Epididymal Distribution of the Vesicles
Using these immunological tools, the origin of the vesicles along the epididymis was investigated. We probed the epididymal fluids from different zones with antibodies against the proteins linked to the vesicles, i.e., phosphodiesterase E-NPP3, neprylisin, lactadherin-PAS 6/7, and protein G-beta (Fig. 6A). We found that E-NPP3, NEP, and the 35-kDa G-beta protein were restricted to the cauda epididymal fluid (zones 6/7 to 9).
We observed that one form (30 kDa) of PAS6/7-lactadherin was mainly associated with the caudal fluid and another (50-55 kDa) was present throughout the epididymis.
The Western blot probed with the anti-actin antibody (Fig. 6B, control) showed the presence of this protein in the fluid throughout the epididymis. Because this might suggest that some of the vesicles were transported from the upper epididymal tract, we submitted these different fluids to high-speed centrifugation. After this treatment, actin was slightly decreased in the high-speed supernatant from zone 7 (Fig. 6B, high speed) as compare with the control (Fig. 6B, control) and was completely removed from the fluid in zones 8 and 9. It was also in these zones that actin was retrieved associated with the pellet (see Fig. 5).
FIG. 6. Regional distribution of the vesicle in the epididymal fluid. A) Equivalent amounts of protein from the rete testis and the different epididymal zones were run on 6-16% SDS-PAGE and transferred to nitrocellulose and probed with antibodies reactive against vesicle proteins. B) An equivalent Western blot, actin was present throughout the epididymal fluid (control); high-speed centrifugation (high speed) decreased the quantity of actin in zone 7 and removed it from the fluid in zones 8/9. C) The fluid of zones 7 and 9 were probed with antibodies reactive against vesicle proteins before (—) or after (+) high-speed centrifugation. After this treatment, the immunoreactive proteins were significantly reduced or removed, indicating they are linked to vesicles.