Department of Physiology, University College London, Gower Street, London, WC1E 6BT, UK
Author for correspondence (e-mail: j.carroll{at}ucl.ac.uk)
Accepted 5 July 2005
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Summary |
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Key words: Ca2+ transient, Mitosis, Inositol (1,4,5)-trisphosphate, Two-photon microscopy
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Introduction |
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The case for a role for Ca2+ in mitosis is strongest in early sea-urchin embryos. Ca2+ chelators such as EGTA and BAPTA prevent Ca2+ release and block NEBD (Steinhardt and Alderton, 1988; Twigg et al., 1988
). Conversely, NEBD can be induced by treatments that increase [Ca2+]i (Wilding et al., 1996
; Twigg et al., 1988
; Steinhardt and Alderton, 1988
). Similar findings have been made at the metaphase-anaphase transition, which can be reversibly inhibited by Ca2+ buffers or by inhibiting Ins(1,4,5)P3 receptors (InsP3Rs) (Groigno and Whitaker, 1998
). Inhibition of the Ca2+ signalling pathway by interfering with the activity of Ca2+/calmodulin-dependent protein kinase II results in similar phenotypes to Ca2+ buffers, implicating this kinase in transducing many of the effects of Ca2+ in mitosis (Baitinger et al., 1990
; Torok et al., 1998
). Thus, at least in sea-urchin embryos, there is a compelling body of evidence suggesting that Ca2+ is both necessary and sufficient for NEBD and anaphase onset (Whitaker and Larman, 2001
).
In other cell types, some observations suggest that Ca2+ might not be a universal signal for driving mitosis. Most notably, mitosis occurs in the apparent absence of Ca2+ transients in some cell lines (Tombes and Borisy, 1989; Whitaker and Patel, 1990
; Kao et al., 1990
; Whitaker and Larman, 2001
). Also, in cells that do generate mitotic Ca2+ oscillations, inhibition of the Ca2+ increases by intracellular Ca2+ buffers, removal of extracellular Ca2+ or removal of serum does not hinder cell division (Tombes and Borisy, 1989
; Tombes et al., 1990; Kao et al., 1990
).
One hypothesis which has been proposed to explain these apparent inconsistencies is that, in the absence of global Ca2+ transients, mitotic events are driven by spatially restricted Ca2+ microdomains that are inaccessible to conventional epifluorescence microscopy (see Hepler, 1994; Kao et al., 1990
; Whitaker and Patel, 1990
; Kono et al., 1996
). This idea was supported by early reports that [Ca2+] is elevated at spindle poles before anaphase in PTK and endosperm cells (Keith et al., 1985
; Ratan et al., 1986
). More recently, using confocal imaging and ratiometric Ca2+ indicators, localized Ca2+ increases were detected in the perinuclear region just before NEBD in sea-urchin embryos (Wilding et al., 1996
). Consistent with the ability to generate local Ca2+ signals is the observation that, shortly before NEBD, the endoplasmic reticulum aggregates around the nucleus (Terasaki, 2000
). The presence of a large source and sink of Ca2+ around the nucleus might provide a basis for the generation of local Ca2+ gradients. These studies have therefore raised the possibility that Ca2+ is a universal regulator of mitosis but that it can act in a highly spatially restricted manner.
One-cell mouse embryos provide a useful model with which to investigate the role and mechanism of generation of mitotic Ca2+ transients. Several studies have reported Ca2+ oscillations during mitosis (Tombes et al., 1992; Kono et al., 1996
; Day et al., 2000
; Tang et al., 2000
; Gordo et al., 2002
). In some cases, these oscillations have been reported to continue through mitosis, albeit at a low and variable frequency (Tombes et al., 1992
; Kono et al., 1996
; Day et al., 2000
; Marangos et al., 2004). Consistent with the results from sea-urchin embryos, AM loading with the Ca2+ buffer BAPTA inhibits NEBD (Tombes et al., 1990; Kono et al., 1996
). However, recent studies have shown that the NEBD precedes the first mitotic Ca2+ transient and that parthenogenetic embryos produced by activating oocytes using strontium proceed through mitosis in the absence of any detectable Ca2+ transients (Kono et al., 1996
; Marangos et al., 2003
). By analogy with the sea-urchin embryo, one possible explanation for this discrepancy is that progression through mitosis in parthenogenetic embryos is driven by localized Ca2+ elevations.
