Effect of Ca

Cortical deformation and polar body extrusion are the principal events that occur at fertilization in the ascidian egg. We demonstrated that the intracellular Ca2+ concentration ([Ca2+ ]i ) in the fertilized egg of Ciona savignyi increased at egg deformation (main peak) and then several small Ca2+ spikes (1st spikes) appeared before the first polar body extrusion. Brief Ca2+ spikes (2nd spikes), then appeared in the period between the first and second polar body extrusion. When eggs were fertilized in Ca2+ -free artificial seawater, the main peak and 1st spikes appeared, but the 2nd spikes did not, suggesting that the Ca2+ required for the main peak and 1st spikes is released from the intracellular store in this species and that extracellular Ca2+ is required for the 2nd spikes. When [Ca2+ ]i was clamped at a low level (0.03-0.13 μmol/L) by injecting the egg with low-Ca2+ buffers and the egg was then inseminated, deformation, polar body extrusion and pronucleus formation were suppressed. In contrast, egg deformation and first polar body extrusion were induced without insemination when [Ca2+ ]i was 0.9 μmol/L. A higher Ca2+ concentration of 1.2-10.1 μmol/L was required for extrusion of the second polar body and pronucleus formation. These data suggest that sequential Ca2+ increases (i.e. main peak and 1st and 2nd spikes) are prerequisite for the deformation and polar body extrusion of the egg. Furthermore, in eggs arrested at the second meiotic metaphase after first polar body extrusion by the injection of Ca2+ buffer, subsequent injection of excess Ca2+ caused formation of an irregular second polar body-like protrusion, suggesting latent arrest at the second meiotic metaphase in the ascidian egg.