Total sperm extracts were obtained by cell suspension in nonreducing Laemmli buffer and by boiling for 5 min. Before running, samples were supplemented with 5% b-mercaptoethanol when required. The SDS-denaturing gels of different acrylamide concentrations (10% or 15%) were used depending on the protein under study. After electrophoresis, proteins were electroblotted to polyvinylidene fluoride membrane and blocked with 5% skimmed milk. All incubation and washing procedures were done with PBS supplemented with 0.1% Tween 20 (PBST). Membranes were blocked for 1 h at RT and then incubated with the different first antibodies overnight at 48C as previously described [29] . After washing three times for 5 min, the specific peroxidaseconjugated secondary antibody was added, and incubation was carried out for 1 h at RT. The membranes were washed with PBST, and immune complexes were located using ECL Plus and Kodak Biomax light films.