Department of Physiology, UCL, Gower Street, London WC1E 6BT, UK
* Author for correspondence (e-mail: j.carroll{at}ucl.ac.uk)
Accepted 10 December 2002
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SUMMARY |
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Key words: Ca2+ signalling, Fertilization, Pronucleus, Mouse oocytes, Meiosis, Mitosis
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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
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 ß45-462 (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 ß45-462. 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.
Imaging
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 4
µM 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).
Kinase assays
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 3
µl of storing solution (10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 mM
p-nitrophenyl phosphate, 20 mM ß-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 2x
kinase buffer containing 60 µg/ml leupeptin, 60 µg/ml aprotinin, 24 mM
p-nitrophenyl phosphate, 90 mM ß-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 2x 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.
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RESULTS |
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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 inset Fig. 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.
Inhibition of MAP-kinase activity has no effect on Ca2+
oscillations
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).
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Injection of cyclin B1-GFP can lead to persistent Ca2+
oscillations
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.
|
Inhibition of protein synthesis does not affect the generation of
Ca2+ transients
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.
Inhibition of pronucleus formation leads to persistent
Ca2+ oscillations
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.
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Inhibition of importin ß-mediated nuclear transport leads to
prolonged generation of Ca2+ transients
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 ß45-462
(Kutay et al., 1997). This
mutant of importin ß acts as a dominant-negative inhibitor of nuclear
transport because of its ability to bind the nuclear pore complex but not
importin
(Kutay et al.,
1997
). Fertilization of eggs injected with importin
ß45-462 led to the formation of only rudimentary pronuclei,
suggesting a requirement for importin ß-mediated nuclear transport for
full development of pronuclei (data not shown). In the presence of importin
ß45-462, 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.
|
Ca2+ transients at the first mitosis follow NEBD
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.
|
Ca2+ transients are not detected after the formation of
nuclei in two-cell embryos
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.
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DISCUSSION |
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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 ß 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.
Ca2+ transients at mitosis: a role for the nucleus
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.
A nuclear compartmentalization model for the regulation of
Ca2+ signalling in early development
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 (PLC
) 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, PLCß1 and PLC
4, 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 PLC), 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
).
![]() |
ACKNOWLEDGMENTS |
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REFERENCES |
---|
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---|
Avazeri, N., Courtot, A. M., Pesty, A., Duquenne, C. and
Lefevre, B. (2000). Cytoplasmic and nuclear phospholipase
C-beta 1 relocation: role in resumption of meiosis in the mouse oocyte.
Mol. Biol. Cell 11,4369
-4380.
Bavister, B. D. and Squirrell, J. M. (2000).
Mitochondrial distribution and function in oocytes and early embryos.
Hum. Reprod. 15 Suppl.
2, 189-198.
Beckhelling, C., Perez-Mongiovi, D. and Houliston, E. (2000). Localised MPF regulation in eggs. Biol. Cell 92,245 -253.[CrossRef][Medline]
Brind, S., Swann, K. and Carroll, J. (2000). Inositol 1,4,5-trisphosphate receptors are downregulated in mouse oocytes in response to sperm or adenophostin A but not to increases in intracellular Ca(2+) or egg activation. Dev. Biol. 223,251 -265.[CrossRef][Medline]
Carroll, J. (2001). The initiation and regulation of Ca2+ signalling at fertilization in mammals. Semin. Cell Dev. Biol. 12, 37-43.[CrossRef][Medline]
Cheung, A., Swann, K. and Carroll, J. (2000).
The ability to generate normal Ca(2+) transients in response to spermatozoa
develops during the final stages of oocyte growth and maturation.
Hum. Reprod. 15,1389
-1395.
