Vertebrate eggs arrest at metaphase of the second meiotic division before fertilization under the effect of a cytostatic factor (CSF). This arrest is established during oocyte maturation by the MAPK kinase module, comprised of Mos, MEK, MAPKs and p90^Rsk. Maintenance of CSF arrest at metaphase requires inhibitors of the anaphase-promoting complex (APC) like Emi1, which sequesters the APC activator Cdc20. Although it was proposed that the Mos pathway and Emi1 act independently, neither one alone is sufficient to entirely reproduce CSF arrest. Herein we demonstrate that p90^Rsk2 associates with and phosphorylates Emi1 upstream of the binding region for Cdc20, thus stabilizing their interaction. Experiments in transfected cells and two-cell embryos indicate that Emi1 and p90^Rsk2 cooperate to induce the metaphase arrest. Moreover, oocyte maturation was impaired by interfering with the interaction between p90^Rsk2 and Emi1 or by RNA interference of Emi1. Our results indicate that p90^Rsk2 and Emi1 functionally interact during oocyte maturation and that the Mos pathway establishes CSF activity through stabilization of an APC-inhibitory complex composed by Emi1 and Cdc20 before fertilization.

Keywords: Cdc20, CSF, fertilization, metaphase arrest, mouse oocytes

In vertebrate eggs, a cytostatic factor (CSF) is responsible for the stabilization of the cyclinB/cdc2 complex known as maturation-promoting factor (MPF) (Masui and Markert, 1971). CSF prevents the transition to anaphase until fertilization occurs and can induce cytostatic arrest when injected into the cytoplasm of mitotically dividing blastomeres (reviewed in Masui, 2001). A breakthrough in the molecular characterization of CSF came with the identification of Mos, a serine-threonine kinase newly synthesized during oocyte maturation and capable of inducing metaphase arrest in injected blastomeres (Sagata et al, 1988). Mos translation during oocyte maturation establishes CSF arrest (Masui, 2001), whereas Mos depletion by RNAi prevents its establishment (Dupré et al, 2002). Moreover, the mitogen-activated protein kinase (MAPK) pathway is necessary for the biological activity of Mos in both Xenopus and mouse eggs (Haccard et al, 1993; Kosako et al, 1994; Verlhac et al, 2000a). The downstream effector of the Mos-dependent MAPK pathway in Xenopus eggs is p90^Rsk. It was demonstrated that p90^Rsk2 is the predominant Rsk isoform expressed in Xenopus eggs and embryos and that immunodepletion of p90^Rsk2 from cycling egg extracts completely abolished the CSF activity of Mos (Bhatt and Ferrell, 1999; 2000). Furthermore, in the absence of Mos, a CSF effect could also be obtained by microinjection of a constitutively active p90^Rsk1 isoform (Gross et al, 1999), confirming that a p90^Rsk isoform acts downstream of Mos. The role of Mos in the establishment of cytostatic activity appears to be evolutionarily conserved in vertebrate eggs because mice with homozygous deletion of the mos gene ovulate oocytes that do not arrest at metaphase and undergo spontaneous parthenogenetic activation (Colledge et al, 1994; Hashimoto et al, 1994). In addition, both MAPK and p90^Rsk are similarly activated during meiotic maturation of mouse oocytes (Kalab et al, 1996; Verlhac et al, 1996), indicating conservation of the whole pathway.

Most of the data available on the molecular mechanisms of CSF arrest have been gathered using a constitutively active p90^Rsk1. Gross et al (2000) demonstrated that this protein inhibits the ubiquitin–ligase complex known as anaphase-promoting complex (APC), thus preventing the complete destruction of cyclin B at anaphase I and promoting its rapid accumulation during meiosis II. Recent evidence suggests that the Mos pathway inhibits the APC through activation of the spindle checkpoint (Tunquist and Maller, 2003). The kinase Bub1 is phosphorylated and activated by the constitutively active p90^Rsk1 in vitro and in a MAPK-dependent manner during meiotic maturation (Schwab et al, 2001), and other components of this checkpoint, like Mad1 and Mad2, are required for the establishment of the Mos-dependent cytostatic arrest in Xenopus eggs (Tunquist et al, 2002; 2003). However, the function of the spindle checkpoint in CSF maintenance must be distinguishable from that exerted in mitotic cells, because the checkpoint appears active even though in metaphase-arrested oocytes chromosomes are correctly attached with their kinetochores to microtubules of the spindle. Moreover, in eggs, only Mad1 is required for maintenance of CSF, whereas Mad2, the direct inhibitor of Cdc20, is dispensable at metaphase. Thus, the connection between the endogenous p90^Rsk and Mad1 in CSF arrest is still unknown (Tunquist et al, 2003).

