Semen was washed over a discontinuous Percoll density gradient. For this purpose, sperm cells were first layered on top of 35% and 70% isotonic Percoll layers diluted with saline medium as described by Harrison et al. and subsequently centrifuged at 350 X g for 10 min and 20 min at 700 X g at room temperature to remove saline medium and seminal plasma. Traces of Percoll were removed by washing the pellet four times with Hepes buffered saline (HBS; containing 5 mM Hepes, 2.7 mM KCl, 146 mM NaCl, 5 mM CaCl2) (700 X g, 10 min). The final pellet was diluted in HBS and sperm motility was estimated (approximately 85% of the cells were progressively motile). To assess sperm cell concentration, sperm was diluted in fixative (0.5% formaldehyde in saline) and counted in a Biirker-Turk cell chamber. Sperm motility and concentration were estimated under a phase contrast microscope, BH2 Olympus (Tokyo, Japan).
Fresh sperm cells were collected and prepared as described above. Sperm was incubated with one of four media: i) Tyrode medium displaying minimal supportive effects on sperm capacitation (T—BIC; 115 mM NaCl, 3.1 mM KCl, 0.3 mM NaH2PO4, 20 mM Hepes, 2 mM CaCl2, 0.4 mM MgSO4, 21.6 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/ ml BSA, 5 mM glucose, and 1 |xg/ml gentamycin; pH 7.4, 290 mOsm/ kg). ii) Tyrode medium displaying maximal effects on sperm capacitation (T+BIC; 99 mM NaCl, 25 mM NaHCO3, 3.1 mM KCl, 0.3 mM NaH2PO4, 20 mM Hepes, 2 mM CaCl2, 0.4 mM MgSO4, 21.6 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/ml BSA, 5 mM glucose, and 1 |xg/ ml gentamycin; pH 7.4, 290 mOsm/kg). iii) T+BIC supplemented with 10 |xg/ml heparin (T+BIC+Hep). iv) T+BIC+Hep with 10 mM glucose as final concentration; the higher amount of glucose is thought to inhibit capacitation (T+BIC+Hep+Glu).