After 10 min, sperm cells were slowly extruded into a 50-ml polyethylene tube into which protease inhibitors were added. All further isolation steps were performed on ice or at 4°C. This procedure resulted in the exclusive removal of the apical sperm head membrane from sperm cells (cf., porcine sperm cells; data not shown). The cavitate was centrifuged for 10 min at 1000 X g and the pellet was washed with 5 ml HBS and centrifuged for 10 min at 1000 X g. Supernatants were combined and centrifuged for 10 min at 6000 X g. The 6000 X g supernatant was further centrifuged for 70 min at 285 000 X g. The 285 000 X g pellet was washed in cold HBS and centrifuged for 40 min at 285 000 X g. The final pellet, containing SPM, was diluted in cold HBS with protease inhibitors. SPM was frozen in liquid nitrogen and stored at —20°C till use.
The degree of purification of the isolated SPM relative to the total sperm sample was estimated by quantifying their specific SPM and intracellular marker enzyme activities. i) Protein contents were determined according to Lowry, with minor modifications using bovine serum albumin as the standard. Samples were boiled for 10 min in 30 mM SDS, 160 mM Na2CO3, and 12 mM KNa-tartrate in 80 mM NaOH and then incubated for 10 min in 1.5 mM CuSO4.