Electron Microscopy of the Vesicle Pellet
The white-yellowish pellet obtained after ultracentrifugation of the cauda epididymal fluid was analyzed morphologically by electron microscopy (Fig. 2). This pellet was composed of vesicles with a distribution size between 25- and 75-nm diameter (64 ± 22 nm; mean ± SD, n = 292). Some of these vesicles contained electron-dense material and a large majority showed a typical membrane bilayer of an average size of 7.9 ± 1.4 nm (mean ± SD, n = 200), suggesting a homogeneous population.
Characterization of the Vesicle Proteins by 1-D SDS-PAGE
The proteins from these vesicles were analyzed by SDS-PAGE (Fig. 3A). The protein pattern obtained was very specific and different from that of the cauda fluid, particularly bands at 140, 110, and 45 kDa that were strongly enriched in the pellet. To estimate the protein quantity represented by these vesicles, the pellet was resuspended to concentrate the vesicles from the starting caudal fluid 20, 10, 5, 2.5, and 1 times (1 = no concentration).
FIG. 2. Electron micrograph and analysis of the high-speed pellet from the cauda epididymal fluid. Electron microscropy of the high-speed pellet showed small vesicles with a typical bilayer membrane. The diameters of the vesicles from two different preparations (10 different microscopic fields, 292 vesicles) were measured to establish the size distribution of these vesicles.
FIG. 3. Protein pattern of the vesicles. A) SDS-PAGE comparison of the proteins from caudal fluid (CEF), caudal fluid after high-speed centrifugation (CEF-hs), and from epididymal vesicles (vesicles). The same volume of CEF and CEF-hs were loaded while vesicles were resuspended in the initial volume of fluid or concentrated 2.5, 5, 10, or 20 times. B) Protein separation of the vesicle pellet obtained from the seminal plasma (SP Intact) of an intact and a vasectomized (SP Vas.) ram. The same volume of 10-fold concentrated pellet was loaded per lane. 6-16% SDS-PAGE; Coomassie blue-stained gel.