Cell cycle-dependent Ca2+ oscillations in mouse embryos are regulated by nuclear targeting of PLC{zeta}

Mark G. Larman1,2, Christopher M. Saunders2, John Carroll3, F. Anthony Lai2 and Karl Swann1,*

1 Department of Anatomy and Developmental Biology, University College London, London, WC1E 6BT, UK
2 Cell Signalling Laboratory, Wales Heart Research Institute, University of Wales College of Medicine, Cardiff, CF14 4XN, UK
3 Department of Physiology, University College London, London, WC1E 6BT, UK

* Author for correspondence (e-mail: k.swann{at}ucl.ac.uk)

Accepted 19 January 2004


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During the first cell cycle Ca2+ oscillations are regulated in a cell cycle-dependent manner, such that the oscillations are unique to M phase. How the Ca2+ oscillations are regulated with such cell cycle stage-dependency is unknown, despite their importance for egg activation and embryo development. We recently identified a novel, sperm-specific phospholipase C (PLCzeta; PLC{zeta}) that triggers Ca2+ oscillations similar to those caused by sperm. We show that PLC{zeta}-induced Ca2+ oscillations also occur exclusively during M phase. The cell cycle-dependency can be explained by PLC{zeta}'s localisation to the pronuclei, which depends specifically upon a nuclear localisation signal sequence. Preventing pronuclear localisation of PLC{zeta} by mutation of the nuclear localisation signal, or by inhibiting pronuclear formation/import, can prolong Ca2+ oscillations or allow them to occur during interphase. These data suggest a novel mechanism for regulating a PLC through nuclear sequestration and may explain the cell cycle-dependent regulation of Ca2+ oscillations following fertilisation.

Key words: Fertilisation, Phospholipase C, Ca2+ oscillations, Cell cycle


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
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 References
 
Increases in cytosolic Ca2+ concentrations regulate a vast range of cellular processes (Berridge et al., 2003Go). Control of cell cycle divisions by Ca2+ has been shown in a number of species (for reviews, see Stricker, 1999Go; Whitaker and Larman, 2001Go). Probably one of the most striking displays of increases in cytosolic Ca2+ is seen at mammalian fertilisation where the sperm triggers a long-lasting series of Ca2+ oscillations in the egg, which last for several hours and eventually terminate about the time of pronuclear formation when first interphase begins (reviewed by Carroll, 2001Go). These Ca2+ oscillations are both necessary and sufficient for egg activation at fertilisation (Kline and Kline, 1992Go; Miyazaki et al., 1993Go; Swann and Ozil, 1994Go) and later development (Ozil, 1990Go; Bos-Mikich et al., 1997Go; Lawerence et al., 1998; Ozil and Huneau, 2001Go). Generation of the Ca2+ transients is regulated in a cell cycle-dependent manner since no Ca2+ increases are observed during interphase, but oscillations do start again when the zygote enters first mitosis (Tombes et al., 1992Go; Kono et al., 1996Go; Day et al., 2000Go; Marangos et al., 2003Go). The stimulus for the mitotic Ca2+ oscillations appears to be related to some factor(s) from the sperm because they are seen in fertilised zygotes, but not in parthenogenetic embryos that have been artificially activated by strontium or ethanol (Kono et al., 1996Go).

The mechanism for generating these cell cycle-dependent Ca2+ signals has not been established (Carroll, 2001Go). One explanation is that the pronuclei may sequester a sperm-derived factor or cofactor in order to stop it triggering Ca2+ oscillations (Carroll, 2001Go). This idea is consistent with the ability of fertilised one-cell embryos, which contain a pronucleus, to activate metaphase II-arrested (MII) eggs when they are fused together (Zernicka-Goetz et al., 1995Go). Two further studies, using different approaches, have provided direct evidence for the sequestering role of the pronuclei in terminating the Ca2+ oscillations. Nuclear transplantation experiments involving the transfer of pronuclei from fertilised one-cell embryos to unfertilised mouse eggs demonstrated that Ca2+ oscillations followed subsequent pronuclear envelope breakdown in the meiotic metaphase cytoplasm (Kono et al., 1995Go). Secondly, blocking pronuclear formation or nuclear import was found to significantly prolong the fertilisation Ca2+ oscillations (Marangos et al., 2003Go). These data strongly support the idea that a factor responsible for controlling the Ca2+ oscillations may become localised to pronuclei during interphase. Thus, possible candidates for nuclear targeting include the Ca2+-releasing factor, cofactor(s) and substrate (Marangos et al., 2003Go).

