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Our previous study demonstrated the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in transporting bicarbonate that is necessary for sperm capacitation; however, whether its involvement is direct or indirect remains unclear. The present study investigated the possibility of a Cl-/HCO3- exchanger (solute carrier family 26, number 3 [SLC26A3]) operating with CFTR during guinea pig sperm capacitation. Incubating sperm in media with various concentrations of Cl- resulted in varied percentages of capacitated sperm in a concentration-dependent manner. Depletion of Cl-, even in the presence of HCO3-, abolished sperm capacitation and vice versa, indicating the involvement of both anions in the process. Capacitation-associated HCO3--dependent events, including increased intracellular pH, cAMP production, and protein tyrosine phosphorylation, also depend on Cl- concentrations. Similar Cl- dependence and inhibitor sensitivity were observed for sperm-hyperactivated motility and for sperm-egg fusion. The expression and localization of CFTR and SLC26A3 were demonstrated using immunostaining and Western blot analysis. Taken together, our results indicate that Cl- is required for the entry of HCO3- that is necessary for sperm capacitation, implicating the involvement of SLC26A3 in transporting HCO3-, with CFTR providing the recycling pathway for Cl-.

Phospholipase A2 (PLA2) is activated in spermatozoa in response to progesterone and Ca 21 ionophores, but to our knowledge, no study has yet reported zona pellucida (ZP)-induced activation of PLA2. We investigated whether PLA2 is involved in ZP-stimulated acrosomal exocytosis, if Ca 21 is required for activation of PLA2, and signal transduction pathways modulating PLA2 using guinea pig sperm as a model. Spermatozoa were capacitated and labeled in low-Ca 21 medium with [ 14 C]choline chloride or [ 14 C]arachidonic acid and were then exposed to millimolar Ca 21 and various reagents and stimulated with ZP. Precapacitated spermatozoa exposed to millimolar Ca 21 and stimulated with ZP experienced increases in arachidonic acid (AA) and lysophosphatidylcholine (lysoPC) levels and a parallel decrease in phosphatidylcholine level; these changes are indicative of PLA2 activation. Simulation with ZP also led to acrosomal exocytosis in a high proportion of spermatozoa. Lipid changes and exocytosis were prevented if spermatozoa were exposed to aristolochic acid, a PLA2 inhibitor, before treatment with ZP. Stimulation with ZP in medium without added Ca 21 or in medium with millimolar Ca 21 and EGTA or La