In light of these recent observations, we have readdressed the role of Ca2+ in mitosis in mouse embryos. We used multiple strategies to disrupt or impose Ca2+ transients in order to test the role of Ca2+ in progression through mitosis in mouse embryos. In addition, we have used two-photon microscopy in an attempt to reveal evidence for localized Ca2+ release in parthenogenetic embryos. Our data suggest that neither global nor local Ca2+ release is necessary for mitosis in early mouse embryos. However, Ca2+ is sufficient to accelerate mitosis, a function that appears to be conserved from sea-urchin to mammalian embryos.
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Materials and Methods |
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Microinjection
Embryos were pressure injected using a micropipette and Narishige manipulators mounted on a Leica DM IRB inverted microscope (Leica, Wetzlar, Germany). Embryos were placed in a drop of H-KSOM covered with mineral oil to prevent evaporation. Cells were immobilized using a holding pipette while the injection pipette was pushed through the zona pellucida until contact was made with the oocyte plasma membrane. A brief overcompensation of negative capacitance caused the pipette tip to penetrate the cell. Microinjection was performed using a fixed-pressure pulse through a pico-pump (WPI, Sarasota, FL, USA). Injection volumes were estimated at 5% of total cell volume by cytoplasmic displacement.
Whole-cell fluorescence microscopy and photolysis of `caged' Ins(1,4,5)P3
[Ca2+]i was monitored using Fura-2/dextran or Fura-red (Molecular Probes, Eugene, OR, USA). Fura-2/dextran (10 kDa) was microinjected to an estimated final concentration of 2-4 µM. Indicator-loaded embryos were placed in a drop of H-KSOM under oil in a chamber and placed on an Axiovert microscope fitted with a 20x air objective lens (Zeiss, Welwyn Garden City, UK). Illumination was performed using a monochromator (TILL Photonics, Gräfelfing, Germany) to select appropriate wavelengths. Fura-2 was illuminated at 340 nm and 380 nm, and emitted light was collected using a 510 nm dichroic mirror and a 520 nm band-pass filter. Fluorescence was detected using a cooled CCD camera (MicroMax, Princeton Instruments). The monochromator, filter wheel and camera were all controlled using Metafluor software (Universal Imaging, Downington, PA, USA), and background subtraction was performed online. Ca2+ measurements are presented as the ratio of emission in response to illumination at 340 nm and 380 nm. To monitor the presence of nuclei during the second mitotic division, blastomeres were coinjected with Fura-2/dextran and a fluorescein-isothiocyanate-labelled BSA tagged with a nuclear targeting signal (FITC-NLS-BSA; kindly provided by M. Jackman, Gurdon Institute, University of Cambridge, Cambridge, UK). Experiments examining the second mitotic division were performed using an excitation filter wheel; Fura-2 was excited at 340 nm and 380 nm, and fluorescein at 490 nm. As before, emitted light was collected with a 520 nm band-pass filter. Fura-red was used to monitor [Ca2+]i in experiments involving flash photolysis (see below). Embryos were loaded with 4 µM Fura-red-AM for 10 minutes. Furared was illuminated at 427 nm and 490 nm, and emitted light collected with a 600 nm long-pass filter.
