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6 August 2014 Acrosome Reaction and Ca2 Imaging in Single Human Spermatozoa: New Regulatory Roles of [Ca2 ]i
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Abstract

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.

Claudia Sánchez-Cárdenas, Martha Rocio Servín-Vences, Omar José, Claudia Lydia Treviño, Arturo Hernández-Cruz, and Alberto Darszon "Acrosome Reaction and Ca2 Imaging in Single Human Spermatozoa: New Regulatory Roles of [Ca2 ]i 1," Biology of Reproduction 91(3), (6 August 2014). https://doi.org/10.1095/biolreprod.114.119768
Received: 1 April 2014; Accepted: 1 June 2014; Published: 6 August 2014
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