Ca²⁺ oscillations at fertilization in mammals are regulated by the formation of pronuclei

Sperm-egg fusion at fertilization triggers an increase in intracellular Ca²⁺ in all species examined(Stricker, 1999;Runft et al., 2002). The increase in Ca²⁺ is necessary for egg activation, including the resumption (and completion) of meiosis and entry into the first mitotic cell cycle (Kline and Kline, 1992;Swann and Ozil, 1994;Schultz and Kopf, 1995;Runft et al., 2002). Ca²⁺ transients also feature during the early cell division cycles of embryos from a variety of species in which they control nuclear envelope breakdown (NEBD) and anaphase onset(Steinhardt, 1990;Ciapa et al., 1994;Kono et al., 1996;Groigno and Whitaker, 1998). The pattern of Ca²⁺ signalling at fertilization shows a remarkably varied temporal organization, ranging from single monotonic increases inXenopus, sea urchins and starfish to long lasting Ca²⁺oscillations in nemerteans, ascidians and mammals(Stricker, 1999). The temporal organization of Ca²⁺ oscillations at fertilization in mammals can have a dramatic effect on egg activation and embryonic development(Ozil, 1990;Lawrence et al., 1998;Ozil and Huneau, 2001), yet there is little understanding of how sperm-induced Ca²⁺oscillations are regulated.

A number of lines of evidence suggest that the meiotic and mitotic cell-cycle kinases, Cdk1-cyclin B and MAP kinase (M-phase kinases), may be involved in controlling the temporal organization of Ca²⁺transients at fertilization (Carroll,2001; Nixon et al.,2000). Species in which sperm trigger repetitive Ca²⁺transients are fertilized at meiosis I (MI) or meiosis II (MII), when the activity of the M-phase kinases is high. By contrast, those species in which fertilization stimulates a monotonic increase are fertilized at prophase of meiosis or mitosis, when the activity of the M-phase kinases is low(Stricker, 1999). One notable exception to this is in Xenopus eggs where MII is completed and Cdk1-cyclin B activity is reduced soon after the Ca²⁺ wave crosses the egg. The association between Ca²⁺ oscillations and M phase is strongest in ascidian eggs, where fertilization triggers two series of Ca²⁺ oscillations that are tightly coupled to Cdk1-cyclin B1 activity during MI and MII (McDougall and Levasseur, 1998). MAP-kinase activity remains high when Ca²⁺ oscillations stop between the two meiotic divisions,suggesting that it is Cdk1-cyclin B kinase activity, rather than MAP kinase,that regulates sperm-induced Ca²⁺ signalling in ascidians(McDougall and Levasseur,1998). Furthermore, maintenance of Cdk1-cyclin B activity by expressing full-length or non-destructible cyclin B1 leads to the generation of persistent Ca²⁺ oscillations, whereas inhibition of MAP kinase is without effect (Levasseur and McDougall, 2000). These experiments strongly suggest that fertilization-induced Ca²⁺ signals in ascidians are controlled by a cyclin B-dependent kinase activity.

In mammalian eggs, there is also a relationship between sperm-induced Ca²⁺ signalling and an M-phase state. First, maintenance of meiotic arrest with nocodazole or colcemid leads to persistent Ca²⁺oscillations (Jones et al.,1995), mirroring the effects of excess cyclin B1 in ascidian eggs. Second, the Cdk1-cyclin B inhibitor roscovitine inhibits sperm-induced Ca²⁺ transients, although it also inhibits Ca²⁺ release by Ins(1,4,5)P₃ and the Ca²⁺-pump inhibitor thapsigargin (Deng and Shen,2000). Third, similar to ascidians, fertilization of mouse oocytes leads to two sets of Ca²⁺ transients that occur only in an M-phase cytoplasm. The first occurs at fertilization and continues until entry into interphase (Jones et al.,1995; Day et al.,2000; Nixon et al.,2002); the second starts some 12 hours later at NEBD of the first mitotic division (Kono et al.,1996; Day et al.,2000). It remains uncertain how many transients are generated during the first mitotic division. A single transient has been reported in many cases but multiple transients have been reported in others(Day et al., 2000;Kono et al., 1996;Tombes et al., 1992). Irrespective of the number of transients, the evidence strongly suggests that in mammals, like ascidians, the sperm-induced Ca²⁺ transients are regulated by the activity of Cdk1-cyclin B. However, there is one important exception to the correlation of Cdk1-cyclin B activity and Ca²⁺oscillations. At fertilization, Cdk1-cyclin B activity declines at the time of second polar body extrusion (Moos et al.,1995; Verlhac et al.,1994), whereas the Ca²⁺ transients persist for a further 2 hours until the pronuclei form(Jones et al., 1995).