'Caged' Ins(1,4,5)P3 [cIns(1,4,5)P3; Molecular Probes] was used to trigger Ca2+ release in one-cell embryos. cIns(1,4,5)P3 was microinjected to an estimated final concentration of 50 µM. Photorelease was performed 30-60 minutes after microinjection by a brief, timed illumination at 360 nm. The duration of ultraviolet (UV) exposure was controlled using the Metafluor software. We and others have shown previously that using this protocol, repeated UV exposures trigger Ca2+ transients of similar amplitude (Jones and Nixon, 2000; FitzHarris et al., 2003
). In experiments in which cIns(1,4,5)P3 was used to compare the sensitivity of Ca2+ release between populations of embryos, both experimental groups were placed on the stage together and simultaneously exposed to UV light. Thus, comparisons were made between groups that were injected with the same pipette of cIns(1,4,5)P3, loaded with Fura-red and illuminated at the same time and under identical conditions.
Two-photon imaging
In experiments involving two-photon imaging, embryos were microinjected with Ca2+-Green/dextran (10 kDa), rhodamine-dextran (10 kDa) or a mixture of Ca2+-Green/dextran (10 kDa) and a larger rhodamine-dextran (70 kDa), each to an estimated final concentration of 50-100 µM. Injected embryos were placed in a chamber shortly before the expected time of NEBD and maintained at 37°C. The zona pellucida was removed by brief exposure to acidified Tyrode's solution (Sigma) to permit adherence to the coverslip. The chamber was mounted on the stage of a Nikon E600 FN upright microscope equipped with a 60x water immersion objective (Nikon CFI Fluor 60xW) and the one-cell parthenogenetic embryos were imaged with a BioRad two-photon laser-scanning microscope. This consisted of an MRC 1024 scan head mounted on the upright microscope and a Spectra-Physics Tsunami titanium-sapphire mode-locked laser pumped by a Millennia V green laser. Fluorescence was excited with a wavelength of 775 nm. The exciting beam was focussed via the 60x objective, which also collected the emitted fluorescence. The intensity of excitation was adjusted by means of neutral-density filters and was generally less than 7 mW at the specimen. Emitted fluorescence was detected by a photomultiplier mounted as a non-scanning external detector. For experiments that used two dyes, the emitted light was split into its red and green components by a dichroic mirror before being collected by separate photomultipliers. As for single-dye experiments, these photomultipliers were mounted as non-scanning external detectors. In some experiments, bright-field images were also collected via a photodiode mounted beneath the microscope stage. For each experiment, a sequence of x-y axis images in a single plane were collected at 5-10 second intervals. Images were analysed after acquisition with Metamorph (Universal Imaging). Fluorescence intensity was measured from specific regions of interest (ROIs) as indicated, with background subtraction as necessary. In the two-dye experiments, there was no significant contamination of the green signal from the rhodamine but there was a significant bleed through of the green signal into the red channel.
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Results |
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Precocious mitosis entry can be initiated by photorelease of Ins(1,4,5)P3
Mitosis entry can be triggered in sea-urchin embryos and fibroblasts by treatments that stimulate an increase in [Ca2+]i (Steinhart and Alderton, 1988; Twigg et al., 1988; Kao et al., 1990
). However, microinjection of Ca2+-EGTA solutions is apparently not sufficient to stimulate NEBD in the mouse zygote (Tombes et al., 1992
). Here, we addressed this question using photolysis of cIns(1,4,5)P3, which has the advantage of allowing Ins(1,4,5)P3 to be increased in a population of embryos simultaneously and at a time divorced from the microinjection procedure. Photorelease of cIns(1,4,5)P3 28-29 hours after hCG, when less than 10% of embryos had undergone NEBD, had no effect upon the timing of mitosis entry in fertilized embryos (not shown). Previous studies on sea-urchin embryos demonstrated that Ins(1,4,5)P3 was effective only during a brief window before NEBD (Twigg et al., 1988
). In mouse embryos, there is considerable asynchrony in the population, resulting in a window of several hours during which the population undergoes mitosis. Therefore, to increase the proportion of embryos in which NEBD might have acquired a sensitivity to Ca2+, we released Ins(1,4,5)P3 when half of the embryos had already undergone NEBD (32-34 hours after hCG). First, we verified that addition of cIns(1,4,5)P3 resulted in an increase in [Ca2+]i and that [Ca2+]i was not affected by the UV flash (Fig. 2A). Monitoring the timing of NEBD revealed that photorelease of Ins(1,4,5)P3 induced an increase in the rate of NEBD in this population of embryos (Fig. 2B). Thus, an increase in [Ca2+]i close to the time of NEBD is sufficient to accelerate entry into mitosis in one-cell mouse embryos.
