Effect of Ca2+ on deformation, polar body extrusion and pronucleus formation in the egg of the ascidian, Ciona savignyi

Abstract

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.

Keywords: Ca2+ injection; ascidian egg; egg deformation; meiotic division; polar body extrusion.