A role for pronuclei in regulating Ca²⁺ release at fertilization is suggested by nuclear transfer and cell-fusion experiments. Pronuclei from fertilized embryos trigger Ca²⁺ release and egg activation when fused with MII oocytes (Kono et al.,1995). This activity is apparently sperm derived and nuclear associated because pronuclei from parthenogenetic embryos or cytoplasts are without effect. Similar results have been obtained by fusing fertilized and parthenogenetic embryos (Zernicka-Goetz et al., 1995). The only way of furnishing nuclei in parthenogenetic embryos with Ca²⁺-releasing activity is by stimulating activation by microinjection of Ca²⁺-releasing sperm extracts(Kono et al., 1995). The same paternally derived activity appears to regulate mitotic Ca²⁺transients. Reciprocal transfer of pronuclei between fertilized and parthenogenetic one-cell embryos shows that Ca²⁺-releasing activity at NEBD is independent of the origin of the cytoplasm and always follows the pronuclei from fertilized embryos (Kono et al., 1996). Together, these suggest that localization of a sperm-derived Ca²⁺-releasing activity to the pronuclei contributes to the cessation of Ca²⁺ transients, whereas release from the pronuclei induces their return at NEBD. Data that do not support a role for pronucleus formation also exist. Nucleate and anucleate halves of one-cell embryos bisected prior to pronucleus formation cease to oscillate about the same time, suggesting that, in bisected embryos, pronucleus formation is not necessary for the cessation of sperm-induced Ca²⁺ transients(Day et al., 2000).

The experiments in mouse and ascidian oocytes described above suggest two main mechanisms: first, that Ca²⁺-releasing activity is regulated directly or indirectly by M-phase kinases; and second, that activity is inhibited as a consequence of pronucleus formation. We describe experiments in mouse oocytes that distinguish between these two mechanisms. By dissociating pronucleus formation from the mitotic kinase activities, we show that Ca²⁺ transients continue after M-phase kinases are inactivated, and pronucleus formation or nuclear transport is inhibited. Furthermore, in mitotic one-cell embryos we find that Ca²⁺ transients start after the pronuclear membranes become permeable and stop before the nuclei form in two-cell embryos. These findings reveal a new compartmentalization-mediated mechanism for regulating the release of intracellular Ca²⁺ in early mammalian development.

MII oocytes were recovered from MF1 mice that had previously been administered, by intra-peritoneal injection, 7.5 International Units (U) of pregnant mare's serum gonadotrophin and 5 U of human chorionic gonadotrophin(hCG; Intervet) at a 48-52 hour interval. To collect the oocytes the mice were killed by cervical dislocation 13-15 hours post-hCG injection and the oviducts removed to HEPES-buffered KSOM (H-KSOM)(Lawitts and Biggers, 1993;Summers et al., 2000). Using a dissecting microscope, the cumulus masses were released from the oviducts into H-KSOM containing hyaluronidase (150 U/ml; embryo tested grade, Sigma). The oocytes were collected and washed three times in fresh H-KSOM. For production of one-cell embryos, hormone-primed mice were housed with a single male of the same strain immediately after injection of hCG. The oviducts were recovered 27-28 hours post-hCG injection. The one-cell embryos were released into H-KSOM and washed to remove any adherent cumulus cells before placing in a drop of H-KSOM under oil.

For in vitro fertilization, sperm from the epididymis of proven fertile MF1 mice were released into 1 ml T6 medium and allowed to disperse for 20 minutes as described previously (Halet et al.,2002). The suspension was diluted into a total of 5 ml of the same medium and incubated for 1.5-3.0 hours to allow capacitation to take place. All manipulations and incubations of sperm, oocytes and embryos were at 37°C.

Microinjection was performed using Narishige manipulators mounted on an inverted Leica microscope. Micropipettes with an internal filament were pulled and back-filled with ∼1-2 μl of the appropriate reagent (see below)made up in injection buffer (120 mM KCl, 20 mM HEPES, pH 7.4). Oocytes were immobilized using a holding pipette and the injection pipette was pushed through the zona pellucida until it was in contact with the oocyte plasma membrane. The pipette was inserted into the oocyte by a brief overcompensation of negative capacitance. A pressure pulse was applied using a PicoPump pressure injection system (WPI) to inject 0.5-5% of the egg volume, depending on what was being injected (Halet et al.,2002). The final concentrations or amounts of a given molecule that were injected were: fura 2-dextran, 2-4 μM; cyclin B1-GFP (provided by Jonathon Pines), 20-40 pg; wheat germ agglutinin (WGA), 200-500 μg/ml;fluorescein isothiocyanate-labelled bovine serum albumin (BSA) tagged with a nuclear localising signal (FITC-NLS-BSA; provided by Mark Jackman), 20 μM;and importin β^45-462 (provided by Dirk Gorlich), 5-10 μM. To minimize possible risks of performing multiple injections, a number of reagents were co-injected, including, fura 2-dextran and FITC-NLS-BSA, or fura 2-dextran and importin β^45-462. In experiments that aimed to determine the timing of NEBD relative to the Ca²⁺ transient, one of the pronuclei was injected with FITC-dextran (77 kDa; Sigma). After microinjection, the oocytes were removed to the hot block in fresh H-KSOM and allowed to recover for at least 10 minutes.

