Coordinated CAII and CAIV establish a dual pH-regulatory axis essential for CatSper activation during sperm maturation and capacitation

Abstract

Carbonic anhydrase II (CAII) and IV (CAIV) orchestrate pH regulation essential for male fertility through distinct cellular compartments, with CAII functioning intracellularly and CAIV GPI-anchored extracellularly in epididymal and vas deferens epithelia as well as on spermatozoa. These enzymes control pH environments required for sperm maturation and capacitation by regulating bicarbonate availability, which triggers soluble adenylyl cyclase/cAMP signaling, elevates intracellular pH (pHi), and enables motility activation. However, the specific contributions of CAII and CAIV to luminal pH and sperm pHi regulation remain unclear. Here, we show that genetic ablation of either CAII or CAIV in mice impairs normal luminal acidification in the male reproductive tract and unexpectedly lowers the basal pHi of sperm. This lower basal pHi blunts subsequent intracellular alkalinization, thereby diminishing the activation of pH-sensitive CatSper Ca2+ channels essential for hyperactivated motility. Mechanistically, we demonstrate that this basal pHi reduction occurs independently of the CatSper channel itself, but it is phenocopied in K+channel Slo3-deficient sperm, suggesting a homeostatic link between flagellar pH and membrane potential. Super-resolution imaging reveals that CAII is located within the flagellum near the CatSper channel in the principal piece, while CAIV is distributed along the entire flagellar membrane. Together, our findings demonstrate that CAII and CAIV employ spatially coordinated mechanisms to regulate pH. By maintaining an acidic luminal environment for sperm maturation and priming the subsequent alkalinization of sperm pHi, these enzymes modulate downstream ion channel activity to govern sperm motility and fertility. One-sentence summarySpatially coordinated carbonic anhydrases II and IV govern sperm motility by establishing the dual-compartment pH environments necessary to activate downstream flagellar ion channels.