Article |
Address correspondence to Guillaume Halet, Department of Physiology, University College London, Gower Street, London WC1E 6BT, England, UK. Tel.: 0207-679-3229. Fax: 0207-383-7005. email: g.halet{at}ucl.ac.uk; or John Carroll, Department of Physiology, University College London, Gower Street, London WC1E 6BT, England, UK. Tel.: 0207-679-3229. Fax: 0207-383-7005. email: j.carroll{at}ucl.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: calcium; PKC; oscillations; GFP; influx
Abbreviations used in this paper: BIM, bisindolylmaleimide I; cPKC, conventional protein kinase C; DiC8, 1,2-dioctanoyl sn-glycerol; InsP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; SOC, store-operated channel; SOCE, store-operated calcium entry.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
At fertilization, hydrolysis of PIP2 and generation of InsP3 by PLC activity triggers the Ca2+ signal necessary for egg activation and the initiation of embryonic development (Stricker, 1999). In mammalian eggs, fertilization is characterized by the generation of low frequency Ca2+ oscillations due to the opening of the InsP3-sensitive Ca2+ release channels in the ER (Miyazaki et al., 1993). The mechanism underlying these Ca2+ oscillations has recently been suggested to require a novel sperm-borne PLC, PLC (Saunders et al., 2002). Fertilization is thus expected to activate a classical PIP2 hydrolysis pathway leading to the generation of InsP3 and DAG.
Fertilization-induced Ca2+ oscillations proceed for several hours and are the primary trigger for cortical granule exocytosis, exit from metaphase II arrest, and entry into the first mitotic division (Kline and Kline, 1992a; Xu et al., 1994). The number of Ca2+ oscillations has recently been proposed to differentially regulate these events (Ducibella et al., 2002). How the amplitude and frequency of these Ca2+ oscillations are decoded into specific activation events by the fertilized egg remains unclear. In somatic cells, PKC is a major downstream effector of Ca2+ signals, decoding Ca2+ oscillations into corresponding bursts of PKC substrate phosphorylation (Oancea and Meyer, 1998; Violin et al., 2003). In mammalian eggs, although biochemical assays have reported an increase in PKC activity as early as 10 min after insemination (Tatone et al., 2003), little is known about the kinetics and role(s) of PKC activation at fertilization.
PKCs form a large family of serine/threonine kinases involved in a multitude of cellular functions from cell growth and differentiation to secretion, gene expression, and regulation of other signaling pathways. 10 mammalian PKC isotypes have been described and classified into three major subfamilies, according to their structure and cofactor requirements (Mellor and Parker, 1998; Newton, 2001, 2003): conventional PKCs (cPKCs)namely PKC, ßI, ßII, and
are activated by negatively charged phospholipids and DAG in a Ca2+-dependent manner; in contrast, novel PKCs (
,
,
, and
) do not require Ca2+ for activation, but are regulated by anionic lipids and DAG, whereas atypical PKCs (
and
/
) require neither Ca2+ nor DAG for activation, but do require negatively charged phospholipids. Activation of PKCs requires the release of an autoinhibitory interaction between the NH2-terminal pseudosubstrate motif and the COOH-terminal catalytic core (Oancea and Meyer, 1998; Newton, 2001). According to current models, activation of cPKCs involves the sequential binding of Ca2+ and DAG to their respective binding sites on the kinases, the C2 and C1 domains (Oancea and Meyer, 1998; Violin et al., 2003). The binding of Ca2+ ions to the C2 domain increases its affinity for phosphatidylserine (Verdaguer et al., 1999; Stahelin et al., 2003) and results in the translocation of cPKCs to the plasma membrane, where DAG binding to the C1 domain provides maximal kinase activity. cPKC translocation to the plasma membrane is therefore regarded as a sign of cPKC activation (Oancea and Meyer, 1998; Newton, 2001; Violin et al., 2003).
Numerous PKC isotypes have been identified in mouse eggs at the mRNA or protein level, including cPKCs and
(Luria et al., 2000; Pauken and Capco, 2000; Tatone et al., 2003; Viveiros et al., 2003). Immunolocalization or staining with a fluorescently labeled PKC inhibitor have revealed the translocation of some PKC isoforms to the cortex of fertilized mammalian eggs (Gallicano et al., 1995, 1997; Luria et al., 2000; Eliyahu and Shalgi, 2002; Fan et al., 2002), raising the possibility that PKC could be a major downstream effector of Ca2+ oscillations at fertilization. However, these data were obtained in populations of eggs fixed some time after sperm addition, and they provided little information on the kinetics and regulation of PKC activation in living eggs.
