Sperm was incubated in capacitating media (all T+BIC media) for 4 h in a humidified atmosphere at 38°C and 5% CO2, and sperm diluted in T—BIC medium was incubated in a humidified atmosphere at 38°C in air (containing 0.05% CO2) to prevent bicarbonate buffering of CO2. Sperm concentration in the media was 20 X 106 sperm/ml. Following incubation, 10-|xl aliquots from the incubated sperm samples were used to assess the capacitation status of the sperm cells by CTC staining. The remaining part of the incubated samples was concentrated by centrifugation at 800 X g for 10 min at room temperature. The obtained pellets were resuspended in 10 ml HBS (room temperature) and recentrifuged at 800 X g for 10 min, and this washing procedure was repeated three times. The resulting pellets were resuspended in HBS and plasma membranes were isolated (see below).
Sperm Plasma Membrane Isolation
Bull sperm plasma membranes (SPM) were isolated according to a method described for boar sperm, with slight modifications. Briefly, sperm cells (approximately 5 X 108 cells/ml) were subjected to a nitrogen pressure of 45 Bar in a cell-disruption bomb (Parr Instrument Company, Moline, IL) on ice.