Inactivation of MAPK in mature oocytes triggers progression into mitosis via a Ca²⁺-dependent pathway but without completion of S phase

The mitogen-activated protein (MAP) kinase (MAPK) family member ERK2 (extracellular regulated kinase 2) is well known to control meiotic maturation in oocytes (Abrieu et al., 2001). In eggs of starfish (Tachibana et al., 2000; Kishimoto, 2004) and Xenopus (Tunquist and Maller, 2003), fertilization results in the inactivation of this MAPK. High levels of activated MAPK prevent eggs from entering a program of mitotic divisions, as well demonstrated in starfish (Tachibana et al., 2000). ERK activity in unfertilized oocytes has also been suggested to prevent DNA synthesis but results obtained in starfish seem to contradict this hypothesis. In oocytes of the starfish A. pectinifera, where fertilization occurs at G1 after meiosis, ERK inactivation has been correlated with stimulation of DNA synthesis (Tachibana et al., 1997; Tachibana et al., 2000). On the contrary, in oocytes of the starfish M. glacialis and A. aranciacus, MAPK inactivation was neither required for premature DNA replication after the first meiotic cell cycle nor for DNA replication after completion of meiotic maturation (Picard et al., 1996). In those species, the calcium ionophore A23187 could inactivate ERK in oocytes arrested at G2 without inducing DNA synthesis (Fisher et al., 1998).

What is the situation in sea urchin eggs fertilized after completion of meiosis, in G1 phase? Our previous data (Chiri et al., 1998; Zhang et al., 2005) and those reported by others (Carroll et al., 2000; Kumano et al., 2001) strongly suggest that, at this stage, unfertilized eggs contain a highly active ERK-like protein (ERK-LP) that is inactivated after fertilization. Inactivation of a Ca²⁺-sensitive MAP kinase kinase (MEK) pathway has been proposed to inactivate ERK-LP at fertilization (Kumano et al., 2001). This situation looks similar to that described for the starfish A. pectinifera because Carroll et al. also showed that treatment of eggs with the specific MEK inhibitor PD98059 triggers initiation of DNA synthesis (Carroll et al., 2000). However, one group found opposite results; Philipova and Whitaker reported that unfertilized sea urchin eggs do not contain active ERK (Philipova and Whitaker, 1998). Rather, fertilization after the activation of ERK-LP is required for S-phase onset and cell-cycle progression, whereas inactivation of this MAPK by XCL100 (a MAPK phosphatase) or the MEK inhibitor U0126 does not induce DNA synthesis in unfertilized eggs (Philipova et al., 2005a). Whether DNA synthesis depends on ERK inactivation at G1 and how the MEK-ERK cascade is involved in cell-cycle progression after fertilization of sea urchin eggs are then two important issues that need to be clarified.

We report here the behavior of unfertilized eggs in which ERK-LP dephosphorylation is induced with U0126 or PD98059 (Davies et al., 2000). These eggs showed microtubule polymerization, chromatin condensation and nuclear envelope breakdown (NEB), which corresponds to entry into mitosis. However, no division and no real development were observed. Since a transient increase in intracellular free Ca²⁺ (Ca²⁺_i), triggers entry in mitosis in sea urchin embryos (Wilding et al., 1996), we tested whether inactivation of ERK-LP leads to changes in Ca²⁺_i. We found that M-phase entry is indeed inhibited in eggs deprived of phosphorylated ERK-LP after injection of EGTA. Altogether, our results suggest that inhibition of the MEK-ERK pathway in unfertilized sea urchin eggs generates M-phase entry and recurrent oscillations of the M-phase-promoting factor (MPF) by altering the Ca²⁺_i level, but does not induce complete DNA replication.

We first investigated whether inactivation of the highly phosphorylated ERK-LP present in unfertilized sea urchin eggs can drive eggs into early parthenogenesis, as it is the case in starfish oocytes arrested in meiosis I (MI) expressing an antisense-Mos (Tachibana et al., 2000). We hypothesized that the situation is different in these two species, because unfertilized sea urchin eggs arrested at G1 do not contain any centrosome, whereas MI-arrested starfish oocytes contain two centrosomes (Schatten, 1994; Kato et al., 1990).