To measure intracellular Ca²⁺ while monitoring pronucleus formation, oocytes were loaded with fura 2 using a 10 minute incubation with 0.2-0.5 μM fura 2 AM at 37°C or were microinjected with fura 2-dextran as described above. For monitoring mitotic Ca²⁺ transients and NEBD, embryos were loaded with fura red using a 10 minute incubation in 4μM fura red AM for 10 minutes at 37°C. Ca²⁺ transients during cytokinesis were monitored after microinjection of one-cell embryos that had undergone NEBD with fura 2-dextran as described above.

For fertilization experiments, the zona pellucida was removed by a brief treatment with acidified Tyrode's medium. The zona-free oocytes were placed in a heated chamber with a coverglass base containing 0.5 ml H-KSOM without BSA. After 3-5 minutes, 0.5 ml complete H-KSOM was added to the chamber and the media was then covered with oil to prevent evaporation. Capacitated sperm were added to the chamber as described above. For monitoring NEBD, using NLS-FITC-dextran, and associated Ca²⁺ transients, the injected embryos were loaded with fura-red as described above and placed in a 20 μl drop of H-KSOM under oil in the heated chamber on the microscope stage.

Illumination of the indicator-loaded oocytes was performed using a monochromator (Till, Germany) to provide appropriate excitation wavelengths:340 nm and 380 nm for fura 2; 440 nm and 490 nm for fura red; and 490 nm for FITC-dextran. In some experiments, we monitored fura 2-dextran and FITC-NLS-BSA simultaneously, taking advantage of the broad excitation spectrum of FITC such that the FITC emission could be readily observed using the fura 2 excitation wavelengths. A 510 nm dichroic mirror was used for all experiments and the required wavelengths were collected using a 520 nm long-pass filter for fura 2, a 600 nm or 665 nm long pass filter for fura red and a 520 nm band pass for FITC-dextran. To monitor fura red and fluorescein dextran in the same embryo, the emission filters were situated in a filter wheel sited in front of the camera. The emitted light from the fluorochromes was collected using a cooled CCD Camera (MicroMax, Princeton Instruments). The monochromator, the emission filter wheel and the CCD camera were controlled using MetaFluor software (Universal Imaging).

Histone H1 and myelin basic protein (MBP) kinase assays were performed in order to measure MPF and MAP-kinase activity respectively. The MBP assay has been shown previously to correlate with MAP-kinase activity in mouse oocytes as determined by more specific gel- or immunoprecipitation-based assays(Verlhac et al., 1994). The protocol was similar to that described elsewhere(Moos et al., 1995;Kubiak et al., 1993). Five eggs (unless stated otherwise) in 2 μl of H-KSOM were transferred in 3μl of storing solution (10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 mM p-nitrophenyl phosphate, 20 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 5 mM EGTA) and immediately frozen on dry ice. After three thaw-freeze cycles, the samples were diluted twice by the addition of 2×kinase buffer containing 60 μg/ml leupeptin, 60 μg/ml aprotinin, 24 mM p-nitrophenyl phosphate, 90 mM β-glycerophosphate, 4.6 mM sodium orthovanadate, 24 mM EGTA, 24 mM MgCl₂, 0.2 mM EDTA, 4 mM NaF, 1.6 mM dithiothreitol, 2 mg/ml polyvinyl alcohol, 40 mM MOPS, 0.6 mM ATP, 2 mg/ml histone H1 (HIII-S from calf thymus, Sigma), 0.5 mg/ml MBP (from guinea pig brain, Sigma) and 0.25 mCi/ml [³²P]ATP. The samples were then incubated at 30°C for 30 minutes. The reaction was stopped by the addition of 2× SDS-sample buffer (0.125 M Tris-HCl, 4% SDS, 20% glycerol, 10%mercaptoethanol, 0.002% Bromophenol Blue) and boiled for 3-5 minutes. The samples were then analysed with SDS-PAGE followed by autoradiography. The autoradiographs were imaged using the Fuji Bas-1000 phosphorimager system and analysed with TINA 2.0 software.

The cessation of Ca²⁺ transients in relation to pronucleus formation has been assessed using two techniques. For both, Ca²⁺was monitored using fura 2-dextran and different approaches were used to monitor pronucleus formation. In the first approach, while recording Ca²⁺, we viewed the fertilizing eggs using bright-field optics every 10-15 minutes. The first sign of pronucleus formation was taken as a small spot in the cytoplasm, which is evident some 20-30 minutes before the pronuclei become marked by an obvious nuclear-cytoplasmic border(Fig. 1A,Bi,C). The second approach used a fluorescent nuclear-targeted marker, FITC-NLS-BSA, that accumulates in the developing pronuclei, thereby allowing simultaneous imaging of Ca²⁺ and pronucleus formation(Fig. 1Bii,C).

