Modulation of bovine sperm signalling pathways: correlation between intracellular parameters and sperm capacitation and acrosome exocytosis

Abstract

In the present study, the viability, intracellular pH (pH_i), cAMP ([cAMP]_i), calcium concentration and protein phosphotyrosine content were evaluated in relation to the acrosomal and capacitation status of freshly ejaculated bull spermatozoa. These parameters were evaluated before and after incubation with the capacitation inducer heparin, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), the phosphotyrosyl-protein phosphatase inhibitors phenylarsine oxide (PAO) and sodium orthovanadate, and hydrogen peroxide. The results obtained were integrated to address the physiological interactions between the different signalling events affecting sperm capacitation and acrosome reaction. As expected, heparin promoted the expression of the ‘B’ pattern of chlortetracycline binding, increased pH_i, [cAMP]_i and the phosphotyrosine content of sperm proteins. The effects of heparin were enhanced by IBMX. Both PAO and sodium orthovanadate stimulated protein phosphotyrosine content and acrosomal exocytosis, although only PAO affected pH, Ca²⁺ and cAMP levels. Intracellular pH was increased while both Ca²⁺ and [cAMP]_i were decreased. Physiological concentrations of H₂O₂ increased [cAMP]_i and promoted acrosomal exocytosis. A significant positive correlation was found between sperm capacitation, protein phosphotyrosine content and stored Ca²⁺ concentration, whereas the acrosome reaction was correlated with pH_i and Ca²⁺ concentration. This study presents the first global analysis of the major elements individually described during sperm capacitation and acrosome reaction signalling pathways, supported by statistical correlations.