Treatment with the MEK inhibitors U0126 or PD98059 induced changes in the general morphology of unfertilized eggs. Observation under a light microscope showed that eggs of the sea urchin P. lividus that had been treated with 1-2 μM U0126 or with 2.5-5 μM PD98059 underwent NEB (93±3%; n=13 experiments or 50±5%; n=15 experiments, respectively) 3 hours after administration of the drugs (Fig. 1A, top panels), indicating entry into mitosis. At this time, 5-20% and 7-25% of eggs, depending on the batch of eggs treated with UO126 and PD98059, respectively, showed figures of constriction and blisters more or less accentuated (Fig. 1A, top panels). Very similar results were obtained using another sea urchin species L. pictus (Fig. 1A, bottom panels). Changes in morphology took longer to occur when concentrations of MEK inhibitor higher than 50 μM were used (not shown). Although a few eggs showed attempts of division, complete cytokinesis was never observed and parthenogenesis did never occur. Finally, some P. lividus eggs treated with 2.5 μM PD98059 (four out of 15 batches) or with 1 μM U0126 in (9 out of 13 batches) showed a very thin fertilization membrane (5-10% or 4-28%, respectively) (Fig. 1A, top panels). Similar results were obtained in L. pictus eggs (Fig. 1A, bottom panels).

P. lividus eggs treated or not with 1 μM U0126 (Fig. 1B) and fixed at different times for microtubule and chromatin staining showed the following kinetics of events: A fast microtubule polymerization step took place in the cortical area within 30 minutes of treatment (Fig. 1Bb) that later led to formation of arrow-shaped bundles generated from several points of `nucleation' at the periphery of the egg (Fig. 1Bc). Microtubule polymerization then continued, showing fewer bundles but a deeper and denser network that either remained cortical (Fig. 1Bd,e) or was both cortical and localized at the periphery of condensed chromatin (Fig. 1Bd,f). Chromatin progressively condensed, until individualization of chromosomes within 2 hours of treatment (Fig. 1Bd). Chromosomes were then dispersed and microtubules entered the nuclear space, indicating that NEB had occurred between 2 and 3 hours of treatment (Fig. 1Be,f). Even though some kind of chromosome attachment to microtubules - as well as a number of bipolar spindles - was occasionally observed, we never observed a real metaphase spindle that led to anaphase entry. Very similar results were obtained with eggs treated with 2.5 μM PD98059 (supplementary material Fig. S1, Table S1).

Several conclusions can be drawn from these results. (1) Unfertilized eggs deprived of MEK activity enter mitosis. (2) In contrast to starfish oocytes arrested in meiosis I, early development cannot be induced in G1-arrested sea urchin eggs by only inactivating MEK. (3) Elevation of a thin fertilization envelope in some eggs that had been treated with MEK inhibitor suggests changes in Ca²⁺_i levels, because cortical exocytosis is Ca²⁺-dependent (Zimmerberg et al., 1999).

Since unfertilized sea urchin eggs whose MEK activity was blocked entered mitosis, we thought they first went through S phase. Therefore, DNA replication by BrdU incorporation (see Materials and Methods) was estimated in unfertilized eggs treated with 1 μM U0126. Under these conditions, we observed some BrdU incorporation; however, it was similar in the presence or in the absence of aphidicolin, an inhibitor of DNA polymerase α (Sheaff et al., 1991) (Fig. 2). No detectable incorporation of BrdU was measured in untreated unfertilized eggs at this point in time (data not shown). Therefore, BrdU incorporation induced by U0126 was not due to complete replication of DNA. Block of the MEK-ERK pathway induced entry into mitosis without completion of S phase.

We then questioned whether a lack of MPF fluctuations, as reported in starfish oocytes (Tachibana et al., 2000), explains the absence of early development in sea urchin eggs after inactivation of the MEK-ERK pathway.

To compare our results with those obtained by Philipova et al. who used L. pictus (Philipova et al., 2005a; Philipova et al. 2005b), we first verified that all antibodies we used to detect ERK recognized the same protein in P. lividus and L. pictus. Results obtained in unfertilized eggs are reported in Fig. 3A. In both species, the anti-ERK and the anti-panERK antibodies labeled a doublet of proteins at approximately 44 kDa, widely accepted to represent the non-phosphorylated (lower band) and the phosphorylated form (upper band) of ERK (Fig. 3Aa), and which we therefore named ERK-LP. As a matter of fact, the upper band of the doublet and the protein labeled by the antibody against phosphorylated MAPK (anti-phosphoMAPK antibody) migrated at the same molecular mass. Furthermore, the proteins revealed in both species had the same molecular mass (Fig. 3Aa). We observed that the anti-panERK antibody also revealed a protein at a molecular mass of approximately 81 kDa, in L. pictus (Fig. 3Aa) as well as in P. lividus, depending on the exposure of the gel during ECL procedure (see Materials and Methods) (Fig. 3Aa vs Ab right panel). As shown in our previous report (Zhang et al., 2005), a decrease in the phosphorylation of ERK-LP occurred in P. lividus eggs after treatment with PD98059 (Fig. 3Ab left panel). This decrease in phosphorylation was not due to a change in the total amount of ERK-LP because labeling intensity did not vary when using the anti-panERK antibody (Fig. 3Ab right panel) and because the decrease in phosphorylation was also observed after treatment with alkaline phosphatase (Zhang et al., 2005). Finally, a very faint signal was detected with the anti-phosphoMAPK antibody at approximately 81 kDa, which did not vary after addition of PD98059 (Fig. 3Ab left panel), similarly to the signal detected at this molecular mass with the anti-panERK antibody under the same conditions (Fig. 3Ab right panel). Altogether, we are confident that the ERK-LP detected in our assays in eggs of P. lividus and L. pictus is the same and that the anti-phosphoMAPK antibody is specific against the phosphorylated form of ERK-LP.