The two techniques generated similar results, although the FITC-NLS-BSA reported pronucleus formation ∼20 minutes earlier than bright-field microscopy (see Fig. 1C). Ca²⁺ oscillations ceased in a window of time either side of pronucleus formation such that 50% (FITC-NLS-BSA) and 55% (bright field) of fertilized eggs stopped generating Ca²⁺ transients 15 minutes either side of pronucleus formation. Such a window is not surprising given that the mean interspike interval between the last two Ca²⁺oscillations is 29±7 minutes, and both techniques report that ∼80%of eggs stop generating Ca²⁺ transients within 30 minutes of pronucleus formation (Fig. 1B). In these conditions only 2 out of 38 eggs (both techniques) stopped generating Ca²⁺ transients more than 30 minutes before pronuclei formed. Of those that continue to generate Ca²⁺ oscillations, 4 out of 18 eggs(FITC-NLS-BSA) generated two transients after evidence of pronucleus formation and one egg generated three transients.

Consistent with previous studies, we find that Cdk1-cyclin B activity declines about the time of second polar body extrusion, whereas MAP-kinase activity decreases around the time of pronucleus formation (see insetFig. 1A). The close association between pronucleus formation and the cessation of Ca²⁺ transients suggests a correlation with MAP-kinase activity rather than Cdk1-cyclin B.

To test the hypothesis that MAP-kinase activity is responsible for maintaining Ca²⁺ transients at fertilization, we have used the well characterized MAP-kinase inhibitor UO126(Duncia et al., 1998;Gross et al., 2000;Favata et al., 1998;Gross et al., 2000). We first measured MAP kinase and Cdk1-cyclin B activity in eggs incubated in 50 μM UO126 for 1 hour. Incubation in UO126 inhibited MAP-kinase activity levels to 10-15% of control levels with little effect on Cdk1-cyclin B(Fig. 2A). Continued treatment with UO126 during fertilization inhibited MAP-kinase activity throughout the 6 hours up to pronucleus formation (Fig. 2A). Cdk1-cyclin B activity decreased after polar body extrusion in a manner similar to controls (Fig. 1C).

The inhibition of MAP kinase in UO126-incubated eggs provided a system with which to investigate the role of MAP kinase in the generation of Ca²⁺ transients at fertilization. Measurement of Ca²⁺ at fertilization of UO126-treated eggs revealed that Ca²⁺ oscillations were initiated in the same way as in control oocytes and that they continued for several hours (Fig. 2B). Correlating the time that the oscillations stopped with the time of pronucleus formation revealed a pattern similar to controls (seeFig. 1Bi), with Ca²⁺oscillations stopping in 55% of eggs 15 minutes either side of pronucleus formation and within 30 minutes in around 74% of eggs (n=38)(Fig. 2C;Table 1). Thus, MAP-kinase activity is not required for the generation of Ca²⁺ oscillations at fertilization.

In the experiments described above, no correlation between the generation of Ca²⁺ transients and the activity of Cdk1-cyclin B or MAP kinase has been established. This is surprising given the observation that maintenance of meiotic arrest (and the kinase activities) using nocodazole leads to long-lasting Ca²⁺ oscillations(Jones et al., 1995). To ensure this is a specific effect of nocodazole on the maintenance of the activity of the M-phase kinases, we microinjected a cyclin B1-GFP fusion protein (Clute and Pines, 1999)to maintain arrest at MII by another method. Eggs injected with cyclin B1-GFP and then fertilized did not extrude polar bodies or form pronuclei (as indicated in the schematic diagrams in Fig. 3A), indicating that the exogenous cyclin B1 leads to the maintenance of Cdk1-cyclin B activity at a level that prevents egg activation. To determine if maintenance of meiotic arrest using cyclin extends Ca²⁺ oscillations in a manner similar to nocodazole,Ca²⁺ was recorded in cyclin-injected eggs. These studies revealed that Ca²⁺ oscillations carried on for a mean time of 9.5±1.5 hours (n=15), compared with 4.2±0.5 hours in controls(mean±s.d.; n=18; P<0.001)(Fig. 3). This shows that maintenance of M-phase arrest using exogenous cyclin B has a similar effect to nocodazole and supports the generation of sperm-induced Ca²⁺transients (Table 1). Thus,maintenance of high Cdk1-cyclin B activity appears to be sufficient but, as seen at fertilization, not necessary for the generation of Ca²⁺transients at fertilization.

