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Sanchez-Cardenas, Claudia; Servin-Vences, Martha Rocio; Jose, Omar; Trevino, Claudia Lydia; Hernandez-Cruz, Arturo; Darszon, Alberto (2014)

ACROSOME REACTION AND CA2+ IMAGING IN SINGLE HUMAN SPERMATOZOA: NEW REGULATORY ROLES OF [CA2+]I

Biol. Reprod. 91(3):
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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.