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Ins(1,4,5)P3R downregulation prevents mitotic Ca2+ release
A second strategy of preventing mitotic Ca2+ transients involved depletion of Ins(1,4,5)P3Rs. Adenophostin A (Ad-A) is a potent Ins(1,4,5)P3R agonist that has previously been found to trigger extensive Ins(1,4,5)P3R downregulation within 4 hours of microinjection (Takahashi et al., 1993; He et al., 1999
; Brind et al., 2000
). In previous experiments, we have shown that injection of Ad-A during oocyte maturation prevents Ca2+ release at fertilization. Here, we used Ins(1,4,5)P3R-depleted embryos to investigate the role of Ins(1,4,5)P3Rs in mitosis.
First, we used cIns(1,4,5)P3 to confirm that Ad-A treatment inhibits Ca2+ release in one-cell embryos. As expected, Ins(1,4,5)P3-induced Ca2+ release was dramatically inhibited in Ad-A-treated embryos compared with controls (Fig. 4A). Furthermore, Ad-A-injected embryos (n=9) showed no evidence of mitotic Ca2+ transients during mitosis (Fig. 4B). As before, global Ca2+ transients were recorded in all controls (n=12; 2.3±0.5 transients per embryo). Despite abolishing all measurable Ca2+ release during mitosis, Ad-A-injected embryos proceeded through mitosis with kinetics indistinguishable from controls (Fig. 4Ci). To confirm that anaphase had proceeded, the resultant two-cell embryos were loaded with Hoechst 33342. Each blastomere in all of the embryos examined was found to contain chromatin, indicating that chromosome disjunction at anaphase had taken place (Fig. 4Cii; control, n=19; Ad-A, n=12). These experiments show that mitotic Ca2+ transients are dependent on the Ins(1,4,5)P3R, and that, in fertilized embryos, progression through mitosis can take place in the absence of measurable Ca2+ transients.
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Removal of extracellular Ca2+ prevents mitotic Ca2+ release
A third way to manipulate Ca2+ release during mitosis is to remove extracellular Ca2+. It has been shown previously that mouse embryos are not dependent on extracellular Ca2+ for progression through the first mitotic division (Tombes et al., 1992). However, it was not known whether removal of extracellular Ca2+ inhibits mitotic Ca2+ transients. We therefore monitored [Ca2+]i during mitosis in Ca2+-free medium and found no Ca2+ transients in any of the embryos examined (Fig. 5A; n=14). In addition, we found the timing of mitosis was not affected by Ca2+-free medium and that normal chromatid separation had taken place despite the absence of any Ca2+ transients (Fig. 5B; n=13).
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Mammalian embryos are susceptible to photodamage by visible light (Daniel, 1964; Hegele-Hartung et al., 1991
) and we have previously found that time-lapse confocal microscopy prevents mitosis entry in zygotes in almost all cases (G.F. and J.C., unpublished). Two-photon microscopy uses two-photon excitation of the fluorophore, allowing the use of longer-wavelength light and restricting excitation to the focal point. This technique has been demonstrated to be less damaging than confocal microscopy when used with two-cell hamster embryos (Squirrell et al., 1999
). We have established conditions in which mitosis proceeds during imaging with a two-photon microscope (sampling frequency, 5-10 seconds) (Fig. 6A). In this example the fertilized embryo undergoes three Ca2+ transients, each transient resulting in a threefold increase in fluorescence of Ca2+-Green/dextran. We have used this set up to investigate whether parthenogenetic mitotic embryos generate local perinuclear Ca2+ transients during NEBD.