Ciapa, B., Pesando, D., Wilding, M. and Whitaker, M. (1994). Cell-cycle calcium transients driven by cyclic changes in inositol trisphosphate levels. Nature 368,875 -878.[CrossRef][Medline]
Clute, P. and Pines, J. (1999). Temporal and spatial control of cyclin B1 destruction in metaphase. Nat. Cell Biol. 1,82 -87.[CrossRef][Medline]
Colledge, W. H., Carlton, M. B., Udy, G. B. and Evans, M. J. (1994). Disruption of c-mos causes parthenogenetic development of unfertilized mouse eggs. Nature 370, 65-68.[CrossRef][Medline]
Cox, L., Larman, M. G., Saunders, C. M., Hashimoto, K., Swann,
K. and Lai, F. A. (2002). Sperm phospholipase C from
humans and cynomolgus monkeys triggers Ca2+ oscillations,
activation and development of mouse oocytes.
Reproduction 124,611
-623.
Day, M. L., McGuinness, O. M., Berridge, M. J. and Johnson, M. H. (2000). Regulation of fertilization-induced Ca(2+)spiking in the mouse zygote. Cell Calcium 28, 47-54.[CrossRef][Medline]
Deng, M. Q. and Shen, S. S. (2000). A specific
inhibitor of p34(cdc2)/cyclin B suppresses fertilization-induced calcium
oscillations in mouse eggs. Biol. Reprod.
62,873
-878.
Duncia, J. V., Santella, J. B., III, Higley, C. A., Pitts, W. J., Wityak, J., Frietze, W. E., Rankin, F. W., Sun, J. H., Earl, R. A., Tabaka, A. C. et al. (1998). MEK inhibitors: the chemistry and biological activity of U0126, its analogs, and cyclization products. Bioorg. Med. Chem. Lett. 8,2839 -2844.[CrossRef][Medline]
Faenza, I., Matteucci, A., Manzoli, L., Billi, A. M., Aluigi,
M., Peruzzi, D., Vitale, M., Castorina, S., Suh, P. G. and Cocco, L.
(2000). A role for nuclear phospholipase C beta 1 in cell cycle
control. J. Biol. Chem.
275,30520
-30524.
Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J.,
Stradley, D. A., Feeser, W. S., van Dyk, D. E., Pitts, W. J., Earl, R. A.,
Hobbs, F. et al. (1998). Identification of a novel inhibitor
of mitogen-activated protein kinase kinase. J. Biol.
Chem. 273,18623
-18632.
FitzHarris, D. G., Marangos, P. and Carroll, J. (2003). Cell cycle-dependent regulation of the structure of the endoplasmic reticulum and InsP3-induced Ca2+ release in mouse oocytes and embryos. Mol. Biol. Cell (in press).
Georgi, A. B., Stukenberg, P. T. and Kirschner, M. W. (2002). Timing of events in mitosis. Curr. Biol. 12,105 -114.[CrossRef][Medline]
Groigno, L. and Whitaker, M. (1998). An anaphase calcium signal controls chromosome disjunction in early sea urchin embryos. Cell 92,193 -204.[Medline]
Groisman, I., Huang, Y. S., Mendez, R., Cao, Q., Theurkauf, W. and Richter, J. D. (2000). CPEB, maskin, and cyclin B1 mRNA at the mitotic apparatus: implications for local translational control of cell division. Cell 103,435 -447.[Medline]
Gross, S. D., Schwab, M. S., Taieb, F. E., Lewellyn, A. L., Qian, Y. W. and Maller, J. L. (2000). The critical role of the MAP kinase pathway in meiosis II in Xenopus oocytes is mediated by p90(Rsk). Curr. Biol. 10,430 -438.[CrossRef][Medline]
Halet, G., Tunwell, R., Balla, T., Swann, K. and Carroll, J.
(2002). The dynamics of plasma membrane PtdIns(4,5)P(2) at
fertilization of mouse eggs. J. Cell Sci.
115,2139
-2149.
Hashimoto, N., Watanabe, N., Furuta, Y., Tamemoto, H., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. et al. (1994). Parthenogenetic activation of oocytes in c-mos-deficient mice. Nature 370,68 -71.[CrossRef][Medline]
Huang, J. and Raff, J. W. (1999). The
disappearance of cyclin B at the end of mitosis is regulated spatially in
Drosophila cells. EMBO J.
18,2184
-2195.
