Effect of Spermine-NONOate on acrosome reaction and associated protein tyrosine phosphorylation in Murrah buffalo (Bubalus bubalis) spermatozoa

The present study was conducted to know the role of Nitric Oxide (NO) on the acrosome reaction (AR) in Murrah buffalo (Bubalus bubalis) spermatozoa. Ejaculated buffalo spermatozoa were washed, suspended in sp-TALP media containing 6 mg BSA/mL and cell concentration was adjusted to 50 × 106 cells/mL. The cells were incubated for 6 h in the absence or presence of heparin (10 μg/mL) to induce capacitation. Fully capacitated spermatozoa were incubated in presence of 100 μg/mL Lysophosphatidyl choline (LPC, T1) or 100 μM Spermine-NONOate (T2) or 100 mM l-NAME (T3) or 100 μM Spermine-NONOate + 100 mM l-NAME (T4) or 1 mM db-cAMP + 0.1 mM IBMX (T5) or 100 μM H-89 (T6) or 100 μM Spermine-NONOate + 100 μM H-89 (T7) in combination to induce acrosome reaction. The extent of AR was assessed by dual-staining of spermatozoa with trypan blue/Giemsa stain. AR-associated tyrosine-phosphorylated proteins were detected by SDS-PAGE followed by immunoblotting using monoclonal anti-phosphotyrosine antibody. Significant (P < 0.05) number of spermatozoa were acrosome reacted in Spermine-NONOate (T2) treated cells but it was significantly (P < 0.05) lower than LPC (T1) induced AR. Addition of Spermine-NONOate + l-NAME (T4) resulted in non significant (P > 0.05) decrease in acrosome reaction. On addition of H-89 + Spermine-NONOate (T7) to sperm culture medium, resulted in significant (P < 0.05) decrease in the percent acrosome reaction. Conversely, addition of db-cAMP + IBMX (T5, cAMP analogue) resulted in the significantly (P < 0.05) higher number of acrosome reacted spermatozoa. Pattern of sperm protein tyrosine phosphorylation was also different in NO induced acrosome reaction compared to that of LPC. The present study concluded that nitric oxide is involved in acrosome reaction of buffalo spermatozoa by causing the tyrosine phosphorylation of proteins mainly p17 and p20 and through activation of cAMP/PKA pathway.