Sperm suspensions were centrifuged at 500 3 g for 10 min and washed with WH medium. The pellet was resuspended in 400 ll of TEN buffer (25 mM Tris-HCl, 150 mM NaCl, 5 mM edetic acid, pH 7.3) supplemented with 0.5% Triton X-100 and a protease inhibitor cocktail (1 mM PMSF, 1 mM NaF, 2 mM sodium orthovanadate, 20 lg/ml of leupeptin, 15 lg/ml of pepstatin, 0.8 mM aprotinin, 10 mM benzimidine, 3 lg/ml of TLCK, 1 mM AEBSF, 40 lM bestatin, and 14 lM E-64). This suspension was Dounce homogenized and sonicated with five brief bursts of 1 sec each. Samples were kept on ice for 5 min and then rotated at 48C for 45 min. Lysates were adjusted to 40% sucrose with the addition of 400 ll of 80% sucrose in TEN buffer and placed in the bottom of a 2-ml Beckman centrifuge tube. This suspension was gently overlaid with 800 ll of 30% sucrose in TEN buffer, followed by 400 ll of 5% sucrose in TEN buffer. The samples were then centrifuged at 200 000 3 g for 18 h in a TLS 55 swinging bucket rotor in a Beckman Optima-TLX ultracentrifuge. After centrifugation, 200-ll fractions were carefully collected from the top to the bottom of the gradient. Fractions were prepared for SDS-PAGE by the addition of 0.2 volumes of 53 nonreducing Laemmli buffer, boiled for 5 min, and kept frozen until use.