Cdk1-cyclin B activity is maintained in MII-arrested mouse eggs by cyclin synthesis (Kubiak et al.,1993) and by a brake on cyclin destruction by the mos/MAP kinase pathway (Colledge et al., 1994;Hashimoto et al., 1994). After polar body extrusion, persistent cyclin synthesis in the presence of MAP kinase may provide a mechanism of maintaining levels of Cdk1-cyclin B activity sufficient to promote Ca²⁺ oscillations. We have investigated this possibility by inhibiting cyclin synthesis using the protein synthesis inhibitor cycloheximide (CHX) (Moos et al., 1996; Moses and Kline,1995). Preliminary experiments demonstrated the effectiveness of this approach, as 80% of aged MII eggs incubated in CHX underwent parthenogenetic activation (data not shown). The addition of CHX to eggs that had extruded the second polar body had no marked effect on the correlation of Ca²⁺ transients and pronucleus formation. In 47% of eggs,Ca²⁺ transients stopped 15 minutes either side of pronucleus formation, while 82% of eggs stopped within 30 minutes(Fig. 4A;Table 1).

We investigated further the possibility that low levels of Cdk1-cyclin B may be present during the period after polar body extrusion when MAP kinase remains. Cdk1-cyclin B activity was measured in larger samples of 50 eggs/assay. This number was chosen to account for the possibility that the Cdk1 activity may be oscillating with a peak just prior to the generation of the Ca²⁺ transient. Assuming a random distribution of the generation of a Ca²⁺ transient during a 10 minute period (the mean interspike interval), on average 10 out of the 50 eggs will be within 2 minutes of generating a Ca²⁺ transient. We can detect Cdk1-cyclin B activity (as measured by H1 kinase activity) in one or two eggs, suggesting that even if the activity was significantly lower in only a few of the eggs,we would detect the activity. These experiments showed that the level of Cdk1 activity was similar in the period after polar body extrusion but before pronucleus formation, when MAP kinase remains high, and after pronucleus formation, when MAP kinase activity is low(Fig. 4B). These data suggest that, at least within the sensitivity of the available assays, persistent or oscillating Cdk1-cyclin B activity is unlikely to explain the generation of Ca²⁺ oscillations after polar body extrusion.

Our experiments using cyclin B have shown that maintenance of M-phase kinase activity is sufficient to maintain Ca²⁺ oscillations but, in conditions where the kinase activity is inhibited, we have found no consistent correlation between the activities of the M-phase kinases and the generation of Ca²⁺ transients. More consistent is the observation that, in a variety of experimental manipulations, the cessation of Ca²⁺oscillations takes place within a window of time either side of pronucleus formation. To investigate a role for pronucleus formation itself, a protocol that inhibits pronucleus formation without influencing the normal pattern of inactivation of Cdk1-cyclin B and MAP kinase was devised. WGA binds with high affinity to nucleoporins, which leads to the inhibition of pronucleus formation in bovine oocytes (Sutovsky et al., 1998). We confirmed that injection of WGA inhibits pronucleus formation after fertilization of mouse oocytes(Fig. 5A). Importantly,WGA-mediated inhibition of pronucleus formation did not affect the timecourse of inactivation of Cdk1-cyclin B and MAP kinase(Fig. 5B). This provides an experimental system with which to examine the role of pronucleus formation in the cessation of Ca²⁺ oscillations, independent of the M-phase kinases. Monitoring Ca²⁺ in WGA-injected eggs in which pronucleus formation was inhibited revealed that Ca²⁺ oscillations were generated for an average of 9.9±2.5 hours (n=16) compared with 4±0.5 hours in controls (mean±s.d.; n=13;P<0.01; Fig. 5C,D). These data suggest that the formation of pronuclei play a direct role in the cessation of Ca²⁺ oscillations at fertilization.

To test the hypothesis that nuclear transport was leading to the sequestration of factor(s) required for the generation of Ca²⁺oscillations we microinjected importin β^45-462(Kutay et al., 1997). This mutant of importin β acts as a dominant-negative inhibitor of nuclear transport because of its ability to bind the nuclear pore complex but not importin α (Kutay et al.,1997). Fertilization of eggs injected with importinβ ^45-462 led to the formation of only rudimentary pronuclei,suggesting a requirement for importin β-mediated nuclear transport for full development of pronuclei (data not shown). In the presence of importinβ ^45-462, Ca²⁺ transients continued for a mean duration of 12.5±3.5 hours (n=19), compared with 3.8±0.3 (n=12) for controls (P<0.01;Fig. 6). These data are consistent with the hypothesis that factors promoting Ca²⁺ release are transported to the developing pronuclei.

