Coordinated and compartmentalized functions of CAII and CAIV establish a dual pH-regulatory mechanism essential for sperm maturation and capacitation

The intracellular carbonic anhydrase II (CAII) and GPI-anchored extracellular carbonic anhydrase IV (CAIV) are distinctly compartmentalized enzymes that regulate bicarbonate (HCO3-) and acid-base homeostasis. Located in the epithelial cells of the epididymis, vas deferens, and on spermatozoa, these enzymes are positioned to regulate the pH environment critical for sperm maturation and capacitation, crucial for male reproduction. This regulation is essential for early sperm activation, as bicarbonate (HCO3-) triggers the soluble adenylyl cyclase (sAC)/cAMP signaling pathways and leads to an increase in intracellular pH (pHi), a prerequisite for motility. However, the specific contributions of CAII and CAIV to luminal pH and to sperm pHi regulation remain unclear. Here, we show that genetic ablation of either CAII or CAIV in mice results in a loss of normal luminal acidification in the male reproductive tract but, paradoxically, also lowers the basal pHi of sperm. This lower basal pHi reduces the effectiveness of subsequent intracellular alkalinization, thereby diminishing the activation of pH-sensitive sperm CatSper Ca2+ channels essential for capacitation and male fertility. We further establish a direct link between CAIV and the Slo3 K+ channel, as Slo3-deficient sperm exhibit reduced pHi and decreased CAIV protein levels. In contrast, sperm lacking the CatSper Ca2+ channel show no change in CAII, CAIV, or pHi. 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. Our study demonstrates that CAII and CAIV employ coordinated and spatially distinct mechanisms to regulate pH. By maintaining an acidic luminal environment for sperm maturation and triggering the subsequent alkalinization of sperm pHi, these enzymes modulate ion channel activity, thereby regulating sperm motility and fertility.