We then analyzed the variations of ERK-LP phosphorylation after treatment of eggs with the MEK inhibitors. Unfertilized eggs from the sea urchin P. lividus were first treated with 2.5 μM PD98059. As expected, this induced a rapid dephosphorylation of the ERK-LP. Surprisingly, a transient peak of ERK-LP phosphorylation was observed at approximately 180 minutes, and a second peak was observed approximately 240 minutes after addition of PD98059 (Fig. 3Ba, upper panel). Unfertilized eggs contained a substantial amount of Cdc2 phosphorylated at tyrosine residue 15 (Cdc2-TyrP) and a low H1 kinase activity, corresponding to a low MPF activity. Addition of PD98059 induced a rapid decrease in the Tyr phosphorylation of Cdc2 that was followed by two peaks of phosphorylation, occurring at times earlier than those observed for P-MAPK, namely 60 minutes and 140 minutes after drug addition (Fig. 3Ba, upper panel). Concomitantly, H1 kinase activity increased gradually, but recurrent partial inactivation was observed when phosphorylation of Cdc2-TyrP was increased (Fig. 3Ba lower panel). The level of H1 kinase activity reached after a 3-hour treatment with the MEK inhibitor, corresponded to a third of activity measured at mitosis in fertilized eggs (De Nadai et al., 1998) (data not shown). Similar results were obtained in L. pictus (data not shown).

These results could be explained by a gradual loss of activity of PD98059 present in the external medium. A complete loss of the drug activity was measured at approximately 1-2 hours after dilution (Fig. 3Bb), which explains the fact that ERK-LP is phosphorylated again 1-2 hours after drug addition (Fig. 3Bb). However, this cannot explain the later peak of ERK-LP phosphorylation.

Experiments with 1 μM U0126 gave results similar to those obtained with PD98059. At 1 μM, U0126 induced inactivation of the ERK-LP, which was followed by two peaks of reactivation 150 minutes and 270 minutes after addition of the drug in L. pictus (Fig. 3Ca) and in P. lividus (data not shown). The initial decrease in phospho-ERK-LP was not due to a loss or degradation of the protein itself during that period, because no change of total ERK was observed when using the pan-ERK antibody. As observed for PD98059, the level of Cdc2-TyrP progressively decreased to reach a very low level 1 hour after addition of U0126; peaks of Cdc2-TyrP were then observed 150 minutes and 240 minutes after addition of the drug. These data indicate that inhibition of the MEK-ERK cascade is sufficient to trigger MAPK and MPF oscillations in unfertilized eggs. Therefore, the question arises whether both types of oscillations are linked to each other?

Oscillations in the phosphorylation of ERK were not always seen in experiments using U0126. In some experiments, ERK was dephosphorylated and no reactivation was seen thereafter, whereas oscillations of Cdc2-TyrP still occurred (Fig. 3Cb). A progressive decrease in Cdc2-TyrP was first observed under these conditions and reached a low 30 minutes after addition of U0126. Two later peaks of Cdc2-TyrP were then detected, after approximately 100 minutes and 200 minutes (Fig. 3Cb upper panel). As observed with PD98059, a gradual increase in H1 kinase activity, showing transient waves, was measured after addition of U0126, and lower activity was observed after approximately 60 minutes and 200 minutes when Cdc2-TyrP was high (Fig. 3Cb lower panel).

As shown above for PD98059, a gradual loss of U0126 activity was achieved at 1 hour and a complete loss 2-3 hours after dilution (Fig. 3Cc). This loss of activity of the MEK inhibitor thus explains that ERK-LP could be phosphorylated again within 3 hours in U0126-treated eggs.

In conclusion, oscillations in MPF activity can be observed in eggs treated with U0126 that did not show any rephosphorylation of ERK-LP. Therefore, MPF oscillations triggered by an initial inactivation of MAPK appear to be independent of MAPK.