A role for pronucleus formation and nuclear transport in the cessation of Ca²⁺ oscillations raises the possibility that Ca²⁺oscillations may be generated when the pronuclear membranes break down at NEBD of the first mitotic division. This is consistent with the finding that fertilized embryos generate Ca²⁺ transients during mitosis(Kono et al., 1996;Day et al., 2000). The precise relationship between the Ca²⁺ transient associated with NEBD and NEBD itself has not been determined and, as such, it is unclear whether Ca²⁺ drives NEBD or whether NEBD leads to the Ca²⁺transients. To address this question, we have carefully investigated the temporal relationship of NEBD and the associated Ca²⁺ transient. The permeability of the pronuclear membrane to large molecular weight molecules was determined by monitoring fluorescence of a 77 kDa FITC-dextran that had been injected into one of the pronuclei. Ca²⁺ was recorded in the same embryos using fura red, thereby allowing simultaneous recording of NEBD and intracellular Ca²⁺.Fig. 7 shows that the first indication of NEBD, as indicated by a loss of fluorescence from the pronucleus, precedes the peak of the Ca²⁺ transient by 9.1±1.4 minutes (n=24). Thus, the temporal sequence of events suggests that the global Ca²⁺ transient at NEBD is a result of NEBD, rather than its cause.

If Ca²⁺ transients generated during the first mitotic division are regulated similarly to those at fertilization, they should cease close to the time of nucleus formation in the two cell embryo. To address this question, we injected mitotic one-cell embryos with fura 2-dextran to monitor Ca²⁺, and FITC-NLS-BSA, to monitor the formation of the nuclei in the two-cell embryo. Ca²⁺ transients were detected in 13 out of 14 mitotic embryos (Fig. 8). The Ca²⁺ transients were apparent during cytokinesis, as defined by evidence of a cleavage furrow, with 10 out of the 13 embryos generating a transient within 20 minutes of the first evidence of nucleus formation. In one embryo, a single Ca²⁺ transient was detected about 1 minute after the nuclei had formed in the two-cell embryo. The relationship between the presence of a nucleus and the cessation of Ca²⁺ transients is strictly maintained during the first mitotic division.

In mammals, the main evidence for a role for the M-phase kinases in regulating Ca²⁺ release is that maintaining kinase activity with microtubule inhibitors (Jones et al.,1995; Day et al.,2000) or cyclin B1 (present study) leads to long-lasting Ca²⁺ transients. However, these treatments also inhibit pronucleus formation. We have used two approaches to test the relative roles of the M-phase kinases and pronuclei in the regulation of sperm-induced Ca²⁺ signalling: first, we have inhibited pronucleus formation independently of the M-phase kinases; and second, we have measured and manipulated the M-phase kinases in order to correlate activity with the generation of Ca²⁺ oscillations.

A number of observations presented here show that the temporal pattern of Ca²⁺ signalling at fertilization is regulated by pronucleus formation. First, Ca²⁺ oscillations stopped in ∼80% of eggs within 30 minutes of pronucleus formation. The window over which pronucleus formation takes place in relation to when Ca²⁺ transients stop is,in part, a reflection of the fact that the mean interspike interval between the last two Ca²⁺ transients is nearly 30 minutes. In addition, the presence of an abortive spike after the last transient suggests that decrease in Ca²⁺-releasing activity appears not to be a switch-like mechanism, but rather a gradual decrease in activity. This, in combination with the regenerative process of Ca²⁺ signalling, will provide for a variability in when the process is dulled sufficiently to stop the generation of Ca²⁺ transients. Additional factors that influence the sensitivity of Ca²⁺ release, such as Ins(1,4,5)P₃ receptor degradation(Brind et al., 2000;Jellerette et al., 2000) and oocyte quality (Cheung et al.,2000), may also influence the precise timing that Ca²⁺oscillations stop. The limitation with these correlative data is that, like the experiments with microtubule inhibitors(Jones et al., 1995;Day et al., 2000) and cyclin B(present study), it is not possible to distinguish between a role for the interdependent activities of the M-phase kinases and pronucleus formation.

We used two independent techniques to elucidate the relative roles of M-phase kinases and pronucleus formation in the pattern of Ca²⁺signalling at fertilization. First, we used the finding that sequestration of nuclear pore complexes with WGA inhibits pronucleus formation(Sutovsky et al., 1998). We extended these findings and have shown that, when pronucleus formation is inhibited, the activities of Cdk1-cyclin B and MAP kinase decrease in the normal time course. Second, we used a dominant-negative importin β to inhibit nuclear transport. The ability of Ca²⁺ oscillations to persist when Cdk1-cyclin B and MAP-kinase activities are low suggests that the formation of the pronuclei and nuclear transport, rather than the activity of the cell-cycle kinases, is the main player in determining when the Ca²⁺ oscillations stop at fertilization.