Cell cycle Ca2+ changes are also seen in embryos of other species, such as sea urchins (Whitaker and Patel, 1990Go; Whitaker and Larman, 2001Go). Biochemical assays in sea urchin embryos suggest that the cell cycle Ca2+ changes are due to endogenously generated increases in inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] production (Ciapa et al., 1994Go). This suggests that an endogenous phospholipase C (PLC) is being activated during the cell cycle but the mechanism and identity of the PLC(s) involved is unclear. We recently identified a mammalian sperm-specific PLC (PLC{zeta}) (Cox et al., 2000Go; Saunders et al., 2002Go). Injecting PLC{zeta} cRNA into mouse eggs, at a concentration that produces PLC{zeta} protein in the same range as that found in a single sperm, triggers Ca2+ oscillations during meiosis exit that are indistinguishable from those observed at fertilisation. Through immunodepletion, PLC{zeta} appears to be the Ca2+-releasing factor present in sperm (Saunders et al., 2002Go).

We investigated if the localisation of PLC{zeta} can provide an explanation for cell cycle-dependency of the Ca2+ signals during the first division in mouse embryos. We first report that PLC{zeta}-induced Ca2+ oscillations only occur during M-phase. By using an epitope-tagged PLC{zeta} we then demonstrate that PLC{zeta} localises to the pronuclei and that preventing nuclear sequestration by mutation of the PLC{zeta} nuclear localisation signal (NLS) or inhibiting pronuclear formation/import prolongs the Ca2+ oscillations. We present a model of PLC{zeta} nuclear localisation that offers a simple and novel explanation for the cell cycle-dependent Ca2+ oscillations during the first cell cycle.


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Materials
All reagents used were from Sigma, unless stated otherwise. The fluorescently tagged nuclear localisation signal (FITC-BSA-NLS) was a kind gift from Mark Jackman (Wellcome/CRC, UK) and wheat germ agglutinin (WGA) from Calbiochem. Complementary RNA (cRNA) was synthesised from the open reading frame of mouse PLC{zeta} and c-myc tagged-PLC{zeta} (Saunders et al., 2002Go). Each batch of cRNA produced was checked for in vitro protein expression using a reticulocyte lysate system (Promega), as previously described (Saunders et al., 2002Go).

Preparation and handling of eggs and embryos
Female MF1 mice were superovulated by an injection of 5 IU of pregnant mare's serum gonadotropin (PMSG; Intervet). This was followed by an injection of human chorionic gonadotropin (HCG; Intervet) 48 hours later. Eggs were collected after a further 13.5-14.5 hours, as previously described (Lawrence et al., 1997Go) and maintained in 100 µl droplets of H-KSOM (HEPES-buffered potassium simplex optimized medium) (Summers et al., 2000Go) under mineral oil at 37°C. For experiments on MII-arrested eggs cRNA injections were carried out between 14.5 and 15.5 hours after HCG injection. To prepare parthenotes for interphase (G1), eggs were treated with 20 mM Sr2+ in Ca2+-free H-KSOM (Kline and Kline, 1992Go). Interphase (G2) parthenotes were obtained by exposing eggs to H-KSOM containing 7% ethanol for 7 minutes at room temperature (Cuthbertson, 1983Go) 24 hours after HCG and culturing for a further 9-12 hours (Tang et al., 2000Go). All activations were carried out in H-KSOM containing 2 µM cytochalasin D to prevent second polar body extrusion, making the embryos diploid.

Microinjection and measurement of intracellular calcium changes
Eggs and embryos were injected as previously described (Marangos et al., 2003Go). cRNA solutions were diluted with an RNase-free injection buffer (0.5x PBS; Ambion). The volume injected was estimated from the diameter of cytoplasmic displacement caused by the bolus injection. Depending on the reagent, injection volumes ranged from 0.5-5% of the egg volume. The amount of PLC{zeta} protein expressed after microinjection with a pipette concentration of 0.02 mg/ml cRNA (3-5% egg volume) initiates Ca2+ oscillations with a comparable amount of PLC{zeta} that is present in a single sperm (Saunders et al., 2002Go). All experiments were carried out with 0.02 mg/ml, unless stated otherwise. In experiments that required a number of reagents to be introduced into the cell, to reduce possible damage caused by multiple injections, some reagents were co-injected (Marangos et al., 2003Go). Intracellular calcium changes were measured with Fura-dextran 10,000 Mr (Molecular Probes; pipette concentration 0.5 mM) made up in injection buffer (120 mM KCl, 20 mM Hepes, pH 7.4). Briefly, calcium measurements were carried out on CCD-based imaging systems using a Zeiss Axiovert 100 microscope with illumination from a monochromator (Photonics) controlled by MetaFluor v4.0 (Universal Imaging Corp.) (Marangos et al., 2003Go). Pseudo-simultaneous imaging of Ca2+ (2-4 µM Fura-dextran final concentration) and FITC-BSA-NLS (1-3 µM final concentration) was carried out using an excitation filter wheel (Sutter, UK; 350/380 nm for Fura-dextran and 490 nm for FITC-BSA-NLS), a 510 nm dichroic mirror and 520 nm long pass emission filter.