We tested the hypothesis that, in oocytes, active MAPK prevents entry into mitotic cycles by maintaining a low level of Ca²⁺_i, which can be overcome by the fertilization Ca²⁺_i signal (Tunquist and Maller, 2004). Experiments were performed using P. lividus eggs. Although no modification in the level of Ca²⁺_i was recorded in control eggs during 150 minutes, the usual fertilization Ca²⁺_i transient was observed and addition of U0126 induced a slow and progressive rise in Ca²⁺_i levels (Fig. 4A). Small oscillations seemed to occur during the slow rise because the average values of approximately 62 minutes (0.46±0.02, n=9) and 106 minutes (0.50±0.03, n=5), were significantly higher (Student's test, P<0.05) than the average value measured at 82 minutes (0.42±0.04, n=10) (Fig. 4A). The increase in Ca²⁺_i levels after 3 hours of U0126 treatment could reach a level corresponding to the smallest (or half of the largest) fertilization Ca²⁺_i signal that we measured (Fig. 4A). This corroborates the appearance of a fertilization membrane in a small number of eggs (Fig. 1A).

Is this increase in Ca²⁺_i necessary to trigger entry in mitosis in eggs treated with MEK inhibitors? We first determined the accurate amount and concentration of EGTA buffer that needs to be injected to inhibit activation after fertilization (data not shown). Injection of EGTA buffer did not induce any activation by itself (Fig. 4B). It blocked NEB in 17 of 22 egg batches that were injected and then treated with PD98059 but it reduced the number of eggs entering mitosis in only five of 22 egg batches (Table 1). Similar results were obtained with U0126 (Table 1). An experiment that led to inhibition of NEB (Fig. 4B) showed that all EGTA-injected eggs that were further treated with U0126 showed neither NEB nor mitotic figures, whereas non-injected eggs did.

These changes in Ca²⁺_i levels could either be due to modifications in Ca²⁺ permeability of the endoplasmic reticulum or an influx of external Ca²⁺ into the egg. Therefore, we tested whether the sensitivity to inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃] was modified in eggs treated with 2 μM U0126 or with 10 μM PD98059. Ins(1,4,5)P₃ injection activated unfertilized eggs in a concentration-dependent manner. Treatment with each inhibitor reduced the percentage of activated eggs, when less than 10 μM Ins(1,4,5)P₃ was injected (Fig. 4C). This suggests that a block in MEK activity reduced the sensitivity of intracellular compartments to Ins(1,4,5)P₃.

Finally, the absence of external Ca²⁺ did not inhibit entry in mitosis in eggs treated with U0126, because eggs treated at a low external Ca²⁺ concentration showed NEB and morphological alterations that seemed even more severe (Fig. 4D). Similar results were obtained with PD98059 (data not shown).

All these results suggest that entry in mitosis, triggered by a block of the MEK-ERK cascade in unfertilized eggs, depends on an increase of Ca²⁺_i. Moreover, they also suggest that variations in Ca²⁺_i triggered by MEK inhibition were due to modifications of Ca²⁺ transport inside the egg and not to a stimulation of Ca²⁺ influx into the egg.

The mitotic events that are induced after ERK-LP dephosphorylation could be due to an increase in intracellular pH (pH_i), as it is the case after fertilization or after ammonia treatment. We used the DMO technique, which has been widely used to determine variations of pH_i in sea urchin eggs (Johnson and Epel, 1981) and gives results similar to those obtained using fluorescent probes (Dube and Eckberg, 1997). Treatment of eggs with concentrations of PD98059 as high as 50 μM did not trigger any increase in pH_i and eggs behaved like untreated eggs. An increase of 0.35 pH units was, as expected, detected after fertilization (Payan et al., 1983) but was not altered by PD98059 (Fig. 5A).

Does an increase in pH_i that is normally induced by ammonia (Dube and Epel, 1986) trigger ERK-LP dephosphorylation? Although complete ERK-LP dephosphorylation was observed after fertilization or, as expected, after treatment of eggs with the Ca²⁺ ionophore A23187 (Carroll et al., 2000), no modification was seen (even after 1 hour) when eggs where treated with 10 mM ammonia (Fig. 5B).

Altogether, these results strongly suggest that, in unfertilized sea urchin eggs, mitotic events triggered by ERK dephosphorylation are independent of the pH_i status.

