the protein vesicles. Equivalent amounts of epididymal cauda sperm before (—) and after percoll wash (+), cytoplasmic droplets, cauda epididymal fluid (CEF), and purified vesicles were separated by 616% SDS-PAGE. After Coomassie blue staining, except for sperm before and after percoll washing, a very different pattern of proteins was observed for each type of sample. An equivalent gel was transferred to nitrocellulose and probed with a ram anti-acrosin antibody. Sperm were strongly reactive and a slight reaction was observed in the cauda fluid.