Immunostaining and confocal imaging
Eggs were microinjected with either 0.2 mg/ml PLC{zeta} cRNA or c-Myc-PLC{zeta} cRNA and cultured in H-KSOM medium containing 2 µM cytochalsin D. Once the embryos had formed pronuclei they were washed in PBS containing 4 mg/ml bovine serum albumin (BSA) for 5 minutes. Fixation was carried out by transferring the embryos into 4% paraformaldehyde (in PBS alone) for 15 minutes. Embryos were washed for a further 5 minutes before permeabilisation was achieved with 2% Triton X-100 (in PBS/BSA) for 10 minutes. The blocking step was carried out overnight in a solution of 50 mM glycine (in PBS/BSA) before the c-Myc primary antibody (9E10; Santa Cruz Biotechnology) was used at 0.4 ng/µl (in PBS/BSA) and applied for 16 hours. This was followed by another PBS/BSA wash and then the embryos were transferred into the secondary antibody solution (10 µg/ml) for 2 hours (goat anti-mouse Alexa Fluor® 488; Molecular Probes, USA).

Confocal microscopy was carried out on a Zeiss Axiovert 100TV microscope equipped with Biorad µRadiance. Using the 488 nm laser line of an Argon laser, the emission of Alexa Fluor® 488 was collected using a 520 nm (40 nm bandwidth) bandpass filter.

Generation of NLS mutant, Myc-PLC{zeta}K377E
Mutation of 377Lys to 377Glu within the putative PLC{zeta} nuclear localisation signal sequence to produce Myc-PLC{zeta}K377E was carried out using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), as described previously (Saunders et al., 2002Go). The K377E mutant of Myc-PLC{zeta} was generated using the primer pair 5'-gtcaagaaaaggaaggagaagatgaaaatagcc-3' and 5'-ggctattttcatcttctccttccttttcttgac-3', with the previously described Myc-PLC{zeta} as template (Saunders et al., 2002Go). Incorporation of the mutated sequence in Myc-PLC{zeta}K377E was verified by nucleotide sequence analysis performed on an ABI Prism® 3100 Genetic Analyzer.


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 Materials and Methods
 Results
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PLC{zeta}-induced Ca2+ oscillations are cell cycle-dependent
Expression of PLC{zeta} protein in mouse eggs, through microinjection of PLC{zeta} cRNA, produces a series of long lasting Ca2+ oscillations indistinguishable from those at fertilisation (Saunders et al., 2002Go). During the first cell cycle, sperm-triggered oscillations appear to be controlled with a cell cycle dependence (review by Carroll, 2001Go). To test if PLC{zeta}-induced Ca2+ oscillations are also cell cycle dependent, mouse eggs were injected with PLC{zeta} cRNA and Fura-dextran at three different stages of the first cell cycle. PLC{zeta} injection into MII eggs triggers a series of long lasting Ca2+ oscillations that terminate as the embryo enters interphase (Fig. 1a). In contrast, eggs that had been Sr2+-activated and allowed to form pronuclei (G1 interphase) did not show any increase in Ca2+ following PLC{zeta} injection, during the 4-hour observation period (Fig. 1b).



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Fig. 1. PLC{zeta}-induced Ca2+ oscillations are cell cycle-dependent. PLC{zeta} cRNA was injected into MII-arrested mouse eggs and at two different stages of the first cell cycle following parthenogenetic activation: interphase G1 and late G2. Eggs were microinjected with Fura-dextran and PLC{zeta} cRNA. Intracellular Ca2+ was monitored by changes in fluorescence excitation ratio. Time zero indicates approximately the point at which PLC{zeta} was microinjected. (a) A series of Ca2+ oscillations occurs during MII that ceases as the egg enters interphase (n=20). (b) MII eggs were activated with Sr2+ in the presence of cytochalsin D. 2 hours after pronuclear formation the eggs were injected with Fura-dextran and PLC{zeta}. No Ca2+ transients were detected in these parthenotes (n=20). (c) Parthenogentically activated eggs were cultured until interphase (G2), then 2-6 hours before NEB was due to occur embryos were injected with Fura-dextran and PLC{zeta} cRNA (n=15). Ca2+ oscillations again occur.

