Calmodulin-dependent protein kinase II, and not protein kinase C, is sufficient for triggering cell-cycle resumption in mammalian eggs

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/cyclosome. 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. We have used a combination of pharmacological inhibitors and constitutively active cRNA constructs of PKCalpha and CamKIIalpha 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 the resumption of meiosis but caused an immediate and persistent elevation in intracellular Ca2+ when store-operated Ca2+ entry was stimulated. With respect to resumption of meiosis, 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 resumption of meiosis, 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.

The γ isoform of CaM kinase II controls mouse egg activation by regulating cell cycle resumption

“…CaMKII inhibitors inhibit cell cycle progression in both parthenogenetically activated and fertilized mouse eggs (12,25,26). Conversely, expression of a truncated, constitutively active form of CaMKIIα triggers cell cycle resumption (27), reduction in MAPK and MPF activities, PN formation, and maternal mRNA recruitment, all in the absence of Ca 2+ oscillations (28). Interestingly, CG exocytosis is abnormal in these eggs (28).…”

Transitioning from egg to embryo: Triggers and mechanisms of egg activation

“…Current data suggest that release from CSF arrest in mouse occurs in a similar manner to that in Xenopus. For instance, upon fertilization, elevated Ca 2ϩ levels activate CaMKII, whose activity is important for the activation of the APC (Nixon et al, 2002;Markoulaki et al, 2004;Madgwick et al, 2005). In addition, Emi2 is rapidly degraded in response to Ca 2ϩ .…”

Polar body emission

“…Inhibition of CaM kinase II inhibit second polar body emission [Johnson et al, 1998]. Conversely, constitutively activated CaM kinase II mimic sperm in inducing second polar body emission without triggering calcium waves [Madgwick et al, 2005]. A recent study complements these findings by demonstrating that calcium influx at fertilization, in addition to its role in refilling intracellular calcium stores due to simultaneous calcium efflux, is specifically required for second polar body emission, likely by activating CaM kinase II.…”