One puzzling feature of the elusive CSF of vertebrate eggs is that while the Mos/MAPK/p90^Rsk pathway participates in its establishment during meiosis II, it becomes dispensable afterwards. Indeed, immunodepletion of p90^Rsk2, or of Bub1 kinase, from Xenopus egg extracts is not sufficient to release them from the metaphase arrest (Bhatt and Ferrell, 1999; Tunquist et al, 2002). A possible explanation is that these kinases act upstream of the real effectors of the cytostatic activity. In this regard, it has been proposed that a novel regulator of the APC, Emi1, is directly responsible of the metaphase arrest of Xenopus eggs (Reimann and Jackson, 2002). Emi1 interacts with the substrate-binding region of Cdc20 and stabilizes mitotic cyclins in Xenopus egg extracts (Reimann et al, 2001a; 2001b). Moreover, immunodepletion of Emi1 from these extracts caused degradation of MPF and release from the CSF arrest, whereas an excess of Emi1 blocked release of CSF arrest by Ca⁺⁺ or activation of CamKII (Reimann and Jackson, 2002), events that mimic egg activation at fertilization (Lorca et al, 1993; Markoulaki et al, 2003). Thus, since Emi1 appeared both necessary and sufficient to produce CSF activity in Xenopus egg extracts, it was proposed that this protein was the long-sought CSF responsible for the metaphase II arrest of vertebrate eggs (Reimann and Jackson, 2002).

Surprisingly, the activity of Emi1 in egg extracts did not require the Mos/MAPK/p90^Rsk pathway, suggesting that Emi1 acts independently (Reimann and Jackson, 2002). However, it remains unclear why in the absence of Mos the cytostatic activity of vertebrate eggs does not develop (Colledge et al, 1994; Hashimoto et al, 1994; Dupré et al, 2002) and why mitotic cells, which express Emi1 in the G2 phase like the oocytes, do not arrest at metaphase (Hsu et al, 2002). Emi1 accumulates in the S and G2 phases of mitotic cycles and its destruction at the onset of the M phase allows progression through mitosis. Phosphorylation of Emi1 by Cdc2 in prometaphase promotes its interaction with the substrate adaptor protein βTrCP and its degradation by the SCF (Skp1/Cullin/F-box) ubiquitin–ligase complex (Margottin-Goguet et al, 2003), dictating the timing of entry into mitosis. However, in meiosis, Emi1 is stable throughout maturation of Xenopus oocytes (Reimann and Jackson, 2002) but cyclin B stabilization and metaphase arrest occur only in the second division, after full activation of the Mos pathway. Mos being the only component of CSF selectively expressed during the meiotic divisions (Masui, 2001), it is possible that its pathway functionally interacts with Emi1 during maturation when CSF appears.

Herein we provide evidence that phosphorylation of Emi1 by p90^Rsk2 stabilizes its interaction with the APC activator Cdc20 and that the two proteins cooperate to establish CSF arrest during mouse oocyte maturation. Our studies provide a direct link between components involved in the establishment and maintenance of CSF activity before fertilization.

The CSF activity present in vertebrate oocytes requires activation of the Mos/MAPK/p90^Rsk2 pathway during oocyte maturation and APC^Cdc20 inhibitory regulators, such as Emi1 and Mad1, to be maintained once it is established (Reimann and Jackson, 2002; Tunquist et al, 2003). However, the connection between the factors required for the establishment and the maintenance of CSF is still unclear. Herein, we report that p90^Rsk2, the effector of the Mos pathway, directly interacts with and phosphorylates Emi1, an essential inhibitor of APC at metaphase (Reimann and Jackson, 2002). Moreover, we show that functional interaction between p90^Rsk2 and Emi1 potentiates the ability of Emi1 to bind to Cdc20 and to induce cytostatic arrest both in mouse blastomeres and in transfected cells. Thus, our results establish a first direct connection between proteins involved in the establishment and in the maintenance of cytostatic activity in vertebrate eggs.