 

Following fertilisation no further Ca2+ transients are detected until mitosis (Kono et al., 1996Go; Day et al., 2000Go). This second phase of oscillations then lasts until nuclear envelope reformation (NER) in the daughter cells (Marangos et al., 2003Go). These Ca2+ oscillations are unique to eggs that have been fertilised or activated by sperm extract injection (Kono et al., 1996Go; Tang et al., 2000Go). Eggs activated through chemical-parthenogenetic activation do not display Ca2+ oscillations during mitosis (Kono et al., 1996Go). However, when ethanol-activated embryos were injected with PLC{zeta} cRNA approximately 2-6 hours before nuclear envelope breakdown (NEB) or simply activated with PLC{zeta} and then allowed to progress through the first cell division, they underwent a series of Ca2+ oscillations, similar to those produced by PLC{zeta} in unfertilised eggs, which appeared to correlate with the duration of mitosis (Fig. 1c).

To determine exactly when mitosis entry and nuclear membrane reformation (NER) occurs, a probe for nuclear membrane integrity was required. Specific transport of proteins into the nucleus occurs via the recognition of a nuclear localisation signal (NLS) within the protein by the importin {alpha}/ß heterodimer, which interacts with and then permits passage of the protein through the nuclear pore complex (NPC) (Gorlich, 1998Go). During G2 interphase embryos were injected with 1-3 µM FITC-labelled BSA that had been conjugated to a NLS sequence (FITC-BSA-NLS) (Jackman et al., 2002Go), in addition to Fura-dextran and PLC{zeta} cRNA. Within 10-20 minutes the intact centred pronuclei were visible under fluorescence. Fig. 2a shows the pronuclei fluorescence of FITC-BSA-NLS. The time scale indicates how many minutes had elapsed after injection of PLC{zeta} cRNA. The images of NEB with FITC-BSA-NLS and Ca2+ changes were acquired simultaneously. Measuring the decrease in fluorescence of each pronuclei and increases in Ca2+ clearly shows that one pronucleus begins to undergo NEB before the first increase in Ca2+ (Fig. 2b). We found this to be the case in all 12 experiments carried out, with the initial decrease in FITC-BSA-NLS fluorescence occurring 6.3±4.1 minutes before the peak of Ca 2+.



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Fig. 2. PLC{zeta}-induced Ca2+ oscillations at mitosis are controlled by nuclear membrane integrity. As in Fig. 1c, MII eggs were parthenogentically activated in the presence of cytochalsin D and cultured until interphase (G2). To monitor the exact timing of NEB and NER, embryos were injected (2-6 hours before NEB) with a fluorescent probe for nuclear membrane integrity (FITC-BSA-NLS) in addition to Fura-dextran and PLC{zeta} cRNA. (a) Two pronuclei can be seen with FITC-BSA-NLS. At approximately 416 minutes (415.5) after injection of PLC{zeta} cRNA the first pronucleus begins to break down. By 430 minutes both pronuclei have broken down and the FITC-BSA-NLS can no longer be detected. Measuring the decrease in nuclear FITC-BSA-NLS intensity of both pronuclei (red and blue lines) it was found that NEB of the first pronucleus occurred 6.3±4.1 minutes before the initial Ca 2+ transient (n=12) that was indicated by the Fura-dextran fluorescence ratio (black line). (b) NER was also monitored with FITC-BSA-NLS. Measuring the accumulation of FITC-BSA-NLS into the reforming nuclei showed that the first indication of NER occurred just prior to the last Ca2+ transient (14.3±4.3 minutes). As in a, the red and blue lines represent fluorescence from the FITC-BSA-NLS regions and the black line is the Fura-dextran fluorescence ratio.