Jellerette, T., He, C. L., Wu, H., Parys, J. B. and Fissore, R. A. (2000). Down-regulation of the inositol 1,4,5-trisphosphate receptor in mouse eggs following fertilization or parthenogenetic activation. Dev. Biol. 223,238 -250.[CrossRef][Medline]
Jones, K. T. and Whittingham, D. G. (1996). A comparison of sperm- and IP3-induced Ca2+ release in activated and aging mouse oocytes. Dev. Biol. 178,229 -237.[CrossRef][Medline]
Jones, K. T., Carroll, J., Merriman, J. A., Whittingham, D. G.
and Kono, T. (1995). Repetitive sperm-induced Ca2+
transients in mouse oocytes are cell cycle dependent.
Development 121,3259
-3266.
Kline, D. and Kline, J. T. (1992). Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. Dev. Biol. 149, 80-89.[Medline]
Kono, T., Carroll, J., Swann, K. and Whittingham, D. G.
(1995). Nuclei from fertilized mouse embryos have
calcium-releasing activity. Development
121,1123
-1128.
Kono, T., Jones, K. T., Bos-Mikich, A., Whittingham, D. G. and Carroll, J. (1996). A cell cycle-associated change in Ca2+ releasing activity leads to the generation of Ca2+ transients in mouse embryos during the first mitotic division. J. Cell Biol. 132,915 -923.[Abstract]
Kubiak, J. Z., Weber, M., de Pennart, H., Winston, N. J. and Maro, B. (1993). The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF. EMBO J. 12,3773 -3778.[Abstract]
Kutay, U., Izaurralde, E., Bischoff, F. R., Mattaj, I. W. and
Gorlich, D. (1997). Dominant-negative mutants of
importin-beta block multiple pathways of import and export through the nuclear
pore complex. EMBO J.
16,1153
-1163.
Lawitts, J. A. and Biggers, J. D. (1993). Culture of preimplantation embryos. Methods Enzymol. 225,153 -164.[Medline]
Lawrence, Y., Ozil, J. P. and Swann, K. (1998). The effects of a Ca2+ chelator and heavy-metal-ion chelators upon Ca2+ oscillations and activation at fertilization in mouse eggs suggest a role for repetitive Ca2+ increases. Biochem. J. 335,335 -342.[Medline]
Levasseur, M. and McDougall, A. (2000).
Sperm-induced calcium oscillations at fertilisation in ascidians are
controlled by cyclin B1-dependent kinase activity.
Development 127,631
-641.
Liu, N., Fukami, K., Yu, H. and Takenawa, T.
(1996). A new phospholipase C delta 4 is induced at S-phase of
the cell cycle and appears in the nucleus. J. Biol.
Chem. 271,355
-360.
Maller, J. L., Schwab, M. S., Gross, S. D., Taieb, F. E., Roberts, B. T. and Tunquist, B. J. (2002). The mechanism of CSF arrest in vertebrate oocytes. Mol. Cell. Endocrinol. 187,173 -178.[CrossRef][Medline]
McDougall, A. and Levasseur, M. (1998).
Sperm-triggered calcium oscillations during meiosis in ascidian oocytes first
pause, restart, then stop: correlations with cell cycle kinase activity.
Development 125,4451
-4459.
Moos, J., Visconti, P. E., Moore, G. D., Schultz, R. M. and Kopf, G. S. (1995). Potential role of mitogen-activated protein kinase in pronuclear envelope assembly and disassembly following fertilization of mouse eggs. Biol. Reprod. 53,692 -699.[Abstract]
Moos, J., Kopf, G. S. and Schultz, R. M.
(1996). Cycloheximide-induced activation of mouse eggs: effects
on cdc2/cyclin B and MAP kinase activities. J. Cell
Sci. 109,739
-748.
