Many studies over several years have implicated the c-Mos/MEK/MAPK pathway in the metaphase arrest of the two most widely studied vertebrates, frog and mouse. Murine downstream components of this cascade are not known but in frog involve members of the spindle assembly checkpoint, which act to inhibit the APC/C. Interesting these downstream components appear not to be involved in the arrest of mouse eggs, suggesting a lack of conservation with respect to c-Mos targets. However, the recent discovery of Emi2 as an egg specific APC/C inhibitor whose degradation is Ca²⁺ dependent has greatly increased our understanding of MetII arrest. Emi2 is involved in both the establishment and maintenance of metaphase II arrest in frog and mouse suggesting a conservation of metaphase II arrest. Its identity as the physiologically relevant APC/C inhibitor involved in Cytostatic Factor arrest prompted us to re-evaluate the role of the c-Mos pathway in metaphase II arrest.

Maturation (or M-Phase) Promoting Factor (MPF; CDK1/cyclin B) [6,7] activity drives somatic cells into mitosis and eggs into meiosis (for reviews see [8-10]). MPF is regulated during meiosis, oscillating in time with entry to, and exit from MI and MII (Fig ​(Fig1b).1b). The activity of MPF can be regulated by both CDK1 phosphorylation and cyclin B degradation (for reviews see [2,11]). Hereafter cyclin B is used to denote any B-type cyclin degraded at metaphase, in frog this constitutes B1, B2, B4 and B5, while in mammals B1 and B2 [12,13]. Most is known about cyclin B1 and B2 in frog and cyclin B1 in mammals. Mammalian cyclin B1 appears to be particularly important for eggs in Met II arrest, whereas B2 is non-essential and in all of this review, the use of cyclin B in the context of mammalian eggs actually refers to work carried out using cyclin B1 [8,14,15]. At M-phase exit, as would occur at fertilization, loss of MPF is normally associated with the rapid destruction of cyclin B by anaphase [16] since CDK1 has no catalytic function without its regulatory partner [17]. Cyclin B is degraded by a Destruction-box motif (D-box) in its primary sequence, which is recognized by the E3 ligase Anaphase Promoting Complex/Cyclosome (APC/C). The APC/C polyubiquitinates key cell cycle proteins such as cyclin B, targeting them for immediate proteolysis by the 26S proteasome [18-20].

Eggs arrest at MetII with high MPF due to CSF activity (Fig ​(Fig1c).1c). The long-term stability of MPF is unique to eggs since in a mitotic metaphase, the APC/C would be active and cyclin B degraded. Although it is possible to exit CSF mediated MetII arrest by inhibiting the CDK1 component of MPF [21,22], physiologically a sperm Ca²⁺ signal induces loss of cyclin B rather than CDK1 inactivation. Interestingly in mouse eggs, the APC/C is not completely inhibited during MetII arrest, such that eggs rely on continual cyclin B synthesis to maintain arrest [3,23,24]. Similarly in frog eggs, the APC/C remains active enough to degrade cyclin B [25]. At fertilization, Ca²⁺ stimulates APC/C activity, in mouse about 6-fold [23], such that cyclin B degradation results in MPF loss. Blocking cyclin B degradation by D-box mutation prevents exit from MetII despite a Ca²⁺ signal [26,27].

SAC components have been implicated as the downstream effectors the c-Mos/MEK/MAPK/p90rsk pathway, long thought to be essential for frog CSF arrest. c-Mos (pp39mos), a proto-oncogene from a family of kinases functioning in signal transduction regulating cell growth and differentiation [36], is highly expressed during germ cell maturation, and has proposed roles throughout frog oocyte maturation [37-43]. Functioning as a MAPK kinase kinase (MEKK), c-Mos is important for activation of the MAPK kinase, MEK1 [44-46]. MEK1 serves as the upstream activator of MAPK [47-49], which switches on the 90-kD ribosomal protein S6 kinase (p90rsk [50]). At fertilization c-Mos is degraded [43], whilst MEK1, MAPK and p90rsk are inactivated shortly afterwards [51,52].

The c-Mos ...p90rsk signaling cascade has been shown to aid directly MPF activation and stabilization [53-55] making it an ideal CSF candidate. Microinjection of c-Mos RNA into two-cell embryos results in metaphase arrest, and immunodepletion of c-Mos causes a loss of cleavage-arresting activity [43]. Similarly, an injection of an active form of MAPK [56] or constitutively-active rsk [57], into blastomeres of two-cell embryos arrests the injected blastomere in metaphase. Indeed in frog, p90rsk has been suggested to be the only MAPK substrate needed for cyclin B re-accumulation on entry to MetII, MetII spindle formation, and CSF arrest [57,58]. This is supported by the fact that c-Mos protein is unable to establish CSF arrest in frog egg extracts immunodepleted of p90rsk [59].

The SAC component Bub1 is phosphorlyated and activated by p90rsk [60]. Bub1 [34], Mad1 and Mad2 [35] all appear to be required downstream of c-Mos given that the immunodepletion of these proteins blocked the establishment of CSF arrest by c-Mos in frog egg extracts. Such studies suggest a model in which CSF arrest by c-Mos is mediated by Bub1, Mad1 and Mad2 proteins.

