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).
This result thus confirmed that these proteins are mainly associated with the vesicles.
Presence of Epididymal Hydrophobic Proteins Within the Vesicles
To investigate whether hydrophobic proteins cosediment with the vesicles, we used a similar immunoblotting approach (Fig. 5). The first protein sought was the 17-kDa hydrophobic protein that has previously been described in rams.
Several other membrane-linked proteins were identified such as lactadherin-PAS6/7 protein, chaperone heat shock protein-71 (HSC71), vacuolar ATPase (V-ATPase), protein G-beta (G-beta) and NAP22/BASP1. One protein expressed in testis (HPAST1-testilin-EHD1) at the germ cell level was also identified. The metabolic enzymes creatine kinase (CreaKin), pyruvate kinase (PyrKin), glutamate carboxy-peptidase-like-1 (named carnosinase 1; CARN1), alpha-enolase (Enolase), aldehyde and isocitrate dehydrogenase (Ald-dh and Sorb-dh, respectively) and aldose reductase (Ald-red) were also present.
Fragments matching several proteins were obtained in the major spots at about 140 and 120 kDa, ie: dipeptidyl peptidase IV (DPPIV) and neprilysin (NEP) (140 kDa), and phosphodiesterase-I (E-NPP3) and beta-mannosidase (120 kDa). These proteins were also found when the 140 kDa and 120 kDa bands from a one-dimensional gel were processed in the same way.
The total protein quantity of the different lanes analyzed by a digital system indicated that these proteins were less than 3% of the total protein of the caudal fluid. This value was similar to that obtained by the Bradford assay using BSA as the standard and was also demonstrated by the lack of difference in the pattern of proteins of the cauda fluid before (CEF) or after ultracentrifugation (CEF-hs).
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.
The identification of the major protein spots from two-dimensional gels was obtained by mass spectrometry. Coomassie blue-stained spots were obtained from at least two different bidimensional gels from two different separately treated preparations. The spots were cut into small blocks with a sterile scalpel blade. The blocks were rinsed, then reduced and alkylated with iodoacetamide, and incubated overnight at 37°C in a microtube with 12.5 ng/|xl trypsin (sequencing grade; Roche, Meillan, France) in 25 mM NH4HCO3 as previously described.
The anti-lactadherin (P47/PaS-6/7/MfG-E8) rabbit polyclonal antibody was a gift from Dr. J.T. Rasmussen. The anti-phosphodiesterase I rabbit polyclonal (B10; nucleotide pyrophosphatase 3; E-NPP3) was a gift of Dr. M. Maurice. Anti-CD10 (neprilysin/CALLA/neutral endopeptidase) and anti-protein G-beta (against a peptide mapping at the carboxy-terminus of human G-beta 1 and recognizing G-beta 1, 2, 3, and 4) were rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-actin was a monoclonal antibody (Sigma, Saint Quentin Fallavier, France, and Prolabo, Fontenay-sous-Bois, France).
SDS 6-16% gradient polyacrylamide gels were used for protein separation, and iso-electrofocalization was performed as previously described. The gels were Coomassie blue-stained before spot cutting and mass-spectrometry analysis or silver stained when better visualization of the protein spots was needed. For protein quantification, the Coomassie blue-stained gels were scanned and the total protein content of each lane analyzed using the 1D-Elite software package (Amersham-Pharmacia Biotech AB, Uppsala, Sweden).