In vitro binding experiments, supported also by co-immunoprecipitation experiments, indicate that phosphorylation of the C-terminal region of Emi1 by p90^Rsk2 potentiates four-fold its ability to bind Cdc20. Notably, Reimann and Jackson (2002) showed that cytostatic arrest was released when recombinant Cdc20 was added to Xenopus egg extracts at 3 μM concentration, but not at 1 μM concentration, and that it was prevented by addition of equimolar amounts of Emi1. Their results indicate that CSF arrest is maintained by a strict balance between Emi1 and Cdc20 concentrations, and that changing this balance by as little as three-fold triggers metaphase-to-anaphase transition. Thus, we propose that one of the functions of the Mos pathway during oocyte maturation is to reinforce the cytostatic activity of Emi1 through phosphorylation by p90^Rsk2 (Figure 9). Our conclusion is supported by the observations that coinjection of Emi1 with p90^Rsk2 in mouse blastomeres exerts a stronger cytostatic effect than either protein alone. In addition, substitution of the evolutionarily conserved ser246/thr251 residues, which are phosphorylated by p90^Rsk2, suppresses the cooperation between the two proteins in vivo and the ability to stabilize the Cdc20/Emi1 interaction in vitro. Our results also show that injection of GST-Emi1_236–313, which interacts with p90^Rsk2 and is efficiently phosphorylated by the kinase but is unable to bind Cdc20 efficiently, blocks the cytostatic activity of constitutively active p90^Rsk2 in two-cell embryos and it interferes with meiosis II progression and cytostatic arrest in maturing mouse oocytes. Since similar defects were observed when Emi1 expression was impaired by RNAi, it seems that the effects are due to alterations of Emi1 function. These results suggest that GST-Emi1_236–313 competes for the interaction of p90^Rsk2 with endogenous Emi1 and that this interaction is necessary for the establishment of cytostatic arrest.

The morphological examination of oocytes matured in the presence of GST-Emi1_236–313, or where Emi1 expression was decreased by RNAi, revealed that they closely resemble mos−/− oocytes (Choi et al, 1996; Verlhac et al, 2000a; 2000b), with some oocytes extruding a second polar body and others with abnormal spindles and misaligned chromosomes or abnormal cytokinesis. Beside stabilization of MPF activity through inhibition of the APC^Cdc20, the Mos/MAPK/p90^Rsk pathway is also important for stabilization of the spindle through post-translational modifications of microtubule-associating proteins like MISS and DOC1R (Verlhac et al, 1996; Lefebvre et al, 2002; Terret et al, 2003). The spindle abnormalities in oocytes injected with GST-Emi1_236–313 or Emi1dsRNA suggest that the functional interaction between Emi1 and p90^Rsk2 contributes also to this aspect of meiosis II. Interestingly, it has been shown that MISS protein is stable only in metaphase II-arrested oocytes, once the cytostatic activity has been established. Since the spindle defects observed after in vivo depletion of MISS (Lefebvre et al, 2002) closely resemble those obtained by interference with Emi1, it would be interesting to determine whether MISS is a substrate for the APC^Cdc20 and whether Emi1 prevents its degradation in meiosis.

The only proteins shown to be required for maintenance of the cytostatic arrest in eggs are the APC^Cdc20 inhibitors Emi1 and Mad1 (Reimann and Jackson, 2002; Tunquist et al, 2003). In both cases, the crucial experiments employed an immunodepletion approach from Xenopus egg extracts, which are typically arrested at metaphase with high MPF and MAPK activity. Depletion of either Mad1 or Emi1 led to activation of the APC, cyclin B degradation and mitotic exit. Since addition of either recombinant Mad1 or Emi1 was sufficient to block these events, in both works it was concluded that these proteins were necessary and sufficient to maintain the CSF arrest of Xenopus eggs. One possibility to reconcile this apparent paradox is that Mad1 and Emi1 associate in a complex and depletion of either one causes mitotic exit because of depletion of the inhibitory complex. However, it was not checked whether or not Emi1 co-immunoprecipitates with Mad1 in the immunodepletion experiments (Reimann and Jackson, 2002; Tunquist et al, 2003). Because of the limiting volume of mouse oocytes, such approaches cannot be applied to our system. Nevertheless, our in vivo competition and/or depletion approaches suggest that Emi1 participates in cytostatic arrest also in mouse oocytes.

Our observations suggest that the metaphase-to-anaphase transition in vertebrate eggs is controlled by a relay of phosphorylation events that fine-tune the reciprocal affinity of regulatory components of the APC. In our model, p90^Rsk2 is activated by the Mos pathway during oocyte maturation and phosphorylates Emi1, increasing its affinity for Cdc20 and preventing activation of the APC (Figure 9). After metaphase is reached, the Mos pathway becomes dispensable (Tunquist and Maller, 2003), possibly because phosphorylated Emi1 is stable in the oocyte environment. This might be due to the absence of a counteracting phosphatase or to inaccessibility of Emi1 when complexed to Cdc20. In agreement with this second hypothesis, it was noted that Emi1 has a stronger affinity for the APC activator Cdh1 than for Cdc20, and it is rapidly degraded when Cdh1 disappears (Reimann et al, 2001b; Hsu et al, 2002). Since only Cdc20 is present in oocytes (Lorca et al, 1998), it is possible that phosphorylation by p90^Rsk2 prolongs Emi1 life in meiosis through the stabilization of the normally loose interaction with Cdc20. At fertilization, activation of CaMKII triggers activation of APC^Cdc20 and degradation of cyclin B (Lorca et al, 1993). It remains to be established whether CaMKII acts directly through phosphorylation of Emi1 or Cdc20 and whether phosphorylation causes dissociation of the complex. In this regard, our preliminary data suggest that at least Emi1 is not a direct in vitro substrate for CamKII (MP Paronetto and C Sette, unpublished observation).