Moses, R. M. and Kline, D. (1995). Release of mouse eggs from metaphase arrest by protein synthesis inhibition in the absence of a calcium signal or microtubule assembly. Mol. Reprod. Dev. 41,264 -273.[Medline]
Nixon, V. L., McDougall, A. and Jones, K. T. (2000). Ca2+ oscillations and the cell cycle at fertilisation of mammalian and ascidian eggs. Biol. Cell 92,187 -196.[CrossRef][Medline]
Nixon, V. L., Levasseur, M., McDougall, A. and Jones, K. T. (2002). Ca(2+) Oscillations Promote APC/C-Dependent Cyclin B1 Degradation during Metaphase Arrest and Completion of Meiosis in Fertilizing Mouse Eggs. Curr. Biol. 12,746 -750.[CrossRef][Medline]
Ozil, J. P. (1990). The parthenogenetic development of rabbit oocytes after repetitive pulsatile electrical stimulation. Development 109,117 -127.[Abstract]
Ozil, J. P. and Huneau, D. (2001). Activation
of rabbit oocytes: the impact of the Ca2+ signal regime on
development. Development
128,917
-928.
Perez-Mongiovi, D., Beckhelling, C., Chang, P., Ford, C. C. and
Houliston, E. (2000). Nuclei and microtubule asters stimulate
maturation/M phase promoting factor (MPF) activation in Xenopus eggs and egg
cytoplasmic extracts. J. Cell Biol.
150,963
-974.
Runft, L. L., Jaffe, L. A. and Mehlmann, L. M. (2002). Egg activation at fertilization: where it all begins. Dev. Biol. 245,237 -254.[CrossRef][Medline]
Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J.,
Royse, J., Blayney, L. M., Swann, K. and Lai, F. A. (2002).
PLCzeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo
development. Development
129,3533
-3544.
Schultz, R. M. and Kopf, G. S. (1995). Molecular basis of mammalian egg activation. Curr. Top. Dev. Biol. 30,21 -62.[Medline]
Steinhardt, R. A. (1990). Intracellular free calcium and the first cell cycle of the sea-urchin embryo (Lytechinus pictus). J. Reprod. Fertil. Suppl. 42,191 -197.[Medline]
Stricker, S. A. (1999). Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev. Biol. 211,157 -176.[CrossRef][Medline]
Summers, M. C., McGinnis, L. K., Lawitts, J. A., Raffin, M. and
Biggers, J. D. (2000). IVF of mouse ova in a simplex
optimized medium supplemented with amino acids. Hum.
Reprod. 15,1791
-1801.
Sun, B., Murray, N. R. and Fields, A. P.
(1997). A role for nuclear phosphatidylinositol-specific
phospholipase C in the G2/M phase transition. J. Biol.
Chem. 272,26313
-26317.
Sutovsky, P., Simerly, C., Hewitson, L. and Schatten, G.
(1998). Assembly of nuclear pore complexes and annulate lamellae
promotes normal pronuclear development in fertilized mammalian oocytes.
J. Cell Sci. 111,2841
-2854.
Swann, K. and Ozil, J. P. (1994). Dynamics of the calcium signal that triggers mammalian egg activation. Int. Rev. Cytol. 152,183 -222.[Medline]
Tang, T. S., Dong, J. B., Huang, X. Y. and Sun, F. Z.
(2000). Ca(2+) oscillations induced by a cytosolic sperm protein
factor are mediated by a maternal machinery that functions only once in
mammalian eggs. Development
127,1141
-1150.
Tombes, R. M., Simerly, C., Borisy, G. G. and Schatten, G. (1992). Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+ independent in the mouse oocyte. J. Cell Biol. 117,799 -811.[Abstract]
Verlhac, M. H., Kubiak, J. Z., Clarke, H. J. and Maro, B.
(1994). Microtubule and chromatin behavior follow MAP kinase
activity but not MPF activity during meiosis in mouse oocytes.
Development 120,1017
-1025.
Winston, N. J. (1997). Stability of cyclin B protein during meiotic maturation and the first mitotic cell division in mouse oocytes. Biol. Cell 89,211 -219.[CrossRef][Medline]
Zernicka-Goetz, M., Ciemerych, M. A., Kubiak, J. Z., Tarkowski,
A. K. and Maro, B. (1995). Cytostatic factor inactivation is
induced by a calcium-dependent mechanism present until the second cell cycle
in fertilized but not in parthenogenetically activated mouse eggs.
J. Cell Sci. 108,469
-474.