Differential role of bovine serum albumin and HCO₃^- in the regulation of GSK3 alpha during mouse sperm capacitation

To become fertile, mammalian sperm are required to undergo capacitation in the female tract or in vitro in defined media containing ions (e.g. HCO3-, Ca2+, Na+, and Cl-), energy sources (e.g. glucose, pyruvate) and serum albumin (e.g. bovine serum albumin (BSA)). These different molecules initiate sequential and concomitant signaling pathways, leading to capacitation. Physiologically, capacitation induces changes in the sperm motility pattern (e.g. hyperactivation) and prepares sperm for the acrosomal reaction (AR), two events required for fertilization. Molecularly, HCO3- activates the atypical adenylyl cyclase Adcy10 (aka sAC), increasing cAMP and downstream cAMP-dependent pathways. BSA, on the other hand, induces sperm cholesterol release as well as other signaling pathways. How these signaling events, occurring in different sperm compartments and with different kinetics, coordinate among themselves is not well established. Regarding the AR, recent work has proposed a role for glycogen synthase kinases (GSK3 alpha and GSK3 beta). GSK3 alpha and GSK3 beta are inactivated by phosphorylation of residues Ser21 and Ser9, respectively, in their N-terminal domain. Here, we present evidence that GSK3 alpha (but not GSK3 beta) is present in the anterior head and that it is regulated during capacitation. Interestingly, BSA and HCO3- regulate GSK3 alpha in opposite directions. While BSA induces a fast GSK3 alpha Ser21 phosphorylation, HCO3- and cAMP-dependent pathways dephosphorylate this residue. We also show that the HCO3--induced Ser21 dephosphorylation is mediated by hyperpolarization of the sperm plasma membrane potential (Em) and by intracellular pH alkalinization. Previous reports indicate that GSK3 kinases mediate the progesterone-induced AR. Here, we show that GSK3 inhibition also blocks the Ca2+ ionophore ionomycin-induced AR, suggesting a role for GSK3 kinases downstream of the increase in intracellular Ca2+ needed for this exocytotic event. Altogether, our data indicate a temporal and biphasic GSK3 alpha regulation with opposite actions of BSA and HCO3-. Our results also suggest that this regulation is needed to orchestrate the AR during sperm capacitation.