Respiratory burst and NAD(P)H oxidase activity are involved in capacitation of cryopreserved bovine spermatozoa

Heparin (a glycosaminoglycan) and quercetin (a calcium-ATPase plasma membrane specific inhibitor) induce bovine sperm capacitation. Mitochondria from frozen semen are capable of generating oxidative energy. The aim of the study was to determine oxygen uptake variation and the participation of diphenileneiodonium (DPI)-sensitive oxidases from spermatozoa capacitated with heparin or quercetin. Oxygen uptake was measured polarographically and 2 μM diphenileneiodonium (DPI) was used as a specific inhibitor of NAD(P)H-oxidases. Sperm capacitation was determined by the chlorotetracycline technique. Heparin produced a respiratory burst (17.0 ± 3.2 μL O2/h/108 spermatozoa; mean ± S.D.) versus control (11.3 ± 0.9 μL O2/h/108 spermatozoa; P < 0.05). Oxygen uptake and sperm hypermotility were inhibited by cyanide. Treatment with DPI blocked heparin capacitation and oxygen uptake (cyanide-sensitive) decreased to control levels. Respiration of quercetin-treated samples (cyanide-sensitive; 9.7 ± 0.7 μL O2/h/108 spermatozoa) was not significantly different from the controls; oxygen uptake was not modified by DPI, but quercetin capacitation was inhibited (P < 0.05). The effect of DPI with heparin confirmed that oxidases participate in capacitation induction. The addition of superoxide dismutase and/or catalase to heparin- or quercetin-treated samples, failed to modify oxygen uptake and blocked capacitation (P < 0.05), suggesting that the superoxide anion (O2−) participates in the capacitation induction. High mitochondrial activity from heparin-treated samples indicated that energy requirements, especially for hypermotility, were supported by the respiratory chain. Although a respiratory burst was not produced by quercetin, DPI-sensitive-oxidases (O2− source) were necessary for capacitation. In cryopreserved bovine spermatozoa, heparin- or quercetin-induced capacitation required different levels of mitochondrial energy and DPI-sensitive oxidase activity.