Our present data, which were equally reproducible by using U0126 or PD98059 in the two different sea urchin species P.lividus and L. pictus, show that inactivation of a MEK-ERK cascade in unfertilized eggs triggers oscillations of phosphorylated ERK (ERK-P) and MPF, and leads to entry into mitosis without full DNA replication. All these events were mediated by changes in Ca²⁺_i and not by changes in pH_i. This artificial situation may help to understand the role of MAPK inactivation in cell-cycle progression after fertilization. It is clear that location and activity of MAPK in eggs differ radically before and after fertilization, suggesting not only different targets but also different regulating pathways upstream MEK-MAPK before and after fertilization. (1) The unfertilized egg contains very high levels of active ERK-like protein, which is highly expressed all over the egg, in the cytosol and the nucleus (Zhang et al., 2005), and which, as shown here, prevents eggs to enter a mitotic program. (2) Fertilization induces a rapid decrease in MAPK activity, that is followed at time of mitosis by a very small reactivation, corresponding to a tenth of the unfertilized level (Zhang et al., 2005). ERK-like protein is located at that time in very small areas, in the vicinity of centrosomes and insures formation of a normal mitotic spindle, but controls neither entry into nor exit from first mitosis (Zhang et al., 2005).

In unfertilized eggs, inactivation of the MEK-ERK pathway triggers chromatin condensation. Some DNA replication (measured through BrdU incorporation) occured, which was similar in the presence or in the absence of aphidicolin - an inhibitor of DNA polymerase α, which inhibits the processive elongation of nascent DNA strands but does not prevent the initiation of replication and the formation of short (100-500 bp) primers (Marheineke and Hyrien, 2001; Maiorano et al., 2006). Inactivating MAPK might have led to initiation but not to progression of DNA replication. These results corroborate data by Philipova et al. reported for sea urchin (Philipova et al., 2005a) but contradicts those obtained by Carroll et al., who report to have detected DNA replication (Carroll et al., 2000). We, however, believe they might have detected DNA repair. Our results also corroborate those obtained in Xenopus egg extracts (Abrieu et al., 1997) and oocytes of the starfish M. glacialis and A. aranciacus (Picard et al., 1996; Fisher et al., 1998), but contradict those reported for the starfish A. pectinifera (Tachibana et al., 1997). However, the phosphatase XCL100, used by the latter group to inactivate MAP kinase, might act upstream of DNA synthesis on a target other than ERK and, although injection of constitutively active MEK repressed the initiation of DNA synthesis following fertilization, 30% of these eggs did escape G1 arrest and developed until G2 (Tachibana et al., 1997; Abrieu et al., 1997). Altogether, these results suggest that ERK inactivation is not required for full DNA replication during first mitosis of early sea urchin embryos.

The very opposite hypothesis has been proposed recently by Philipova et al., who suggest that a rise in ERK activity occurring after fertilization is required for S phase (Philipova et al., 2005a). First, the authors detected an ERK protein that is not active in unfertilized eggs, which contradicts the well-established view that “downregulation of MAP kinase is a universal consequence of fertilization in the animal kingdom” (Fisher et al., 1998). Philipova's view could be explained if two types of ERK were expressed in the sea urchin egg. However, this hypothesis seems unlikely because all anti-ERK antibodies used in our study detected a single protein in two sea urchin species, including L. pictus, which was used by these authors (Philipova et al., 2005a; Philipova et al., 2005b; Philipova and Whitaker, 1998; Philipova and Whitaker, 2005). Their so-called ERK1 formed dimers that were detected at a molecular mass of approximately 90 kDa (Philipova and Whitaker, 2005). We also detected a band of approximately 80 kDa with the anti-pan ERK and the anti-phosphoERK antibodies in P. lividus as well as L. pictus, under reducing electrophoresis conditions, but we believe that this high-molecular mass protein is either a non-specific signal or ERK5. Second, 100 μM U0126 were necessary to block DNA replication and MAPK activity (Philipova et al., 2005a), but this huge amount of U0126 might non-specifically alter other components of MEK-MAPK pathways including ERK5 or SAPK2α (stress-activated protein kinase 2α/p38) (Davies et al., 2000), which act on DNA synthesis (Dehez et al., 2001) and on cell-cycle checkpoints (Pearce and Humphrey, 2001). Moreover, it is surprising that PD98059 could not inhibit several biological processes inhibited by UO126 (Philipova et al., 2005a). Third, the MAPK phosphatase XCl 100 used by Philipova et al. to cancel DNA synthesis after fertilization might also, as discussed above, act on targets other than ERK. Our hypothesis is that neither inactivation nor activation of the MEK-ERK cascade act on DNA replication during first mitosis of early sea urchin embryos. Both Ca²⁺_i and pH_i signals triggered by fertilization have been widely described to be upstream regulators of DNA synthesis (Mazia and Ruby, 1974; Nishioka and Magagna, 1981; Schomer-Miller and Epel, 1999), and might upregulate DNA synthesis through pathways others than the MEK-ERK cascade.

