Spatiotemporal Analysis of Ca2+ Waves in Relation to the Sperm Entry Site and Animal–Vegetal Axis during Ca2+ Oscillations in Fertilized Mouse Eggs

Abstract

Fertilized mouse eggs exhibit repetitive rises in intracellular Ca2+ concentration ([Ca2+]i) necessary for egg activation. Precise spatiotemporal dynamics of each [Ca2+]i rise were investigated by high-speed Ca2+ imaging during early development of monospermic eggs. Every [Ca2+]i rise involved a Ca2+ wave. In the first Ca2+ transient, [Ca2+]i increased in two steps separated by a “shoulder” point, suggesting two distinct Ca2+ release mechanisms. The first step was a Ca2+ wave that propagated from the sperm-fusion site to its antipode in 4–5 s (velocity, ∼20 μm/s in most eggs). The second step from the shoulder to the peak was a nearly uniform [Ca2+]i rise of 12–15 s. A slight cytoplasmic movement followed the Ca2+ wave in the same direction and recovered in 25–35 s. These characteristics changed as follows, as Ca2+ oscillations progressed during the second meiosis up to their cessation at the stage of pronuclei formation (∼3 h after fertilization). (1) The duration of Ca2+ transients became shorter. (2) The shoulder point shifted to higher levels and the first step occupied most of the rising phase. (3) The rate of [Ca2+]i rise became greater and wave speeds increased up to 80–100 μm/s or more. (4) The transient cytoplasmic movement always resulted from the Ca2+ wave, although its displacement became smaller. (5) The Ca2+ wave initiation site was freed from the sperm-fusion or -entry site and eventually localized in the cortex of the vegetal hemisphere. Since the shift of the wave initiation site to the vegetal cortex is observed in fertilized eggs of nemertean worms and ascidians, this might be an evolutionarily conserved feature.