In this work, we have imaged GFP fusion constructs of cPKCs and [Ca2+]i simultaneously in living mouse eggs to test the hypothesis that fertilization-induced Ca2+ oscillations are decoded by PKC. We show that fertilization-induced Ca2+ transients trigger the translocation of cPKCs to the egg membrane, and that this translocation is shaped by the frequency and amplitude of Ca2+ release. In addition, we provide evidence for a major role of cPKCs in the sustaining of long-lasting oscillations in fertilized eggs, via the regulation of store-operated Ca2+ influx.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
[Ca2+]i rises to micromolar levels during fertilization-induced Ca2+ spikes
Because of its high affinity for Ca2+ (in vitro Kd reported as 140 nM; Takahashi et al., 1999), it is likely that Fura Red becomes saturated under conditions where [Ca2+]i exceeds micromolar levels, leading to an underestimation of the relative amplitudes of the Ca2+ transients and spikes. To estimate the maximal amplitude of the Ca2+ transients without worrying about indicator saturation, and to investigate the possibility that Ca2+ spikes reach high concentration critical for C2-GFP translocation, we monitored fertilization-induced Ca2+ oscillations with the low affinity Ca2+ indicator Mag-Fura-2 (Kd = 25 µM in vitro; Takahashi et al., 1999). The first fertilization Ca2+ transient appeared as a succession of 34 sharp Ca2+ spikes, whereas subsequent Ca2+ transients were detected as smaller, rather monophasic Ca2+ spikes (Fig. 3 A). Estimation of [Ca2+]i from 420-nm fluorescence changes was done according to Ogden et al. (1995) using a Kd value of 25 µM, and after obtaining the Fmax value with ionomycin (see Materials and methods). On average, [Ca2+]i was found to rise up to 3.1 ± 0.5 µM (n = 7) at the peak of the first Ca2+ spike of the first transient (Fig. 3 B). All subsequent transients were smaller, but transiently reached 12 µM. The Ca2+ plateau during the first transient was estimated to reach up to 1 µM. Overall, the micromolar [Ca2+]i changes observed at fertilization exhibited a pattern very similar to the dynamics of C2-GFP translocation, suggesting that the threshold [Ca2+]i required to recruit the C2 domain at the membrane was in the 13-µM range.
|
|
|
|
|
To examine the contribution of extracellular Ca2+ influx in the acceleration of Ca2+ oscillations after PKC activation, the effect of PMA at fertilization was examined after extracellular Ca2+ had been chelated. Eggs were first fertilized in a normal Ca2+-containing medium (1.8 mM [Ca2+]) to trigger oscillations; then EGTA (3 mM) was added to the medium, resulting in a dramatic decrease in oscillation frequency and ultimately to the arrest of oscillations (Fig. 6 E). Addition of PMA did not stimulate Ca2+ release, nor did it increase the frequency of oscillations (Fig. 6 E), demonstrating that in the absence of extracellular Ca2+, PMA has no stimulatory effect on intracellular Ca2+ release. The effect of EGTA was overcome by raising extracellular [Ca2+] to 6 mM, as indicated by the resumption of Ca2+ oscillations (Fig. 6 E).
Inhibition of PKCs suppresses Ca2+ oscillations at fertilization
The data described above suggest that activation of PKC promotes Ca2+ oscillations at fertilization. To confirm this finding, we examined fertilization-induced Ca2+ transients in eggs treated with the PKC inhibitor BIM. In preliminary experiments, BIM was found to strongly inhibit second polar body emission in fertilized eggs (70% inhibition; unpublished data), confirming an earlier report (Gallicano et al., 1997) and suggesting that PKC plays a major role in cell cycle resumption during egg activation. Gallicano et al. (1997) reported that polar body emission was also inhibited by a membrane-permeant inhibitory peptide mimicking PKC pseudosubstrate; however, we and others (Ducibella and LeFevre, 1997) found that this compound was toxic for the eggs at the concentration required to inhibit polar body emission.
