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

Abstract

Fertilization triggers a rise in intracellular Ca 2+ concentration ([Ca 2+ ] i ) in the egg that initiates a series of events known as egg activation. These events include cortical granule exocytosis that establishes a block to polyspermy, resumption of meiosis, and recruitment of maternal mRNAs into polysomes for translation. Several calcium-dependent proteins, including calcium/calmodulin-dependent protein kinase II (CaMKII), have been implicated in egg activation. However, the precise role of CaMKII in mediating specific events of egg activation and the identity of the isoform(s) present in mouse eggs have not been unequivocally established. Through targeted deletion of the γ isoform of CaMKII, we find that CaMKIIγ is the predominant CaMKII isoform in mouse eggs and that it is essential for egg activation. Although CaMKIIγ −/− eggs exhibit a normal pattern of Ca 2+ oscillations after insemination and undergo cortical granule exocytosis, they fail to resume meiosis or to recruit maternal mRNAs. Surprisingly, we find that the recruitment of maternal mRNAs does not directly depend on CaMKII, but requires elevated [Ca 2+ ] i and metaphase II exit. We conclude that CaMKIIγ specifically controls mouse egg activation by regulating cell cycle resumption.T he transition from a fertilization-competent mammalian egg to a developing embryo entails a sequence of events collectively known as egg activation. Early events of egg activation include modifications of the zona pellucida (ZP) that prevent polyspermy, exit from metaphase II arrest, and completion of meiosis, whereas late events include recruitment of maternal mRNAs into polysomes for translation and formation of male and female pronuclei (1). In all animal species studied to date, a rise in intracellular calcium ([Ca 2+ ] i ) is the universal trigger of all of the events of egg activation (EEA) (2). Release of a spermspecific phospholipase C isoform (PLCζ) likely initiates the inositol 1,4,5-trisphosphate (IP 3 )-mediated increase in [Ca 2+ ] i (3). In mammalian eggs, this increase in [Ca 2+ ] i takes the form of repetitive Ca 2+ transients (oscillations) that last several hours (4, 5). Although Ca 2+ oscillations can induce all of the EEA, individual events require different numbers of [Ca 2+ ] i transients to be initiated and completed, with early events requiring fewer oscillations than late events (6).The pathways that connect the rise in [Ca 2+ ] i to the different EEA have only been partially elucidated. Protein kinases, including calcium/calmodulin-dependent protein kinase II (CaM-KII), and the phosphatase calcineurin have been postulated as integrators of the Ca 2+ signal during egg activation (7-9). However, direct genetic evidence for involvement of these enzymes through loss-of-function studies is lacking, leaving open questions as to whether any single enzyme is essential for all of the EEA or whether these Ca 2+ -dependent events are mediated by different effectors.The CaMKII family of serine/threonine kinases mediates many cellular responses to C...

Ca2+ Signaling During Mammalian Fertilization: Requirements, Players, and Adaptations

“…The role of CaMKII on mammalian egg activation was convincingly shown by studies in the mouse, in which expression of constitutive active forms of CaMKII into eggs initiated all events of egg activation, except CG exocytosis, and promoted development to the blastocyst stage (Madgwick et al 2005;Knott et al 2006). Conversely, depletion of the CaMKIIg isoform abrogated the ability of these eggs to exit MII in response to [Ca 2þ ] i stimulation (Backs et al 2010;Chang et al 2009), causing infertility. Research also implicated Emi2 in MII arrest in the mouse, as inhibition of Emi2 synthesis prevents cyclin B1 accumulation during maturation , which causes spontaneous activation (Shoji et al 2006).…”

“…Importantly, the widespread expression of PKC isoforms in oocytes (Gallicano et al 1997;Eliyahu et al 2001;Page Baluch et al 2004), along with their distinct cellular distribution (Viveiros et al 2001;Page Baluch et al 2004), and the implications of their impact on Ca 2þ influx (Halet 2004), suggest important roles for these enzymes in setting off embryo development. CaMKII was also expected to participate in CG exocytosis, although the aforementioned studies using constitutively active forms of the protein (Knott et al 2006) or eggs devoid of CaMKII have ruled out this possibility (Backs et al 2010;Chang et al 2009). Recent studies have implicated myosin light chain kinase (MLCK), another Ca 2þ -dependent kinase, as being involved in CG exocytosis in mouse fertilization, as pharmacological inhibitors greatly diminished their release in response to Ca 2þ stimulation (Matson et al 2006).…”

“…Thus, based on research in hippocampal neurons, the suggestion was made that pulsatile activation of CaMKII may underlie the enhanced gene expression observed after repeated [Ca 2þ ] i pulses (Ducibella et al 2006). Subsequent research, however, showed that recruitment of mRNAs could occur independently of this kinase (Backs et al 2010). Furthermore, it might not be under the exclusive control of Ca 2þ , as in the absence of cell cycle progression, fertilization-initiated oscillations failed to induce recruitment of mRNAs (Backs et al 2010).…”

Exploring the phosphoproteome profiles duringXenopusegg activation by calcium stimulation using a fully automated phosphopeptide purification system

“…S28 is included in the common region, while T37 and S38 are involved in an alternative splicing region of the enzyme. It is known in mouse eggs that calcium/calmodulin-dependent protein kinase II gamma is activated by calcium/calmodulin through autophosphorylation at T287 and controls egg activation by regulating cell cycle resumption (38). Phosphorylation at T37 or S38 of the enzyme in Xenopus eggs may be involved in the egg activation in an isoform-specific manner, though these are different from the earlier autophosphorylation residues.…”

PKC Regulation of Gametogenesis and Early Development

“…Both PKC and CaMKII have several different isotypes, and the different isotypes have different substrate specifities. The subunit of CaMKII was shown to be effective in the resumption of mouse eggs (Johnson et al, 1998), while others have shown cell cycle resumption to be dependent on the subunit (Change et al, 2009;Backs et al, 2010). A plausible alternative explanation is upon injection the constitutively active CaMKII (Knott et al, 2006) could immediately phosphorylate substrates in the cytoplasm and alter the sequence of events leading to activation.…”