290. Calmodulin-dependent protein kinase II, and not protein kinase C, transduces the Ca²⁺ signal at fertilization

Abstract

Mouse eggs arrest at metaphase II following ovulation and are only triggered to complete meiosis when fertilized. Sperm break the cell cycle arrest by a long-lasting series of Ca²⁺ spikes that lead to an activation of the anaphase-promoting complex. The signal transduction pathway is not fully resolved but both protein kinase C (PKC) and calmodulin-dependent protein kinase II (CamKII) activities increase at fertilization and previous pharmacological studies have implicated both in cell cycle resumption. Here we used a combination of pharmacological inhibitors and constitutively-active cRNA constructs of PKCα and CamKII microinjected into mouse eggs, to show that it is CamKII and not PKC that is the sufficient trigger for cell cycle resumption from metaphase II arrest.

Constitutively active PKC constructs had no effect on meiotic resumption but caused an immediate and persistent elevation in intracellular Ca²⁺ when store-operated Ca²⁺ entry was stimulated. With respect to meiotic resumption, the effects of constitutively-active CamKII on eggs were the same as sperm. Eggs underwent second polar body extrusion and pronucleus formation with normal timings; while both securin and cyclin B1 destruction, visualised by coupling to fluorescent protein tags, were complete by the time of polar body extrusion. Induction of a spindle checkpoint by overexpression of Mad2 or by spindle poisons blocked CamKII-induced meiotic resumption but the Ca²⁺ chelator BAPTA did not. Furthermore direct measurement of Ca²⁺ levels showed that CamKII did not induce exit from metaphase II arrest by raising Ca²⁺. Therefore we conclude that PKCs may play an important role in maintaining Ca²⁺ spiking at fertilization by promoting store-operated Ca²⁺ entry, while CamKII transduces cell cycle resumption, and lies downstream of sperm-induced Ca²⁺ release but upstream of a spindle checkpoint. These data, combined with the knowledge that CamKII activity increase at fertilization, suggest that mouse eggs undergo cell cycle resumption through stimulation of CamKII.