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

Capacitation in mammalian sperm involves the accurate balance of intracellular pH (pH_i), but the mechanisms controlling this process are not fully understood, particularly regarding the spatiotemporal regulation of the proteins involved in 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 capacitation. We found that, upon capacitation induction, sperm cells undergo intracellular alkalization in the head and principal piece regions. The observed localized pH_i increases require the initial uptake of HCO₃^-, which is mediated by several proteins acting consistently with their subcellular localization. Hv1 proton channel (also known as HVCN1) and cAMP-activated protein kinase (protein kinase A, 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 on the complex control mechanisms of pH_i and underscore their importance during human sperm capacitation.This article has an associated First Person interview with the first author of the paper.