 

We then allowed the parthenogenetically activated embryos to complete mitosis to investigate if reformation of the nuclei and entry into the second interphase had a similar effect on PLC{zeta}-induced Ca2+ oscillations to those triggered by sperm. As with NEB, FITC-BSA-NLS was used to monitor NER. Embryos were again injected during interphase (G2) with FITC-BSA-NLS, Fura-dextran and PLC{zeta}. To limit the duration of exposure, fluorescence imaging was not carried out until the embryos had undergone NEB and were approaching cytokinesis. Ca2+ oscillations were detected in all embryos. Ca2+ transients continued through the progression of cytokinesis and it was common that the last Ca2+ increase detected occurred within just one of the daughter cells, suggesting the completion of cytokinesis. By simultaneously measuring the increase in FITC-BSA-NLS as NER occurred it was found that the first indication of NER was detected 14.3±4.3 minutes before the last Ca 2+ spike (Fig. 2bii). NER then proceeded over a period of about 1 hour, during which Ca2+ transients stopped.

Together these data show that PLC{zeta}-induced Ca2+ oscillations during the first cell division have the same cell cycle-dependency as in fertilised and sperm extract-activated eggs. They also support the hypothesis that the precise correlation between the occurrence of Ca2+ oscillations and absence of the pronuclei is due to the free access of PLC{zeta} to the embryo cytoplasm.

PLC{zeta} localises to the pronuclei
If nuclear sequestration of PLC{zeta} does control the Ca2+ oscillations it should be detectable in the pronuclei after PLC{zeta}-induced egg activation. We first attempted to monitor PLC{zeta} localisation by introducing a YFP-tag at the N terminus of PLC{zeta}. When YFP-PLC{zeta} was injected into eggs (pipette concentration up to 2 mg/ml) Ca2+ oscillations were seen, but no YFP fluorescence was detected using whole cell or confocal fluorescent-imaging techniques (data not shown). The lack of signal with this chimeric protein is consistent with the fact that the lower threshold of detection for EGFP above autofluorescence in mouse eggs is approximately 0.6 µM (Medvedev et al., 2002Go), whereas the amount of PLC{zeta} that is required to generate Ca2+ oscillations is 0.3-6 nM. To investigate if PLC{zeta} is indeed sequestered by the pronuclei, a more sensitive tagging method was required.

The localisation of PLC{zeta} was successfully monitored using detection of immunofluorescence from a Myc-tagged PLC{zeta} (Saunders et al., 2002Go). MII-arrested eggs were microinjected with Myc-PLC{zeta} cRNA and cultured in H-KSOM medium containing the actin inhibitor cytochalasin D to create diploid embryos. After pronuclear formation, the embryos were fixed and then labelled using the c-Myc antibody and Alexa Fluor® 488-labelled secondary antibodies. As a control, some eggs were activated with untagged PLC{zeta} (Fig. 3a). Fig. 3 shows brightfield and confocal anti-c-Myc immunostained images that were simultaneously acquired. The nucleoli can be seen as the circular unstained areas, marked with black arrows on the brightfield images. Surrounding the nucleoli is the nuclear membrane, although this is difficult to resolve. In contrast, all eggs activated with Myc-PLC{zeta} possessed clearly fluorescent pronuclei. Representative confocal immunofluorescent images of eggs activated with Myc-PLC{zeta} indicate the striking presence of PLC{zeta} in the nucleoplasm of the pronuclei (Fig. 3b,c). Immunofluorescence of Myc-PLC{zeta}-activated eggs cultured to the two-cell stage also showed nuclear localisation in each of the daughter cells after first mitosis (Fig. 3d). These data demonstrate that PLC{zeta} localises to pronuclei in one-cell embryos and to the nuclei in two-cell embryos.



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Fig. 3. PLC{zeta} localises to the pronuclei. MII eggs were injected with PLC{zeta} tagged with a c-Myc epitope and cultured in the presence of cytochalsin D until pronuclear formation, then they were fixed and stained using the c-Myc (9E10) monoclonal antibody and the anti-mouse Alexa Fluor® 488 secondary antibody. Control eggs were activated with untagged PLC{zeta}. Confocal and brightfield images were simultaneously obtained. The black arrows in brightfield images indicate the nucleoli. Though not easily visible, the nuclear membrane surrounds the nucleolus. The control embryo shows non-specific binding. Eggs activated with Myc-PLC{zeta} show a striking localisation to the nucleoplasm of each pronuclei. Scale bar: 10 µm.

