Capacitation-associated alkalization in human sperm is differentially controlled at the subcellular level

ABSTRACT Capacitation in mammalian sperm involves the accurate balance of intracellular pH (pH i ), but the underlying control mechanisms are not fully understood, particularly regarding the spatiotemporal regulation of the proteins involved in such pH i modulation. Here we employed an image-based flow cytometry technique combined with pharmacological approaches to study pH i dynamics at the subcellular level during sperm capacitation. We found that, upon capacitation induction, sperm cells undergo intracellular alkalization in the head and principal piece regions, but not in the midpiece. The observed localized pH i increases require the initial uptake of HCO 3 - , and it is mediated by several proteins acting in a manner consistent with their subcellular localization. Hv1 proton channel and cAMP-activated Protein Kinase (PKA) antagonists impair alkalization mainly in the principal piece. Na + /HCO 3 - cotransporter (NBC) and cystic fibrosis transmembrane regulator (CFTR) antagonists impair alkalization only mildly, predominantly in the head. Motility measurements indicate that inhibition of alkalization in the principal piece prevents the development of hyperactivated motility. Altogether, our findings shed light into the complex control mechanisms of pH i and underscore their importance during human sperm capacitation. Summary statement Human sperm display differential pH i regulation at the subcellular level upon capacitation, involving the participation of PKA kinase signaling pathway and several membrane transport proteins, culminating in hyperactivation.