Exposure to BIM did not affect the fertilizability of mouse eggs, but dramatically altered the duration of Ca2+ oscillations. Typically, the first transient was followed by 37 small, short-lived Ca2+ oscillations, ending with an aborted Ca2+ transient (Fig. 7 A). The overall duration of the Ca2+ oscillations in the presence of BIM never exceeded 45 min (against 34 h in controls). Interestingly, Ca2+ transients resumed after raising extracellular [Ca2+] to 6 mM (Fig. 7 A), suggesting that the inhibitory effect of BIM was not due to a failure of the Ca2+ release machinery, but rather to a deficit in the supply of Ca2+ to refill the stores. However, this recovery was transient, as oscillations stopped with an aborted Ca2+ transient after a few minutes (Fig. 7 A).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Micromolar Ca2+ spikes trigger cPKC translocation to the egg membrane at fertilization
One enigmatic feature of the fertilization Ca2+ transients in mammalian eggs is the presence of Ca2+ spikes on the top of the transients (Cuthbertson and Cobbold, 1985; Jones et al., 1995; Deguchi et al., 2000). Our data reveal that these Ca2+ spikesmounting up to 3 µM [Ca2+]i during the first transientare the trigger for cPKC translocation to the egg plasma membrane after spermegg fusion. The C2 domain of the kinase was found to act as an amplitude detector, driving translocation selectively when micromolar Ca2+ spikes are generated. Thus, the C2 domain behaved like a low affinity Ca2+ sensor, a property that was retained in the context of the full-length cPKCs.
Interestingly, cPKCs were also found to act as frequency detectors during these rapid Ca2+ spikes, as shown by the cumulative cPKC recruitment at the membrane, resulting in an increase in the amplitude of translocation. In addition, a residual pool of cPKCs remained associated with the membrane for the duration of the Ca2+ transients. These two features were not observed with the isolated C2 domain, suggesting that full-length cPKCs established additional interactions with the plasma membrane after translocation. This difference may be accounted for by interaction of full-length cPKCs with DAG, as suggested by the effect of DiC8 on cPKC dissociation from the membrane (Oancea and Meyer, 1998; Tanimura et al., 2002; present study). However, using C1-GFP, we could not detect the generation of DAG at fertilization. The reason for this may be that DAG production is limited and insufficient to trigger a detectable C1-GFP translocation. In support of this idea, we previously reported that plasma membrane PIP2 hydrolysis could not be detected at fertilization using PH-GFP, arguing for a rather low PIP2 turnover (Halet et al., 2002). An alternative possibility is that PIP2 itself could ensure maximal kinase recruitment and activation in the absence of DAG (Chauhan and Brockerhoff, 1988; Lee and Bell, 1991; Kochs et al., 1993; Pap et al., 1993; Corbalán-García et al., 2003). This possibility is consistent with the Ca2+-dependent increase in plasma membrane PIP2 during fertilization-induced Ca2+ release (Halet et al., 2002). Finally, the interaction with specific anchoring proteins, such as the so-called receptors for activated C-kinase, may localize cPKCs in proximity to their substrates at the plasma membrane (Mochly-Rosen and Gordon, 1998). Further experimental evidence will be needed to explore the possibility of a regulation of cPKC activity by PIP2 or anchoring proteins in mouse eggs.
Because cPKCs seem to play a major role in sustaining Ca2+ oscillations (see below), their activation by high amplitude Ca2+ spikes early after sperm fusion may provide a checkpoint ensuring that oscillations will proceed only in eggs displaying Ca2+ transients with the correct amplitude. Considering that their role has never been investigated before, our observations are the first evidence that these Ca2+ spikes are required to activate Ca2+-sensitive signaling proteins such as cPKCs. Other signaling proteins with a Ca2+-dependent C2 domain may follow a similar activation pattern at fertilization, providing an interesting direction for future analyses examining Ca2+ signaling at fertilization.
cPKCs support fertilization Ca2+ oscillations by regulating Ca2+ influx
We have identified a previously unknown role for PKC at fertilization, that of providing a positive feedback on the generation of Ca2+ oscillations. We show that PKC overexpression or PMA stimulation increases the frequency of Ca2+ oscillations while PMA stimulates SOCE, whereas PKC inhibition leads to a premature arrest of Ca2+ oscillations and the inhibition of SOCE. These data suggest that cPKCs may regulate SOCE, and consequently Ca2+ store refilling at fertilization.
