Spatiotemporal Characterization of Intracellular Ca2+Rise during the Acrosome Reaction of Mammalian Spermatozoa Induced by Zona Pellucida

Abstract

The mammalian sperm acrosome reaction (AR) is an essential event prior to sperm–egg fusion at fertilization, and it is primarily dependent on an increase in intracellular Ca2+concentration ([Ca2+]i). Spatiotemporal aspects of the [Ca2+]iincrease during the AR induced by solubilized zona pellucida (ZP) in hamster spermatozoa were precisely investigated with a Ca2+imaging technique using confocal laser scanning microscopy with two fluorescent Ca2+indicators. A rapid rise in [Ca2+]ioccurred immediately after the application of ZP solution through a micropipette. The rise was always initiated in the sperm head, even when the application was directed toward the tail. The elevated [Ca2+]iwas little attenuated during measurement for 30–40 s. Acrosomal exocytosis was detected as a sudden decrease of fluorescence in the acrosomal vesicle ∼20 s after the onset of the [Ca2+]irise. High-resolution imaging revealed that the [Ca2+]irise in the sperm head began at the region around the equatorial segment and spread over the posterior region of the head within 0.6 s, whereas Ca2+concentration in the acrosomal vesicle appeared to be unaltered. The [Ca2+]irise was completely abolished under Ca2+-free extracellular conditions, indicating that it is totally attributable to Ca2+influx. Nifedipine, an inhibitor of L-type Ca2+channels, did not affect the rising phase of the ZP-induced Ca2+response, but accelerated the decline of the [Ca2+]irise and inhibited acrosomal exocytosis. The present study provides implicative information about the spatial organization of functional molecules involved in the signal transduction in mammalian AR.