The unfertilized P. lividus sea urchin egg contains a non-negligible MPF activity, as well as a pool of Cdc2-TyrP that is dephosphorylated after inactivation of the MEK-ERK cascade. This leads to a general increase in MPF activity without any stimulation of the low rate of protein synthesis that can be measured in unfertilized eggs (data not shown) and also to entry into mitosis. This is in agreement with the idea that a Mos-MEK-MAPK cascade downregulates a mechanism that inactivates cyclin B-cdc2 kinase in matured oocytes (Abrieu et al., 1997). The small amount of MPF may be sufficient (Genevière-Garrigues et al., 1995) and is obviously necessary to trigger entry into M phase in unfertilized eggs deprived of MEK-ERK activity (this report) and in fertilized eggs (Abrieu et al., 2001; Philipova et al., 2005b). Oscillations in MPF activity and Cdc2-TyrP that were then generated, seem to be independent of ERK-LP variations, which corroborates data obtained in Xenopus egg extracts (Takenaka et al., 1997; Guadagno and Ferrell, Jr, 1998); alternatively, these oscillations might control activity of ERK-LP, as suggested by Abrieu et al. (Abrieu et al., 2001).

Full inactivation did not occur between peaks of H1 kinase activity, which could explain why eggs did not undergo complete mitotic cycles, never divided or developed, as reported for starfish oocytes (Tachibana et al., 2000). The presence of a centrosome in immature MI-arrested starfish oocytes can also possibly explain this difference between starfish (MI-arrested) and sea urchin (G1-arrested) oocytes, because the remaining centrosome in immature oocytes is incompetent for reproduction and meant to disappear at the end of meiosis, as it is the case in sea urchin unfertilized eggs (Uetake et al., 2002).

Morphological events, such as egg constrictions, occurred either more rapidly after induction of a faster ERK rephosphorylation, for example after rinsing off the inhibitor or with a delay when concentrations higher than 50 μM were used (data not shown), which probably delays ERK-LP rephosphorylation. MEK inhibitor used at such a high level might act non-specifically on pathways that lead to NEB and mitotic events. In another hypothesis, MAPK re-activation acts as a `facilitator' of mitosis, as reported during the first mitosis of the sea urchin embryo (Zhang et al., 2005) or in somatic cells (Roberts et al., 2002). The longer time that is necessary to get rid of a higher amount of inhibitor can then explain the delay in these two hypotheses. It then seems that reactivation of the MEK-ERK cascade regulates, but does not trigger, mitotic events, which, again, contradicts recent data obtained by Philipova et al. (Philipova et al., 2005b), but questions similar to those pointed above concerning DNA replication can be raised.

Unfertilized sea urchin eggs deprived of MEK-ERK activity can enter M phase without complete replication of DNA. This is in agreement with the fact that aphidicolin does not inhibit MPF activation, and suggests that factors required for both DNA replication and checkpoint activation (Takeda and Dutta, 2005) are present in fertilized sea urchin eggs and absent in unfertilized eggs (Sluder et al., 1995; Genevière-Garrigues et al., 1995; Philipova et al., 2005b) (our unpublished results). Finally, 4N chromosomes and the probable absence of a DNA checkpoint machinery in MI-arrested starfish oocytes might explain the situation in these oocytes where aphidicholin does not block the first cleavages when fertilization is induced in MI (Nagano et al., 1981), and where development can go as far as bona fidae larvae when MAPK is inactivated (Tachibana et al., 2000; Sadler et al., 2004).

Ammonia did not trigger dephosphorylation of ERK-LP, and ERK-LP dephosphorylation did not trigger an increase in pH_i. Dephosphorylation of Cdc2-TyrP induced after inactivation of the MEK-ERK pathway is pH_i-independent, as reported after fertilization (Edgecombe et al., 1991). Microtubule polymerization, chromosome condensation and NEB might not be strictly pH_i-dependent events. Ammonia, which has been reported to stimulate these events (Bestor and Schatten, 1982), might act via a pH_i-independent pathway or might be directly affected by amines, as suggested by others (Dube and Epel, 1986; Rees et al., 1995).

Inactivation of the MEK-MAPK pathway led to a progressive and slow increase in Ca²⁺_i that was necessary for progression of eggs into mitosis. This is consistent with results showing that chromatin condensation (Twigg et al., 1988) and NEB (Philipova et al., 2005b; Wilding et al., 1996) that occur after fertilization are Ca-dependent events. It is unlikely that this increase in Ca²⁺ was due to entry of external Ca²⁺_i, because entry into mitosis was not inhibited in zero-Ca²⁺ external medium. Therefore, the rise in Ca²⁺_i could only be due to alterations in Ca²⁺ transport into and out of intracellular compartments. We cannot rule out the possibility of non-specific effects of the MEK inhibitors on Ca²⁺ channels or transporters. However, to our knowledge, all non-specific effects that have been reported were obtained at concentrations higher than those used in our study (Pereira et al., 2002). Moreover, U0126 does not seem to act non-specifically on store-mediated Ca²⁺ entry, as reported for human platelets (Rosado and Sage, 2001).

