Acrosomal alkalization triggers Ca²⁺ release and acrosome reaction in mammalian spermatozoa

The sperm acrosome reaction (AR), an essential event for mammalian fertilization, involves Ca²⁺ permeability changes leading to exocytosis of the acrosomal vesicle. The acrosome, an intracellular Ca²⁺ store whose luminal pH is acidic, contains hydrolytic enzymes. It is known that acrosomal pH (pH_acr ) increases during capacitation and this correlates with spontaneous AR. Some AR inducers increase intracellular Ca²⁺ concentration ([Ca²⁺ ]_i ) through Ca²⁺ release from internal stores, mainly the acrosome. Catsper, a sperm specific Ca²⁺ channel, has been suggested to participate in the AR. Curiously, Mibefradil and NNC55-0396, two CatSper blockers, themselves elevate [Ca²⁺ ]_i by unknown mechanisms. Here we show that these compounds, as other weak bases, can elevate pH_acr , trigger Ca²⁺ release from the acrosome, and induce the AR in both mouse and human sperm. To our surprise, μM concentrations of NNC55-0396 induced AR even in nominally Ca²⁺ free media. Our findings suggest that alkalization of the acrosome is critical step for Ca²⁺ release from the acrosome that leads to the acrosome reaction.