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_a ) increase by accumulating in the acrosomal lumen of mammalian sperm. This accumulation and pH_a 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_a 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-β-naphthylamide (GPN) and pharmacological tools. Our findings show that Mib and NNC increase pH_a 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_a 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_a alkalinization. Finally, our findings contribute to understanding how pH_a controls acrosomal CA²⁺ efflux and extracellular CA²⁺ 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 CA²⁺ , which is essential for fertilization. However, the molecular identity of CA²⁺ transporters involved in the AR and their mechanisms to regulate CA²⁺ fluxes are not fully understood. In mammalian sperm, acrosomal alkalinization induces intracellular CA²⁺ concentration ([CA²⁺ ]_i ) increase and triggers the AR by unknown molecular mechanisms of CA²⁺ transport. In this study, we explored the molecular mechanisms underlying CA²⁺ signals caused by acrosomal alkalinization using mouse sperm as a model. TPC1 and CRAC channels contribute to [CA²⁺ ]_i elevation during acrosomal alkalinization. Our findings expand our understanding of how the acrosomal pH participates in the physiological induction of the AR.