We show here the induction of mitosis entry in unfertilized eggs, i.e. under conditions where artificial ERK-LP inactivation and MPF stimulation occurred at the same time. Normally, fertilization in sea urchin eggs triggers inactivation of both activities in the first minutes after fertilization (Chiri et al., 1998), and mitosis occurs after completion of S phase, when MPF is stimulated (Meijer and Pondaven, 1988). At fertilization, the decrease in MAPK activity must then occur together with another event that delays stimulation of MPF and entry in mitosis, to allow S-phase completion before entry into mitosis. How to compare the situation triggered by chemical MEK inhibition in unfertilized eggs with that where ERK-LP is dephosphorylated by fertilization (Fig. 6)? The large fertilization Ca²⁺_i peak that induces MEK-ERK initial inactivation is by-passed in eggs treated with MEK inhibitors. Two essential elements must then be considered. The first one concerns the level of Ca²⁺_i, reached in unfertilized eggs after a 30-minute treatment with MEK inhibitor, which is much smaller than the Ca²⁺_i measured at entry in mitosis in fertilized eggs (Ciapa et al., 1994; Philipova et al., 2005b; Wilding et al., 1996). Under similar conditions, this small increase was - although detected - not acknowledged by others (Carroll et al., 2000). The MEK-ERK cascade might act on nuclear (Gerasimenko and Gerasimenko, 2004) or mitochondrial (Bianchi et al., 2004) Ca²⁺ signaling mechanisms to generate the small Ca²⁺ signals that are localized in the peri-nuclear area, have been detected before NEB and might be at the origin of the larger NEB Ca²⁺_i transient (Wilding et al., 1996). The second element refers to the role of a Src-PLCγ pathway that is activated at fertilization and leads to Ins(1,4,5)P₃ synthesis necessary for mitotic Ca²⁺ transients (Ciapa et al., 1994; Shearer et al., 1999). Fertilization strongly stimulates the egg metabolism and various signaling pathways, including a constant increase in tyrosine phosphorylation during the first cell cycle (Ribot, Jr et al., 1984). A large amount of Ins(1,4,5)P₃, due to a high level of upstream tyrosine activity attained at mitosis, must then be compensated by a decrease in Ins(1,4,5)P₃ sensitivity to generate small Ca²⁺ peaks. The decrease in Ins(1,4,5)P₃ sensitivity of intracellular stores in unfertilized eggs after MEK inactivation might be due to an altered phosphorylation status of Ins(1,4,5)P₃ receptors that is known to play a role in the specificity of the Ca²⁺_i signals (Yule et al., 2003). This could also explain why the amount of Ins(1,4,5)P₃ that generates the small and slow mitotic Ca²⁺_i signal at entry of mitosis (i.e. when ERK-LP phosphorylation is low) is much higher than that produced during the large and rapid fertilization Ca²⁺_i signal (Ciapa et al., 1994) (Fig. 6).

In conclusion, we believe that ERK inactivation induced after fertilization sets up an endogenous Ca²⁺_i oscillator, independent of pH_i, which controls MPF oscillations and early embryonic division, as suggested by Swanson et al. (Swanson et al., 1997). The frequency and intensity of the Ca²⁺_i spikes, and full activation of MPF during mitosis, may be regulated by another oscillator system. This second mechanism might be of spermatic origin, stimulated by the fertilization Ca²⁺_i signal and might involve the phosphoinoisitide messenger system (Ciapa et al., 1994) including PLCγ (Shearer et al., 1999) (Fig. 6).

Paracentrotus lividus sea urchins were collected in the bay of Marseille or Villefranche-sur-mer (France) and Lytechinus pictus were collected in California (Marinus Sci. Inc.). Gametes were collected, prepared and fertilized either in natural sea water (NSW) or in artificial sea water (ASW; Reef Crystals Instant Ocean) as described (Chiri et al., 1998; De Nadai et al., 1998).

Eggs were treated or not with PD98059, U0126 or U0124 (inactive analog of U0126) (Calbiochem), which were made as a 15 mM stock in DMSO. The appropriate addition of DMSO to the egg culture was used as a control of all experiments.