 

Pronuclear formation and PLC{zeta} nuclear import are essential for terminating the Ca2+ oscillations
Sperm-triggered oscillations appear to be controlled by pronuclear formation (Marangos et al., 2003Go). To test if PLC{zeta}-induced Ca2+ oscillations are also regulated by formation of the pronuclei, MII-arrested mouse eggs were injected with PLC{zeta} cRNA, Fura-dextran and a lectin (wheat germ agglutinin; WGA) that blocks pronuclear formation (Davis, 1995Go). Parallel control eggs were injected with PLC{zeta} cRNA and Fura-dextran only. PLC{zeta} injection triggers a series of long-lasting Ca2+ oscillations that terminate at interphase (Fig. 4a). In contrast to parallel control eggs, we found that WGA-treated eggs did not form proper expanded pronuclei, as previously reported (Vautier et al., 2001Go; Marangos et al., 2003Go). Significantly, WGA-mediated inhibition of pronuclear formation also prolonged the PLC{zeta}-induced Ca2+ oscillations by about 6 hours (control: mean 444.2±142.0 minutes, n=14; WGA: mean 826.5±104.3 minutes, n=14; Fig. 4a,b).



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Fig. 4. PLC{zeta}-induced Ca2+ oscillations are regulated by nuclear import. MII-arrested mouse eggs were microinjected with Fura-dextran and PLC{zeta} cRNA. Intracellular Ca2+ was monitored by changes in fluorescence excitation ratio. Time zero indicates approximately the point at which PLC{zeta} was microinjected. The trace in a shows a series of Ca2+ oscillations in a control egg injected with PLC{zeta} cRNA. In b, parallel treated eggs were also injected with 10 mg/ml (pipette concentration) WGA, which significantly (P<0.0001) prolonged the oscillations (control: mean 444.2±142.0 minutes, n=14; WGA: mean 826.5±104.3 minutes, n=13). MII eggs were also injected with Fura-dextran and Myc-tagged versions of either control PLC{zeta} or PLC{zeta}K377E to monitor the nuclear localisation. Trace ci shows a series of Ca2+ oscillations in a control egg injected with Myc-PLC{zeta} cRNA. In di, parallel eggs were injected with Myc-PLC{zeta}K377E. Oscillations were significantly (P<0.0001) prolonged with the NLS mutant (control: mean 335.9±93.2 minutes, n=20; K377E: mean 702.2±162.8 minutes, n=22). Embryos were fixed and stained (see Fig. 3). Confocal and brightfield images were simultaneously obtained (cii and dii) showing the absence of Myc-PLC{zeta}K377E in the nucleoplasm.

 

The nuclear pore complex (NPC) offers the only route for nucleo-cytoplasmic exchange (Feldherr et al., 1984Go). Diffusion through the NPC is limited to proteins less than 40-60 kDa. Larger proteins traverse the nuclear membrane through an active-carrier mechanism that requires a nuclear localisation signal, which permits direct import into the nucleus via the importin pathway (Gorlich, 1998Go). Many nuclear proteins have been found to contain a cluster of basic amino acids that act as a NLS (Dingwall and Laskey, 1991Go). The amino acid sequence of mouse PLC{zeta} contains a putative NLS (residues 374-379; KKRKRK) in between the X and Y catalytic domains (Saunders et al., 2002Go). If this NLS is functionally important in transporting PLC{zeta} into the nuclei at pronuclear formation then mutating this region should prolong the Ca2+ oscillations.

To enable us to confirm that the mutation prevented nuclear localisation Myc-PLC{zeta} was used. MII-arrested eggs were injected with the NLS mutant Myc-PLC{zeta}K377E cRNA and parallel control eggs were injected with Myc-PLC{zeta} cRNA. Immunostaining of embryos injected with Myc-PLC{zeta}K377E confirmed that the NLS mutant prevented its nuclear import, whereas parallel control eggs injected with wild-type Myc-PLC{zeta} showed the typical pronuclear localisation (Fig. 4cii and dii). Eggs injected with Myc-PLC{zeta}K377E-generated Ca2+ oscillations that were similar in form to those of control PLC{zeta}-injected eggs, except that the Myc-PLC{zeta}K377E-induced oscillations lasted for about 6 hours longer (control: mean 344.4±87.5, n=20; K377E: 702.2±162.8, n=22; Fig. 4ci-di). These data, therefore, show that either mutation of the predicted NLS in the PLC{zeta} gene or inhibition of pronuclear formation leads to a significantly prolonged series of Ca2+ oscillations.

