Acrosome reaction and Ca²⁺ imaging in single human spermatozoa: new regulatory roles of [Ca²⁺]i.

The spermatozoa acrosome reaction (AR) is essential for mammalian fertilization. Few methods allow visualization of AR in real time together with Ca²⁺ imaging. Here, we show that FM4-64, a fluorescent dye used to follow exocytosis, reliably reports AR progression induced by ionomycin and progesterone in human spermatozoa. FM4-64 clearly delimits the spermatozoa contour and reports morphological cell changes before, during, and after AR. This strategy unveiled the formation of moving tubular appendages, emerging from acrosome-reacted spermatozoa, which was confirmed by scanning electron microscopy. Alternate wavelength illumination allowed concomitant imaging of FM4-64 and Fluo-4, a Ca²⁺ indicator. These AR and intracellular Ca²⁺ ([Ca²⁺]i) recordings revealed that the presence of [Ca²⁺]i oscillations, both spontaneous and progesterone induced, prevents AR in human spermatozoa. Notably, the progesterone-induced AR is preceded by a second [Ca²⁺]i peak and ~40% of reacting spermatozoa also manifest a slow [Ca²⁺]i rise ~2 min before AR. Our findings uncover new AR features related to [Ca²⁺]i.