Preparation of egg samples, separation of proteins by SDS-PAGE and western blotting were performed as described previously (Chiri et al., 1998; De Nadai et al., 1998; Zhang et al., 2005). All the antibodies were diluted in blocking buffer as follows: mouse anti-phospho-MAPK isoforms 42 and 44 (Thr202 and Tyr204, respectively; Cell Signalling) at 1:2000, mouse anti-pan ERK antibody (BD Transduction laboratories) at 1:1000, rabbit anti-phospho-Cdc2 (Tyr15, Cell Signalling) at 1:500, rabbit anti-Cdk1/Cdc2 (PSTAIR, UBI) at 1:1000, the anti-mouse horseradish-peroxidase (HRP)-conjugated goat IgG (ICN) and the anti-rabbit HRP-conjugated goat IgG (ICN) secondary antibodies at 1:1000 and 1:10 000, respectively. Proteins were revealed by enhanced chemoluminescence (ECL, Amersham).

H1 kinase activity was measured in egg extracts obtained before and at different times after addition of MEK inhibitor using the protocolt described by Meijer and Pondaven (Meijer and Pondaven, 1988). Since it has been established that H1 kinase activity essentially reflects MPF activity in eggs and early embryos (Levasseur and McDougall, 2000; Levasseur and McDougall, 2003), the word MPF is used in that sense.

To monitor DNA replication, 5-bromo-2-deoxyuridine (BrdU) was added to unfertilized egg suspension at a concentration of 0.1 mg/ml in presence of 1 mM ATA. Unfertilized eggs were treated with 1 μM U0126 with or without 20 μg/ml aphidicholin and harvested after 3 hours. Control-fertilized eggs were harvested 60 minutes after fertilization. Eggs were transferred to 0.2 mg/ml pronase in seawater to remove the fertilization membrane of fertilized eggs. The pronase reaction was stopped by addition of 1% BSA and eggs were rinsed in Millipore-filtered sea water (MFSW) and fixed in 4 N HCl for 2 hours. After post-fixation in methanol for 30 minutes, eggs were washed in PBS-Tween for 1 hour and incubated in anti-BrdU mouse monoclonal antibodies (Amersham) for 2 hours at room temperature. They were washed in PBS-Tween for 1 hour and then incubated with a 1:250 dilution of FITC-conjugated anti-mouse IgG antibody (Sigma) for 1 hour at room temperature. After washing in PBS-Tween for 1 hour at room temperature, eggs were mounted on pre-washed glass plates in Mowiol and observed by fluorescence microscopy on a Zeiss microscope.

Eggs were cultured in ASW or NSW, treated or not with PD 98059 or U0126, and taken for fixation and labeling.

For microtubule and chromatin labeling, eggs were taken at different times after U0126 or PD 98059 addition, then fixed, labeled and observed by confocal microscopy as previously explained (Zhang et al., 2005). Microtubules were labeled with the monoclonal rat anti-tubulin antibody YL1/2 (Chemicon) and chromatin with Hoechst-33258 dye as described (Zhang et al., 2005).

De-jellied eggs were stuck on Petri dishes coated with polylysine (0.5 mg/ml), and were maintained at 18°C. Injection volumes were 1-2% of the egg volume and were performed under N₂ atmosphere using a Narishige IM 300 microinjector. We used zero-Ca²⁺ injection buffer (10 mM EGTA, 480 mM KCl, 20 mM PIPES, pH 7.0) to avoid Ca²⁺_i changes.

Ca²⁺_i measurements were made in eggs injected as described above with injection buffer (480 mM KCl, 0.1 mM EGTA, 20 mM PIPES, pH 7.0) containing 5 mM 10 kDa-Fura-2 dextran (Molecular Probes). Eggs were then treated with U0126 or PD98059 and Ca²⁺_i fluorescence was detected under a Nikon Eclipse TE300, equipped with a ×20 plan Fluor Nikon objective, a Lambda-10 (Sutter Inst.) shutter and a Hamamastu CCD camera C2400-80. Signals were analyzed using a Dynamic Fluorescence and Ion Imaging software (Axon Instruments, Inc.).

Variations of pH_i were measured following the protocol as previously described in detail (Payan et al., 1983).

This work is dedicated to our colleagues and friends Hidemi Sato, a former Director of the Sugashima Marine Laboratory, Nagoya University, Japan, and Jean Febvre, Université de Nice-Sophia Antipolis, Observatoire Océanologique de Villefranche, France, who died a few weeks ago. Work in B.C.'s group has been supported by Groupe de Recherche (GDR No 2688). This work was supported by the Association pour la Recherche sur le Cancer (ARC), the CNRS, The Université Pierre et Marie Curie, and the French Ministère de la Recherche. S.C. acknowledges receipt of an ARC fellowship, and W.L.Z. a Ministère de la Recherche and a K. C. Wong fellowship.