To obtain further evidence that cytosolic PLC{zeta} is transported into the pronuclei and it is this sequestration that ultimately dictates the Ca2+ oscillation cell cycle dependency, Myc-PLC{zeta} was injected during G1 interphase (following Sr2+ activation) and prevented from entering the pronuclei by either injecting WGA or using the NLS mutant. WGA selectively blocks active nuclear import (Davis, 1995Go). Since PLC{zeta} has a predicted molecular mass of 74 kDa (Saunders et al., 2002Go) it is larger than the ~50 kDa that can pass through the nuclear pore complex (NPC) via passive diffusion (Gorlich, 1998Go). As before no Ca2+ oscillations were observed after injection of PLC{zeta} cRNA during interphase (Fig. 5ai). Fig. 5bi shows that blocking transport thorough the nuclear pores restored Ca2+ oscillations in interphase embryos injected with Myc-PLC{zeta} cRNA. We also confirmed, by immunostaining for Myc-PLC{zeta}, that WGA was blocking transport of PLC{zeta} into the pronuclei. Fig. 5aii and bii shows that PLC{zeta} is localised to the pronuclei when it is injected during interphase, but this localisation pattern is prevented by co-injection of WGA. Blocking the NPC would prevent most, if not all, nuclear import. Hence, to investigate if cytoplasmic retention of PLC{zeta} alone is sufficient to resume Ca2+ oscillations during interphase Myc-PLC{zeta}K377E was injected together with Fura dextran. As before, no Ca2+ increases were observed in embryos injected with wild-type PLC{zeta}. In contrast, mutating the NLS and preventing nuclear import of PLC{zeta}, Ca2+ oscillations were observed that were comparable to those generated by blocking the NPC with lectin.



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Fig. 5. Preventing PLC{zeta} nuclear localisation permits Ca2+ oscillations during interphase. MII eggs were activated with Sr2+ in the presence of cytochalsin D. 2 hours after pronuclear formation the eggs were injected with Fura-dextran and Myc-PLC{zeta}. (a) No Ca2+ transients were detected in these parthenotes (n=20). (b) Parallel parthenotes were also injected with WGA to block nuclear transport. In these embryos Myc-PLC{zeta} triggered Ca2+ oscillations within 1 hour (n=20). Embryos were fixed and stained at the end of the experiment (as in Fig. 2) demonstrating that blocking nuclear pores with WGA, prevents import of PLC{zeta} into the pronuclei (aii,bii). (c) Parthenotes were also injected with Myc-PLC{zeta}K377E (n=10). As with inhibiting nuclear import, preventing nuclear localisation of PLC{zeta} through mutation of the NLS permitted Ca2+ oscillations during interphase. Such Ca2+ oscillations were not observed in parallel controls (n=10).

 


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ca2+ oscillations generated by fertilisation and sperm extract injection have been shown to be cell cycle dependent, only occurring during M-phase (Jones et al., 1995Go; Kono et al., 1996Go; Day et al., 2000Go). Interestingly, no Ca2+ increases are observed during mitosis in eggs activated through chemical parthenogenesis (Kono et al., 1996Go), which suggests that some factor supplied specifically by the sperm ultimately causes the mitotic Ca2+ signals in zygotes. A few key studies have led to the hypothesis that the cell cycle dependency of the Ca2+ oscillations may be due to nuclear sequestration of the Ca2+-releasing factor delivered by the sperm (Kono et al., 1995Go; Zernicka-Goetz et al., 1995Go; Marangos et al., 2003Go)

We have recently shown that PLC{zeta} is the protein responsible for the Ca2+-releasing activity of sperm extracts (Saunders et al., 2002Go). PLC{zeta}-induced Ca2+ oscillations also show cell cycle dependency during the first cell cycle. The current data suggest that the paternally supplied factor is PLC{zeta}. Pronuclear envelope breakdown occurs just before the first mitotic Ca2+ transient in PLC{zeta}-activated eggs. In addition, the reformation of nuclei coincides with cessation of PLC{zeta}-induced Ca2+ transients during mitosis, suggesting that the mitotic Ca2+ transients are dependent on the release and sequestration of the Ca2+-releasing factor. Consequently, one prediction is that PLC{zeta} may localise to the nascent pronuclei in PLC{zeta}-activated eggs. Our results indicate that PLC{zeta} is indeed localised to the pronuclei at interphase following meiosis and mitosis. Furthermore, we show not only that nuclear sequestration plays a causal role in inhibiting the activity of PLC{zeta}, but also that the molecular mechanism of sequestration is a predicted NLS present in the X-Y linker of PLC{zeta}. This NLS appears to be a characteristic feature of PLC{zeta} since, although different in precise sequence, a NLS is also predicted in the X-Y linker region of the human and monkey PLC{zeta} (Cox et al., 2000Go).