Previously, using a different approach, a relationship between pronucleus formation and Ca²⁺ transients was not seen in bisected one-cell embryos. It was found that nucleate and anucleate halves stopped oscillating at about the same time after fertilization(Day et al., 2000). This discrepancy may be a result of perturbations in intracellular Ca²⁺that no doubt occur during embryo bisection, which, together with other mechanisms [such as ER reorganisation(FitzHarris et al., 2003),Ins(1,4,5)P₃ receptor downregulation(Jellerette et al., 2000;Brind et al., 2000) and the decrease in sensitivity of Ins(1,4,5)P₃-induced Ca²⁺ release (Jones and Whittingham, 1996; Brind et al., 2000; FitzHarris et al.,2003)], leads to the premature cessation of Ca²⁺oscillations in the absence of pronucleus formation. Furthermore, as nuclear membranes are continuous with the ER and mitochondria localize around the developing pronuclei (Bavister and Squirrell, 2000), it is possible that the anucleate fragment may receive a diminutive share of the organelles important for the regulation of intracellular Ca²⁺. Thus, the non-invasive approaches to inhibiting pronucleus formation used in the present study may have been an important factor in establishing a relationship between the cessation of Ca²⁺oscillations and pronucleus formation.

The experiments described above indicate a role for pronucleus formation and nuclear sequestration, rather than the M-phase kinases per se, in the temporal organization of Ca²⁺ signalling at fertilization. This conclusion is further supported by the finding that inhibition of MAP kinase using the well-characterized MEK inhibitor UO126 had no effect on sperm-induced Ca²⁺ transients. Similar observations have been reported in ascidian eggs where there is no obvious correlation between MAP-kinase activity and sperm-induced Ca²⁺ oscillations(Levasseur and McDougall,2000; McDougall and Levasseur,1998). The other M-phase kinase, Cdk1-cyclin B, is inactivated at the time of polar body extrusion and, as such, does not correlate with the timing of sperm-induced Ca²⁺ transients in mammals(Verlhac et al., 1994;Moos et al., 1995;Schultz and Kopf, 1995)(present study). Low or oscillating levels of Cdk1-cyclin B activity cannot be completely discounted (Levasseur and McDougall, 2000; Nixon et al.,2000; Carroll,2001), particularly in light of the recent observations that MPF activation and inactivation may be regulated locally(Beckhelling et al., 2000;Perez-Mongiovi et al., 2000;Huang and Raff, 1999;Groisman et al., 2000). We have attempted to account for these possibilities. First, we were unable to measure any Cdk1-cyclin B activity using whole-cell assays but, despite using large sample sizes, it remains possible that local activity of Cdk1-cyclin B was not detected. Second, the data obtained using CHX and MAP kinase suggest that Cdk1-cyclin B activity would have to be sustained in the absence of two processes known to maintain its activity: cyclin synthesis and the stabilizing influence of MAP kinase (Kubiak et al.,1993; Winston,1997; Maller et al.,2002). Thus, at least in mammals, it appears that the main role for the M-phase kinases in the regulation of Ca²⁺ release at fertilization is to determine the time of pronucleus formation.

This lack of correlation between the activity of Cdk1-cyclin B and sperm-induced Ca²⁺ oscillations in mammals contrasts with the situation in ascidians. In ascidian oocytes, sperm-induced Ca²⁺oscillations stop between MI and MII, where there is no nuclear membrane but there is a transient decrease in Cdk1-cyclin B activity(McDougall and Levasseur,1998). The explanation for this difference may lie in species differences in the mechanisms of action of the paternally derived Ca²⁺-releasing sperm factor(s) or in differences in how it is regulated in meiosis and mitosis. In ascidians, the cessation of Ca²⁺ transients between MI and MII takes place in the absence of any change in the sensitivity of Ins(1,4,5)P₃-induced Ca²⁺ release, suggesting that Cdk1-cyclin B activity is required to support Ins(1,4,5)P₃ production(McDougall and Levasseur,1998). By contrast, after fertilization, the loss of Cdk1-cyclin B activity is associated with a desensitization of Ins(1,4,5)P₃-induced Ca²⁺ release(Levasseur and McDougall,2000). In mammals, a similar desensitization of Ca²⁺release is seen in pronucleate stage embryos(Jones and Whittingham, 1996;Brind et al., 2000). This has been attributed to the effects of oocyte aging(Jones and Whittingham, 1996);however, it also indicates that M-phase kinases can influence the sensitivity of Ins(1,4,5)P₃-induced Ca²⁺ signalling as we have shown recently (FitzHarris et al.,2003). Nevertheless, the ability of oocytes to undergo Ca²⁺ oscillations when the activity of the M-phase kinases is low,shows that any sensitization afforded by the kinases is not necessary for the maintenance of the oscillations. Taken together, these observations suggest that, in mammals, the cessation of Ca²⁺ transients is primarily governed by the formation of the pronuclei and nuclear transport, but additional factors, including a cell-cycle-dependent change in the sensitivity of Ins(1,4,5)P₃-induced Ca²⁺ release(FitzHarris et al., 2003) and Ins(1,4,5)P₃ receptor downregulation(Brind et al., 2000;Jellerette et al., 2000), act concomitantly to decrease the sensitivity of Ca²⁺ release after fertilization.