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To determine accurately the timing of these localized fluorescence increases with respect to NEBD, we monitored mitosis entry in parthenogenetic one-cell embryos coinjected with Ca2+-Green/dextran (10 kDa) and a 70 kDa rhodamine-dextran. The 70 kDa rhodamine was excluded from the nucleus during interphase and therefore provided a precise indication of the timing of nuclear permeabilization (Fig. 7A). Analysis of relative fluorescence intensities revealed that the localized increase in Ca2+-Green fluorescence occurs at precisely the same time that the rhodamine-dextran entered the nucleoplasm (Fig. 7B; n=5). Therefore, the local increase in Ca2+-Green fluorescence that we detected at NEBD is Ca2+ independent and occurs concomitantly with, rather than before, permeabilization of the nuclear membrane.
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Discussion |
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Given previous studies in mouse and sea-urchin embryos, this result is surprising. We and others have previously found that BAPTA-AM effectively inhibited NEBD in mouse zygotes. This was the case in fertilized embryos that generate Ca2+ transients and in parthenogenetic embryos that do not (Tombes et al., 1992; Kono et al., 1996
) (present study). Similarly, in sea-urchin embryos and fibroblasts, BAPTA inhibits mitosis despite the absence of detectable Ca2+ transients in many cases (Kao et al., 1990
; Wilding et al., 1996
). These inconsistencies could be reconciled after it was found that local perinuclear [Ca2+] increases, which were not detectable using epifluorescence microscopy, were detectable using ratiometric confocal microscopy in sea-urchin embryos (Wilding et al., 1996
). This result therefore raised the possibility that an increase in [Ca2+] is indeed a universal trigger for progression through mitosis.
In the present study, we have tested this possibility in parthenogenetic mouse embryos, in which we would predict that localized increases in [Ca2+] provide the trigger for mitosis. Despite initial enthusiasm when an increase in fluorescence was seen in the nucleus, we subsequently found that this increase was also detectable with Ca2+-insensitive rhodamine-dextran. Furthermore, the increase did not precede NEBD but was coincident with it. Thus, we could not find any evidence for local Ca2+ transients driving mitosis in the mouse zygote. We cannot discount the possibility that Ca2+ changes are present below the level of detection of our system, but the dynamic range of Ca2+-Green/dextran and the ability to detect small Ca2+-independent changes in fluorescence suggests that any such changes would need to be more localized or more transient than previously described. These data suggest that, although an increase in [Ca2+]i is both necessary and sufficient for triggering mitosis in sea-urchin embryos, it is apparently dispensable for mitotic progression in mouse embryos.
Our data showing that microinjection of BAPTA, Br2BAPTA or EGTA has no effect on mitosis suggest that BAPTA-AM loading has effects independent of cytosolic Ca2+ buffering that are able to inhibit mitosis. One possibility is that compartmentalization of BAPTA-AM into organelles such as the endoplasmic reticulum might disrupt other cellular functions such as protein synthesis (Brostrom and Brostrom, 2003). Indeed, BAPTA-AM has been shown to inhibit protein synthesis, probably as a result of depleting intracellular Ca2+ stores (Preston and Berlin, 1992
; Lawrence et al., 1998
). Because mitosis entry in mouse zygotes is critically dependent upon the manufacture of new proteins (Howlett, 1986
), BAPTA-AM might prevent NEBD by disturbing lumenal Ca2+ homeostasis, rather than by preventing cytosolic Ca2+ changes.
