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Capacitation in mammalian sperm involves the accurate balance of intracellular pH (pHi), but the controlling mechanisms are not fully understood, particularly regarding the spatiotemporal regulation of the proteins involved in pHi modulation. Here we employed an image-based flow cytometry technique combined with pharmacological approaches to study pHi dynamics at the subcellular level during capacitation. We found that, upon capacitation induction, sperm cells undergo intracellular alkalization in the head and principal piece regions. The observed localized pHi increases require the initial uptake of HCO3−, which is mediated by several proteins acting consistently with their subcellular localization. Hv1 proton channel and cAMP-activated Protein Kinase (PKA) antagonists impair alkalization mainly in the principal piece. Na+/HCO3− 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 pHi and underscore their importance during human sperm capacitation.

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