Our data shows that sequestration of PLC{zeta} in the nuclear space is the causal link with inhibition of its activity in the Ins(1,4,5)P3 production that leads to Ca2+ release. The reason for this is unclear, but it may be related to access to its relevant substrate. In contrast to most other PLC isoforms, PLC{zeta} lacks a pleckstrin homology domain (Saunders et al., 2002Go) that targets plasma membrane phosphatidylinositol 4,5-bisphosphate (Ins(4,5)P2) (Halet et al., 2002Go). At present the location of the Ins(4,5)P2 substrate used by PLC{zeta} in eggs is unknown, but it is possible that PLC{zeta} may use Ins(4,5)P2 associated with intracellular organelles (Rice et al., 2000Go). The way PLC{zeta} is targeted within the cytoplasm might also explain why it is distinct from other mammalian PLCs and how this PLC activity in sperm extracts can hydrolyse Ins(4,5)P2 in eggs and egg homogenates, whereas PLCs of the ß, {gamma} and {delta} class are inactive (Jones et al., 2000Go).

It should be noted that there are a number of other factors that have already been shown to affect the ability of mouse eggs and embryos to generate Ca2+ oscillations. For example, it has been shown that Ins(1,4,5)P3 receptors are down-regulated after fertilisation, or sperm extract injection (Brind et al., 2000Go; Jellerette et al., 2000Go). After the start of Ca2+ oscillations at fertilisation there are also changes in the endoplasmic reticulum as seen by a decrease in cortical clusters (Kline et al., 1999Go; Fitzharris et al., 2003Go). Both of these factors may reduce the sensitivity of mouse zygotes to Ins(1,4,5)P3-induced Ca2+ release. They may account for the finding that Ca2+ oscillations eventually terminate after injection of the NLS mutant PLC{zeta} that does not localise to pronuclei. However, the changes in Ins(1,4,5)P3 sensitivity of the zygote do not explain why Ca2+ oscillations normally cease around the time of pronuclear formation, nor do they explain why the oscillations return during the first mitosis. These changes in Ins(1,4,5)P3 receptor sensitivity also fail to explain why Ca2+ oscillations during the first mitosis are only seen in fertilised zygotes and not in parthenogenetic embryos. Since this cell cycle dependency of Ca2+ release is a particular feature of a fertilised zygote, our current data also provides another specific example of PLC{zeta} injection precisely mimicking the sperm from fertilisation through to the first mitotic division. They add further weight of evidence to the idea that PLC{zeta} is the agent used by the sperm to trigger Ca2+ release in eggs at fertilisation (Saunders et al., 2002Go).

Our current data provides a simple molecular explanation for the cell cycle dependency of Ca2+ oscillations in early mouse embryos. In this model (Fig. 6) we suggest that the Ca2+ oscillations at fertilisation are generated by the diffusion of sperm PLC{zeta} into the egg cytoplasm after gamete fusion. As a result of pronuclear formation we propose that PLC{zeta} is then transported into the newly formed pronuclei. This leads to the cessation of Ins(1,4,5)P3 production and Ca2+ release in the interphase zygote cytoplasm. When pronuclear envelope breakdown occurs, as the zygote enters first mitosis, PLC{zeta} is released into the cytoplasm and generates the mitotic Ca2+ oscillations that then cease when the two-cell stage nuclei form. As well as providing a potential explanation for the observed pattern of Ca2+ signals in mammalian zygotes, this localisation could represent a novel mechanism for the regulation of Ins(1,4,5)P3 generation in cells.



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Fig. 6. Model for the regulation of Ca2+ oscillations during the first cell cycle. Model whereby PLC{zeta} (green) is introduced from the sperm into the egg upon fusion. Long-lasting Ca2+ oscillations are maintained while PLC{zeta} remains in the cytosol. During the initial phase of Ca2+ oscillations there is downregulation of InsP3 receptors and a change in the endoplasmic reticulum (ER): a decrease in cortical clusters. Both of these changes may lead to a change in the early phase of Ca2+ oscillations. When the pronuclei form, indicating the start of interphase, PLC{zeta} is transported into the nucleoplasm of the pronuclei terminating the Ca2+ oscillations. No further increases in Ca2+ are observed until the nuclear membranes breakdown upon mitosis entry, releasing PLC{zeta} back into the cytosol. Again, Ca2+ oscillations are sustained until the nuclear membranes reform in the two daughter cells and PLC{zeta} is imported into the nuclei.

 


    Acknowledgments
 
This work was funded by a Wellcome Trust grant awarded to K.S. and by Strategic Infrastructure Fund (SIF) and Knowledge Exploitation Fund (KEF) grants to F.A.L. from University of Wales College of Medicine and Education and Learning for Wales, respectively.


    References
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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