In conclusion, our results indicate that p90^Rsk2 functionally interacts with Emi1 in the establishment of CSF activity in mouse eggs. Our model offers a reconciling view on the connection between two components of CSF that were previously considered independent, leaving puzzling doubts on the relation between the establishment and the maintenance of CSF arrest (Duesbery and Vande Woude, 2002). Moreover, since activation of the MAPK pathway is required also in the response to the spindle- and DNA-damage checkpoints in mitotic cells (Chung and Chen, 2003; Panta et al, 2004), our results may provide a direct link between this pathway and cell cycle arrest through the inhibition of the APC.

Two-cell embryos and GV oocytes were collected from hormonally primed 6- to 7-week-old CD1 female mice (Charles River Italia) and cultured in M16 medium under mineral oil as previously described (Hogan et al, 1994). Oocytes were allowed to undergo GVBD by incubation in the absence of an exogenous cAMP source and used for microinjection either immediately (for dsRNAi) or after GVBD (approximately 2 h after collection). Before injection, oocytes and embryos were washed in M2 medium and then transferred to 50 μl drops of the same medium under mineral oil. Microinjection manipulations were performed as previously described (Sette et al, 1998). Briefly, into the cytoplasm of one blastomere of a two-cell embryo we injected 2–5 pl of a purified p90^Rsk2 (1–5 U, Upstate Biotechnology) together with either GST or GST-Emi1 diluted to a protein concentration of 1 mg/ml in injection buffer (20 mM Hepes, pH 7.4, 120 mM KCl, 100 μM EGTA, 10 mM β-glycerophosphate, 1 mM DTT, 10 μg/ml leupeptin, 10 μg/ml pepstatin). Microinjections were performed using an Olympus invertoscope (Olympus) equipped with Hoffman modulation contrast optics (Modulation optics Inc., Greenvale, NY) and two Leitz mechanical micromanipulators (Leica AG, Heerbrugg, Switzerland). After microinjections, embryos or oocytes were returned to M16 medium drops and cultured at 37°C under a humidified atmosphere of 5% CO₂ in air. At 12–14 h after injection, cells were scored for mitotic or meiotic divisions or processed for immunofluorescence analysis.

dsRNA preparation

To generate templates for dsRNA synthesis, we employed forward and reverse oligonucleotides containing at the 5′ end a T7 promoter sequence. For Emi1 amplification, the following primers were designed: forward 5′-GTAATACGACTCACTACTATAGGGCATGCAGC GAGTCA-3′; reverse 5′-GTAATACGACTCACTACTATAGGGCTCACAAT CTTTGT. These oligonucleotides amplify a region of 447 bp from base 945 to 1392 at the 3′ end of mouse Emi1 (AK011820). For GFP amplification, the following primers were designed: forward 5′-GTAATACGACTCACTACTATAGGGCATGCATA AAGGAG-3′; reverse 5′-GTAATACGACTCACTACTATAGGGCTCAATGC ATTAGTTC-3′. These oligonucleotides amplify a region of 600 bp of the GFP sequence. pCDNA3-mycEmi1 and pCMV5-GFP expression vectors were used as templates for PCR amplification. Amplified bands were gel-purified and used as templates (1 μg) for in vitro RNA transcription in order to obtain sense and antisense RNA sequences as previously described (Svoboda et al, 2000). RNA was extracted and precipitated by standard procedures and dissolved in RNAse-free H₂O. Equimolar amounts of sense and antisense RNA were annealed in DEPC-water supplemented with 1 U/μl of RNasin (Invitrogen) and 4 μg of RNA was boiled for 1 min and allowed to cool down at room temperature before phenol/chloroform extraction and ethanol precipitation. dsRNA was resuspended in H₂O, assayed by agarose electrophoresis and stored at −80°C prior to use.

We thank Drs Francesco Cenci and Federica Capolunghi for their help with FACS analysis, Drs Manuela Pellegrini and Susanna Dolci for critically reading the manuscript and Dr Mortin Frodin for the gift of the pMT2-HA-p90^Rsk2 vector. This work was supported by MIUR Cofin 2002 and 2003.