From the above it appears that a well-defined CSF pathway has been identified in frog. However, studying SAC components maybe somewhat misleading with respect to identifying CSF. Although CSF activity and the SAC are similar in being able to induce metaphase arrest through APC/C inhibition, they may use different signalling pathways. Any arrest must be reversed by Ca²⁺ to prove physiological relevance with respect to CSF. A further issue is how CSF arrest can be achieved at MetII but not at MI metaphase (MetI) since many components of the c-Mos pathway are present and active at MetI. For example, c-Mos, MAPK, p90rsk and Bub1 are essential for suppression of S-phase between meiotic divisions [43,58,61,62] yet do not block eggs at MetI. A possible explanation is the involvement of cyclin E/cdk2, both of which are synthesized during MII [63] and inhibit the APC/C.

In frog eggs cyclin E/Cdk2 activity has been reported to play an essential role in CSF arrest. Cyclin E/Cdk2, like c-Mos, can establish metaphase arrest in egg extracts [34,63]. Cdk2 antisense prevents CSF arrest [64] and recombinant cyclin E/Cdk2 causes metaphase arrest in egg extracts even in the absence of c-Mos [34]. The two pathways (c-Mos and cyclin E/cdk2) are therefore suggested to be independent of each other but both appear to inhibit the APC/C. CSF activity therefore may result from the coexpression of cyclin E/Cdk2 with the c-Mos/MEK/MAPK/p90rsk pathway. However, the role of cyclin E/cdk2 in CSF arrest remains to be fully elucidated since inhibiting cdk2 [65], and ablation of cyclin E [66] have both been reported not disrupt CSF arrest.

Once CSF arrest has been established then many of the above proteins seem no longer required for maintenance (p90rsk, Mad2, Bub1 and cyclin E/cdk2 are all dispensable for maintenance [34,35,59]). This suggests that these proteins act upstream or independently of other effectors of CSF activity. SAC proteins may be essential to improve the efficiency of APC/C inhibition on entry into MetII arrest, yet appear redundant in the maintenance of arrest.

Emi2 is a substrate of a polo-like kinase (Plk), which plays a crucial role in regulating progression through M phase [80], allowing timely activation of the APC/C at the onset of anaphase [81-83]. In frog eggs a role for Plx1 in Ca²⁺-mediated APC/C activation was demonstrated several years ago [81] with the authors proposing the existence of a Plx1-regulated inhibitor of the APC/C active at MetII which was inactivated by Plx1 at fertilization. A later yeast-two hybrid screen for Plx1-interacting proteins identified Emi2. Like CSF activity, Emi2 accumulates during egg maturation, is present and stable in CSF arrested egg extracts, but is rapidly degraded on Ca²⁺ addition [75]. Plx1 is fully active at metaphase [84], yet at MetII it does not remove inhibition of the APC/C until at fertilization. At fertilization the target of Ca²⁺ is calmodulin-dependent protein kinase II (CamKII) [85,86], which acts a priming kinase, directly phosphorylating Emi2 [72]. Plx1 further phosphorylates Emi2 [72-74], to generate a degron which is recognised by the SCF ubiquitin ligase, resulting in Emi2 polyubiquitination and destruction [74]. Therefore APC/C inhibition is only removed once both CamKII and Plx1 are active.

In mammals, like frog, a Ca²⁺ signal breaks MetII arrest through a signalling pathway involving CamKII and activation the APC/C [87-89]. Although the full mechanism of Emi2 degradation has not yet been demonstrated in mouse, like frog, mouse Emi2 contains specific motifs for phosphorylation by both Plk and CamKII. Given that ablation of Emi2 in MetII arrested mouse eggs results in parthenogenetic activation [90], it would appear that the target of CamKII in mouse eggs is also Emi2. Supporting a role in maintaining CSF activity, Emi2 is extremely stable in MetII eggs, yet rapidly degraded by Ca²⁺ [91]. As predicted for an APC/C inhibitor, it follows that on release from MetII, Emi2 destruction precedes that of cyclin B [91]. These findings taken together demonstrate an essential role for Emi2 in the maintenance of MetII arrest.

We propose that MetII arrest is established through Emi2-mediated APC/C inhibition, and maintained both by Emi2 and the c-Mos/MAPK pathway, which acts to stabilise MPF (Fig ​(Fig2).2). The exact nature of the effect of the c-Mos pathway on MPF stability is still to be fully resolved however Yamamoto et al. [94] in frog eggs showed that when MPF activity reaches a critical lower level, the c-Mos/MAPK pathway suppresses cyclin B degradation in order to elevate MPF levels; whilst elevation of MPF beyond a critical upper level activates APC/C dependent cyclin B degradation [94]. This suggests that Mos may help set the level of MPF activity. A CSF-arrested frog egg extract will exit MetII without cyclin B loss or a Ca²⁺ stimulus when Greatwall kinase, known to positively affect MPF activity, is immunodepleted [95]. This illustrates the point that when considering the protacted nature of MetII arrest, one must consider mechanisms in the egg which are designed to respond to the sperm (Emi2 mediated APC/C inactivity) as well as those designed to keep MPF active until the time of fertilization. Our suggestion is that the c-Mos pathway may contribute to this second mechanism.

Bub, Budding uninhibited by benzimidazole

CamKII, Calmodulin-dependent protein kinase II

CSF, Cytostatic Factor

D-box, Destruction-box

Emi2, Early mitotic inhibitor 2

Mad, mitotic-arrest deficient

MI, first meiotic division

MII, second meiotic division

MetI, metaphase I

MetII, metaphase II

MPF, Maturation (M-Phase)-Promoting Factor; CDK1/cyclin B