Two-pore channel 1 and Ca release-activated Ca channels contribute to the acrosomal pH-dependent intracellular Ca 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 Ca 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 Ca channel (CatSper) and induce acrosomal pH (pH ) increase by accumulating in the acrosomal lumen of mammalian sperm. This accumulation and pH elevation increase the intracellular Ca concentration ([Ca ] ) and trigger the AR by unknown mechanisms of Ca transport. Here, we investigated the pathways associated with the pH increase-induced Ca signals using mouse sperm as a model. To address these questions, we used single-cell Ca imaging, the lysosomotropic agent Gly-Phe-β-naphthylamide (GPN) and pharmacological tools. Our findings show that Mib and NNC increase pH and release acrosomal Ca without compromising acrosomal membrane integrity. Our GPN results indicate that the osmotic component does not significantly contribute to acrosomal Ca release caused by pH rise. Inhibition of two-pore channel 1 (TPC1) channels reduced the [Ca ] increase stimulated by acrosomal alkalinization. In addition, blockage of Ca release-activated Ca (CRAC) channels diminished Ca uptake triggered by pH alkalinization. Finally, our findings contribute to understanding how pH controls acrosomal Ca efflux and extracellular Ca entry during AR in mouse sperm. KEY POINTS: The acrosomal vesicle is a lysosome-related organelle located in the sperm head. The acrosome reaction (AR) is a highly regulated exocytic process mediated by Ca , which is essential for fertilization. However, the molecular identity of Ca transporters involved in the AR and their mechanisms to regulate Ca fluxes are not fully understood. In mammalian sperm, acrosomal alkalinization induces intracellular Ca concentration ([Ca ] ) increase and triggers the AR by unknown molecular mechanisms of Ca transport. In this study, we explored the molecular mechanisms underlying Ca signals caused by acrosomal alkalinization using mouse sperm as a model. TPC1 and CRAC channels contribute to [Ca ] elevation during acrosomal alkalinization. Our findings expand our understanding of how the acrosomal pH participates in the physiological induction of the AR.