The search for local nuclear Ca2+ transients has not been performed in many cell types but several recent studies indirectly support the conclusion that Ca2+ transients are dispensable for mitosis. In fertilized embryos, we have shown that the detectable Ca2+ transients start minutes after NEBD (Marangos et al., 2003; Larman et al., 2004
). In addition, the metaphase-to-anaphase transition during meiosis I takes place in the absence of any measurable increase in cytosolic [Ca2+] (Hyslop et al., 2004
; Marangos and Carroll, 2004
). Finally, in some (but not all) fibroblast cell lines, mitosis proceeds normally in the absence of Ca2+ transients. Questions have been raised about potential problems with compartmentalization of indicators, which can confound interpretation of Ca2+ records (al Mohanna et al., 1994
; Carroll et al., 1994
). However, a more recent study using a cytosolic chameleon, which avoids such pitfalls, also failed to report an increase in [Ca2+] during mitosis (Whitaker and Larman, 2001
). Therefore, though there is strong evidence for a necessary role for Ca2+ in mitosis in sea-urchin embryos, it appears that, in mammalian embryos and several cell lines, Ca2+ does not play an obligatory role in mitosis. Ca2+ transients might act as a pacing mechanism for the rapid cell cycles of echinoderms, and this mechanism might have been supplanted in the longer, more complex cell cycles of mammalian cells.
Ca2+ might not be necessary, but is sufficient, to accelerate mitosis entry
A more fundamental role for Ca2+ in embryonic cell cycles is indicated by our observation that the ability of a Ca2+ transient to trigger mitosis entry is conserved between sea-urchin and mouse embryos. This sensitivity to Ca2+ was revealed in mouse embryos by an acceleration of NEBD in response to Ins(1,4,5)P3-induced Ca2+ release and could only be uncovered in a population of embryos that remained in interphase once 50% had entered mitosis. A narrow window of sensitivity is also seen in sea-urchin embryos and suggests that a Ca2+-sensitive pathway becomes available only once NEBD is imminent (Twigg et al., 1988). A good candidate for the Ca2+-sensitive switch is activation of Cdc25. Cdc25 is necessary for the activation of Cdk1/cyclin-B at mitosis entry and has been shown to be sensitive to Ca2+ (Patel et al., 1999
). In sea urchins, the Ca2+-sensitive step acts as a cell-cycle checkpoint, arresting progression into mitosis until certain conditions are established (Whitaker and Larman, 2001
). It therefore remains an intriguing possibility that conserved Ca2+-sensitive events drive NEBD in mammalian embryos when the normal cell cycle is perturbed.
Fluorescence changes do not always mean a Ca2+ transient
These studies reveal some of the potential caveats of using fluorescent probes and high-resolution imaging in dynamic systems. Most remarkably, a compelling increase in fluorescence in the nucleus seen with a Ca2+-sensitive indicator was mimicked by an indicator that does not respond to [Ca2+]. The explanation for the small increase in fluorescence at NEBD remains obscure but it is well known that the fluorescence properties of fluorophores are sensitive to environmental factors such as viscosity and polarity (Tsien, 1989; Poenie, 1990
; Roe et al., 1990
; Busa, 1992
). In addition, some cytoplasmic proteins might modify fluorescence by direct binding to fluorophores (Highsmith et al., 1986
; Konishi et al., 1988
). Because NEBD is accompanied by sweeping changes in cellular organization such as nuclear entry and exit of proteins, and a dramatic reorganization of organelle structure (Terasaki, 2000
; FitzHarris et al., 2003
), the fluorescence changes we observe probably reflect changes in the constitution of the nucleoplasm as it mixes with the cytosol. Convincing spatially restricted perinuclear Ca2+ transients have previously been reported in sea-urchin embryos (Wilding et al., 1996
) and in HeLa cells (Lipp et al., 1997
), the spatial organization of which are clearly distinct from the fluorescence changes we have reported here.
