Two-pore channel 1 and Ca2+ release-activated Ca2+ channels contribute to the acrosomal pH-dependent intracellular Ca2+ increase in mouse sperm

The acrosome is a lysosome-related vesicular organelle located in the sperm head. The acrosomal reaction (AR) is an exocytic process mediated by Ca2+ and essential for mammalian fertilization. Recent findings support the importance of acrosomal alkalinization for the AR. Mibefradil (Mib) and NNC 55-0396 (NNC) are two amphipathic weak bases that block the sperm-specific Ca2+ channel (CatSper) and induce acrosomal pH (pH alpha) increase by accumulating in the acrosomal lumen of mammalian sperm. This accumulation and pH alpha elevation increase the intracellular Ca2+ concentration ([Ca2+](i)) and trigger the AR by unknown mechanisms of Ca2+ transport. Here, we investigated the pathways associated with the pH alpha increase-induced Ca2+ signals using mouse sperm as a model. To address these questions, we used single-cell Ca2+ imaging, the lysosomotropic agent Gly-Phe-beta-naphthylamide (GPN) and pharmacological tools. Our findings showthat Mib andNNCincrease pH alpha and release acrosomal Ca2+ without compromising acrosomal membrane integrity. Our GPN results indicate that the osmotic component does not significantly contribute to acrosomal Ca2+ release caused by pH alpha rise. Inhibition of two-pore channel 1 (TPC1) channels reduced the [Ca2+](i) increase stimulated by acrosomal alkalinization. In addition, blockage of Ca2+ release-activated Ca2+ (CRAC) channels diminished Ca2+ uptake triggered by pH alpha alkalinization. Finally, our findings contribute to understanding how pH alpha controls acrosomal Ca2+ entry during AR inmouse sperm.