Mouse sperm were collected from CD1 retired male breeders (Charles River), euthanized in accord with Institutional Animal Care and Use Committee guidelines and following experimental protocols previously approved by the Animal Care Committee of the University of Massachusetts. For the sperm to swim out, the cauda epididymis from each animal was placed in 0.5 ml of modified Whitten-Hepes medium (WH) containing 100 mM NaCl, 4.7 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 5.5 mM glucose, 1 mM Pyruvic acid, 4.8 mM L(þ)-lactic acid hemicalcium salt in 20 mM Hepes, pH 7.3, at 378C. After 10 min, sperm suspension was diluted into capacitating media or collected and centrifuged at 500 3 g for 5 min at room temperature (RT). For capacitation, 50 ll of the original suspension was diluted into 450 ll of capacitating media (WH supplemented with 20 mM NaHCO 3 and 3 mg/ml of bovine serum albumin [BSA], A-0281; Sigma) and incubated for 60 min as previously described [28] . To induce the acrosome reaction, capacitated sperm were treated with 3 lM calcium ionophore A23187 for 30 min. Total sperm extracts were prepared as detailed in Materials and Methods, separated using 10% (A) or 15% (B) acrylamide gels and analyzed by Western blot using different antibodies directed against the corresponding proteins. Arrowheads represent bands highlighted in Figure 2 .