Nitric oxide involvement in the acrosome reaction triggered by leptin in pig sperm

BACKGROUND Nitric oxide (NO) is a signaling molecule produced by intracellular nitric oxide synthase (NOS) enzymes. This free radical appears to affect sperm capacitation, a maturation step preceding acrosome reaction. Recent studies have reported leptin ability to promote capacitation and acrosome reaction in pig male gametes. METHODS This study has investigated nitric oxide production in leptin-treated pig spermatozoa by fluorescence-activated cell sorting, while the intracellular NOS isoforms were assessed by Western blot analysis. In addition, acrosome status of treated-spermatozoa was evaluated by FITC-PNA staining. RESULTS Significant increases of nitric oxide levels and acrosome reaction extent were detected in leptin-treated spermatozoa, but both the effects were reversed in presence of L-NAME. Furthermore, the immunoblots of sperm extracts have evidenced three bands of ~160 Kd(bNOS), ~130 Kd (iNOS) and ~135 Kd (eNOS). CONCLUSIONS The identification of the three intracellular NOS isoforms suggests that pig spermatozoa could produce NO, while the augmented nitric oxide levels in leptin-treated male gametes indicates the capacity of the hormone to induce nitric oxide production. Furthermore, the inhibitory effect of L-NAME and of Ab-ObR on the promotion of acrosome reaction triggered by leptin suggests a possible involvement of NO in the hormone action.