The role of pronucleus formation in stopping the fertilization-induced Ca²⁺ oscillations provides a tempting lead to suggest that NEBD gives rise to the mitotic Ca²⁺ transients. In previous studies, the relationship between NEBD and the Ca²⁺ transient has been disputed,with some studies suggesting that morphological changes in the nucleus precede the Ca²⁺ transient (Kono et al., 1996) while others suggest that the Ca²⁺ transient precedes NEBD (Tombes et al.,1992; Kono et al.,1996; Day et al.,2000). The use of a 77 kDa fluorescent dextran to monitor nuclear permeability, while simultaneously measuring Ca²⁺ transients,unequivocally demonstrated that the pronuclear membrane was permeable prior to the generation of the global Ca²⁺ transient. This temporal relationship between NEBD and the Ca²⁺ transient suggests that it is NEBD that leads to the generation of Ca²⁺ transients rather than the Ca²⁺ transient driving NEBD.

At the completion of mitosis, our data demonstrate that Ca²⁺oscillations are not detected after the reformation of the pronuclei in the newly formed two-cell embryo. In addition, inhibition of NEBD prevents Ca²⁺ oscillations being generated at mitosis, whereas maintenance of mitotic arrest has been shown to lead to persistent oscillations(Day et al., 2000). These observations suggest that the relationship between the presence of a nucleus and the ability to generate Ca²⁺ oscillations is maintained during the first mitotic division.

The inhibition of Ca²⁺ transients at pronucleus formation and their return after NEBD, together with the association of Ca²⁺-releasing activity with the pronuclei described previously(Kono et al., 1996;Kono et al., 1995;Zernicka-Goetz et al., 1995),suggest a model for the regulation of Ca²⁺ signalling at fertilization in mammals. In this model(Fig. 9)Ca²⁺-releasing factors introduced at fertilization are sequestered to the developing pronuclei, where they are unable to generate Ca²⁺oscillations. Later, at NEBD, the Ca²⁺-releasing activity is released back into the cytosol where Ca²⁺ oscillations can again be generated until the factor is sequestered by the nuclei in the newly formed two-cell embryo.

Recently, a sperm-specific phospholipase C ζ (PLCζ) has been proposed to be the factor in sperm responsible for generating Ca²⁺release at fertilization (Saunders et al.,2002; Cox et al.,2002). A number of PLC isoforms, PLCβ1 and PLCδ4, are localized to the nucleus in somatic cells(Faenza et al., 2000;Sun et al., 1997;Liu et al., 1996) and also in oocytes (Avazeri et al., 2000). This model is consistent with our findings and may also explain a number of previous observations, including changes in cell-cycle dependent Ca²⁺ release (Kono et al.,1996), the finding that inhibition of pronucleus formation with colcemid or nocodazole maintains Ca²⁺ oscillations(Jones et al., 1995;Day et al., 2000), generation of single transients after fertilization in species where the oocytes are arrested in interphase (Stricker,1999), and induction of Ca²⁺ oscillations by mammalian sperm extracts after NEBD but not before(Tang et al., 2000).

This model, which suggests nuclear compartmentalization of a paternal Ca²⁺-releasing activity (possibly PLCζ), is the most reasonable interpretation of our data. However, we cannot discount the possibility that co-factors or substrates of the Ca²⁺-releasing activity are sequestered and released from nuclei such that they can only activate, or be used by, the Ca²⁺-releasing activity in an M-phase state. Irrespective of whether it is the Ca²⁺-releasing activity,co-factors or substrates that are sequestered and released from nuclei, this nuclear compartmentalization-mediated regulation of Ca²⁺ release represents a new mechanism for regulating Ca²⁺ oscillations in cells. In particular, it may prove important in stimulating mitotic Ca²⁺ transients in other systems that could play a role in the coordination of the complex series of events that take place during mitosis(Groigno and Whitaker, 1998;Georgi et al., 2002).

The authors thank all those who contributed reagents for the purposes of this work: Dirk Gorlich (University of Heidelberg) for the importinβ ^45-462, Mark Jackman (Wellcome/CRC, UK) for fluorescein-labelled NLS-BSA and Jon Pines (Wellcome/CRC, UK) for cyclin B1-GFP. Thanks to the anonymous reviewer who suggested the use of importinβ ^45-462. For stimulating discussions and critical reading of the manuscript, we thank Drs Mark Larman and Karl Swann. G. F. is supported by a Reproductive Medicine Studentship from the Department of Obstetrics and Gynaecology and the Assisted Conception Unit at UCL. We thank Charles Rodeck and Paul Serhal for their support. This work is supported by an MRC Career Establishment Grant to J. C.