Previous reports on mammalian eggs have attributed a role to PKC in the regulation of cortical granule exocytosis, resumption of the cell cycle, and second polar body formation at fertilization (Colonna and Tatone, 1993; Gallicano et al., 1997; Luria et al., 2000; Eliyahu and Shalgi, 2002; Fan et al., 2002). However, these reports did not consider the possibility that PKC activation/inhibition could alter the pattern of fertilization-induced Ca2+ release, the primary trigger for these activation events. In addition, Ducibella and LeFevre (1997) demonstrated that PKC inhibition with BIM did not alter cortical granule exocytosis nor cell cycle resumption at fertilization in mouse eggs, raising doubts on the actual involvement of PKC in these particular events. Interestingly, recent data suggest that the stimulatory action of phorbol esters on exocytosis may be mediated by members of the Munc13 family rather than PKCs (Rhee et al., 2002). On the other hand, the effect on the cell cycle may rely on the activation of PKC, which has been found to bind to the meiotic spindle and chromosomes in mouse eggs (Tatone et al., 2003; Viveiros et al., 2003). Thus, our data reveal that one physiological role for cPKCs at fertilization is to control the pattern of Ca2+ signaling by regulating Ca2+ influx.
Mouse eggs possess a Ca2+ influx pathway activated by Ca2+ store depletion, which has been proposed to contribute to store refilling and underlie long-lasting oscillations at fertilization (Kline and Kline, 1992b; McGuinness et al., 1996). Although the molecular nature of this pathway has not yet been investigated, it is likely to correspond to a store-operated channel (SOC; Berridge et al., 2003). In our assay, Ca2+ influx activated by store depletion appeared as a phasic increase in [Ca2+]i, followed by a sustained plateau at an intermediate level. The sustained plateau probably reflects a new equilibrium that is maintained in part by negative feedback by Ca2+ on the SOCs, as has been suggested previously in somatic cells and Xenopus oocytes (Petersen and Berridge, 1994; Berridge, 1995; Louzao et al., 1996). Our finding that the plateau is dramatically increased by activating PKC and abolished by inhibiting PKC suggests that PKC may promote Ca2+ influx by counteracting the negative feedback on SOCs. We suggest that SOCs or some accessory proteins at the egg plasma membrane are substrates for cPKC phosphorylation, with the effect of counteracting the rapid feedback inhibition by Ca2+ (Fig. 8).
|
In conclusion, our work reveals that cPKC activation at fertilization plays a crucial role in the regulation of the Ca2+ release machinery and the generation of long-lasting oscillations. This role for cPKCs at fertilization may have far-reaching effects because the pattern of Ca2+ oscillations dramatically affects activation events and developmental fate of the mouse embryo (Gordo et al., 2000; Ducibella et al., 2002). Further research to find the molecular identity of the SOC should help to elucidate whether it is a direct target of cPKCs, or whether an accessory protein is involved.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gamete collection and fertilization
Ovulated, metaphase II (MII)arrested eggs were recovered from hormone-primed MF1 mice and stored in H-KSOM medium containing BSA, as described previously (Marangos et al., 2003). Sperm from the epididymis of MF1 mice were released into T6 medium for capacitation, as described previously (Halet et al., 2002). In vitro fertilization was performed by adding 1020 µl of the sperm suspension into the incubation chamber containing zona-free MII eggs in 1 ml H-KSOM. All experiments were conducted at 38 ± 0.5°C.
Expression of GFP fusion proteins in mouse eggs
Plasmids encoding PKC-GFP, C1-GFP, and C2-GFP were donated by Tobias Meyer (Stanford University, Stanford, CA). The constructs were provided into the pHiro vector, which contains an SP6 promoter suitable for in vitro transcription (Oancea and Meyer, 1998). The EGFP-PKC
construct was previously described by Mostafavi-Pour et al. (2003), and was subcloned into pcDNA3.1. The resulting construct was verified by sequencing and restriction analysis. The plasmid encoding PH-GFP was provided by Tamas Balla (National Institutes of Health, Bethesda, MD). cRNAs encoding each of these constructs were made in vitro using the mMESSAGE mMACHINETM kit (Ambion). The cRNAs were polyadenylated, purified, and micro-injected into mouse MII eggs as described previously (Halet et al., 2002).
