The previous samples and sperm extracts from zones 2 and 6 were probed with the different antibodies reacting with the vesicle proteins (Fig. 8). The anti-phosphodiesterase and anti-neprilysin antibodies showed reactive bands in the cauda fluid containing the vesicles, but no reaction was obtained with the cauda sperm extract or cytoplasmic droplet. The neprilysin antibody reacted with the caput sperm extract but not with corpus and cauda sperm extracts, but the neprilysin form observed in the fluid showed a lower molecular weight (less than 120 kDa; see Fig. 8, compare lanes Z2 and CEF).
Moreover, this neprilysin fluid form appeared only in zones 6-7, suggesting that it was a different form than the sperm neprilysin. The anti-actin antibody reacted with the sperm extracts but the decrease in the reaction observed in the percoll-washed sperm and the substantial reaction with the cytoplasmic droplets indicated that actin was mainly present in these later (Fig. 8). As expected, the total fluid was reactive because actin was present in the vesicles.
The anti-G beta antibody recognized different bands on sperm extracts, the most reactive at 43 and 35 kDa and a strong doublet at about 20 kDa.
FIG. 8. Common proteins between sperm and vesicles. Equivalent amounts of epididymal percoll-washed sperm from zones 2 and 6, cauda sperm before (—) and after percoll washing (+), purified cytoplasmic droplets, and cauda epididymal fluid (CEF) were separated, transferred, and probed with antibodies reactive to vesicle proteins. The major proteins ENPP3 and neprilysin were not found on sperm from zones 6 and 9 nor on cytoplasmic droplets, while actin, G-beta, and the 43-kDa PAS6/ 7 were mainly associated with cytoplasmic droplets but not with cauda epididymal percoll-washed sperm.