Phospholipase A₂ activation by hydrogen peroxide during <i>in vitro </i>capacitation of buffalo spermatozoa

Abstract

Progressively motile, washed buffalo spermatozoa (50×10⁶ cells in 0.5 ml) were <i>in vitro </i>capacitated in HEPES containing Bovine Gamete Medium 3 (BGM₃) in presence of heparin (10μg/ml), and different concentrations of hydrogen peroxide (10 to 100 μ<i>M</i>)<i>. </i>Spermatozoa (60%) were capacitated in presence of heparin compared to 56% in presence of 25μ<i>M &nbsp;</i>H₂O₂ (optimally found suitable for capacitation). The extent of capacitation was measured in terms of acrosome reaction (AR) induced by lysophosphatidyl choline (100<i> μ</i>g/ml).The acrosome reacted cells were counted after triple staining. Catalase (100 μg/ml) significantly reduced the sperm capacitation to 16-18% when added with H₂O₂, or alone in the capacitation medium. Phospholipase A₂ activity of spermatozoa increased linearly up to 50 μ<i>M &nbsp;</i>H₂O₂concentration included in the assay system. Moreover, significant increase in phospholipase A₂ activity was observed after capacitation by both, the heparin and 25 μ<i>M</i> H₂O₂<i>. </i>The activity was always higher in acrosome reacted cells.