Spermatozoa capacitation and acrosome reaction in vitro

The acrosome reactions were stimulated as previously described (Kumar et al., 2014). In brief, the sperm were capacitated in vitro by thawing of frozen semen at 37°C for 40 s and centrifugation at 1,500 rpm for 25 min. The pellets were reconstituted in 2 ml of a modified Tyrode's Hepes-buffered (Sp-TALPH) washing medium and centrifuged twice, at 1,500 rpm for 10 min. After centrifugation, the pellets were washed with a modified Tyrode's bicarbonate-buffered (Sp-TALP). The pellets were re-suspended in Sp-TALP containing concentration of 25 × 10⁶ spermatozoa in a final volume of 0.5 ml in an Eppendorf tube and capacitation was induced by the presence of 10 μg/ml heparin with an incubation for 4 h at the 38.5°C in 5% CO₂. The capacitated spermatozoa were treated for 30 min with 1 mM calcium ionophore A23187 (Sigma Chemical Co.). The acrosome reaction was confirmed by FITC-PNA staining.