Maternal and paternal contributions to mitotic Ca2+ signalling in mouse embryos
The mechanisms by which mitotic Ca2+ transients are generated in mouse embryos are gradually becoming apparent. Our Ad-A experiments indicate that the Ins(1,4,5)P3 receptor is essential for mitotic Ca2+ release. We have previously shown that there is an increase in the sensitivity of Ins(1,4,5)P3R-mediated Ca2+ release at mitosis entry in both fertilized and parthenogenetic embryos (FitzHarris et al., 2003), suggesting a maternal cell-cycle-related modification of the Ca2+-releasing machinery. Recent studies suggest the cell-cycle-dependent control might be mediated by Ins(1,4,5)P3R phosphorylation (Jellerette al., 2004
).
However, maternal factors are clearly not sufficient for the generation of mitotic transients because oscillations are specific to fertilized embryos (Kono et al., 1996) (present study). Indeed, in several studies, even the mitotic Ca2+ transients are not always able to be detected (Tombes et al., 1992
; Day et al., 2000
; Tang et al., 2000
; Gordo et al., 2002
). Recently, it has emerged that phospholipase C
(PLC
), the sperm-borne factor responsible for initiating Ca2+ oscillations and egg activation (Saunders et al., 2002
; Cox et al., 2002
; Rogers et al., 2004
), becomes compartmentalized within the developing pronuclei following fertilization (Larman et al., 2004
; Yoda et al., 2004
). Given the strict relationship between pronucleus formation and cessation of the oscillations, it has been proposed that sequestration of PLC
terminates the oscillations, perhaps by isolating the enzyme from its substrate, and that mitotic Ca2+ release reflects the subsequent liberation of PLC
back into the cytoplasm (Marangos et al., 2003
; Larman et al., 2004
). This model is consistent with the findings that the first mitotic transient occurs several minutes after the first signs that NEBD is imminent (Kono et al., 1996
; Marangos et al., 2003
; Larman et al., 2004
) and that pronuclei from fertilized, but not from parthenogenetic, embryos possess a Ca2+-releasing activity capable of activating unfertilized eggs (Kono et al., 1995
; Zernicka-Goetz et al., 1995
). Thus, it appears that mitotic Ca2+ transients are generated by the release of PLC
into a highly responsive M-phase cytoplasm. Our data showing that the second mitosis lacks global Ca2+ transients suggest that PLC
might be inactivated by the time of second mitosis or that the cytoplasm is no longer capable of supporting Ca2+ release. PLC
activity appears to be the limiting factor, because nuclei transferred from pronucleate, but not late two-cell- or four-cell-stage, embryos can activate recipient eggs (Kono et al., 1995
; Zernicka-Goetz et al., 1995
). Thus, the Ca2+ transients during the first mitotic division are the final act in a Ca2+ signalling pathway initiated some 16-20 hours earlier, at the time of sperm-egg fusion.
Role of mitotic Ca2+ release in mouse embryos
Although dispensable for mitosis, there is some evidence that mitotic Ca2+ transients serve a further developmental role. Sr2+ exposure during the first mitosis induces oscillations in parthenogenotes and resulting blastocysts have more cells in the inner cell mass (Bos-Mikich et al., 1995). Improved development has also been noted following exposure of one- or two-cell (but not four-cell) embryos to low concentrations of ethanol (Leach et al., 1993), a treatment that might increase [Ca2+]i. A similar phenomenon has been described following fertilization, when Ca2+ oscillation regimen can influence long-term developmental potential (Ozil and Huneau, 2001
; Ducibella et al., 2002
). Because these experiments imply a role for Ca2+ as a determinant of developmental potential several cell divisions later, mitotic Ca2+ transients might modify gene expression via Ca2+-dependent transcription factors (Mellstrom and Naranjo, 2001
). However, it should be realized that blastocyst formation can be achieved by ethanol-activated parthenogenotes (Kaufman and Sachs, 1978
), which we have now shown do not generate mitotic Ca2+ transients, and that the developmental outcome of inhibiting endogenous transients has not yet been established.
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Acknowledgments |
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Footnotes |
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Present address: Colorado Center for Reproductive Medicine, 799 East Hampden Avenue, Englewood, CO 80113, USA
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References |
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