Confocal imaging, [Ca2+]i imaging, and data analysis
23 h after cRNA injection, eggs were loaded with 10 µM Fura Red-AM (for 10 min) and freed of their zona pellucida by incubation in acidic Tyrode's medium at 37°C. Zona-free eggs were transferred in an experimental chamber seated in a heated stage, and containing 1 ml H-KSOM medium without BSA. The changes in the distribution of GFP/EGFP-tagged proteins and [Ca2+]i at fertilization were monitored simultaneously at the equator of the cells using a confocal microscope (model LSM510; Carl Zeiss MicroImaging, Inc.), using a 20x (0.75 NA) lens or a 40x (1.3 NA) oil immersion lens. Excitation was provided by the 488-nm line of an argon laser, with the laser power set at 1% of maximum. GFP/EGFP and Fura Red fluorescence were collected simultaneously using BP505-530 and LP650 emission filters, respectively. Confocal time series were acquired at a rate of 1 frame every 5 s, and confocal settings (pin-hole size, detector gain) were the same in all experiments. Confocal data were analyzed using MetaMorph® (Universal Imaging Corp.) as previously published (Halet et al., 2002). In brief, regions of interest were drawn in the cytosol (C) or around the plasma membrane (PM), and changes in fluorescence intensity were measured during confocal time series. The value of the PM/C ratio was used as an index of membrane localization.
For ratiometric [Ca2+]i imaging, eggs were loaded with 2 µM Fura-2-AM (for 10 min) or 10 µM Fura Red-AM (for 10 min) 23 h after injection of cRNA encoding EGFP-PKC or injection buffer. Eggs were then freed of their zona pellucida and were transferred in a similar heated chamber as used for confocal imaging. Eggs were observed with the 20x (0.75 NA) lens of an inverted microscope (Axiovert; Carl Zeiss MicroImaging, Inc.) fitted with a cooled CCD camera (MicroMax; Princeton Instruments). Excitation wavelengths were adjusted to 340/380 nm (Fura-2) or 440/490 nm (Fura Red) using a monochromator (TILL Photonics). Camera shutter and monochromator settings were controlled using MetaFluor® (Universal Imaging Corp.). Emitted fluorescence was collected using a 520-nm long-pass filter (Fura-2) or a 600-nm long-pass filter (Fura Red) every 5 s. For [Ca2+]i measurements using Mag-Fura-2, eggs were injected with Mag-Fura-2 (1 mM in the injection pipette) at least 30 min before zona removal and recording of fertilization-induced Ca2+ transients. The estimated indicator concentration in the egg cytosol was in the range 2050 µM, according to an injection volume equal to 25% of the egg volume. The indicator was excited at 420 nm (Ogden et al., 1995) using the monochromator, and fluorescence was collected every 5 s using a 520-nm long-pass filter. [Ca2+]i was calculated according to Ogden et al. (1995) using the equation established by Grynkiewicz et al. (1985): [Ca2+]i = Kd (F - Fmin)/(Fmax - F), where Kd is the Mag-Fura-2 dissociation constant for Ca2+ binding, F is the experimentally measured fluorescence intensity, Fmin is the F value for the Ca2+-free indicator, and Fmax is the F value for the Ca2+-saturated indicator. Fmin was chosen as the fluorescence value immediately before the [Ca2+]i change because resting [Ca2+]i does not affect the fluorescence of the low affinity indicator (Ogden et al., 1995), whereas Fmax was obtained by adding ionomycin to saturate the dye with Ca2+. The Kd value was set at 25 µM (Takahashi et al., 1999). Fluorescence data from MetaMorph® or MetaFluor® analyses were exported to Microsoft Excel 2000 to generate line graphs.
SOCE assay
SOCE was monitored in zona-free mouse eggs loaded with Fura-2-AM. Ca2+ stores were first depleted with 10 µM thapsigargin in Ca2+-free H-KSOM. This treatment causes a transient rise in [Ca2+]i that has been observed previously (Kline and Kline, 1992b; McGuinness et al., 1996). SOCE was visualized on the readmission of CaCl2 in the extracellular medium at the final concentration of 1.8 mM.
Online supplemental material
Fig. S1 shows how the PKC inhibitor BIM reverses the stimulatory effect of PMA on the frequency of Ca2+ oscillations. Videos 1 and 2 show the membrane translocation of EGFP-PKC and C2-GFP, respectively, during the first fertilization Ca2+ transient, as illustrated in Fig. 1 A and Fig. 2 A. Videos 3 and 4 show the translocation of C1-GFP and PH-GFP, respectively, when ionomycin is added to unfertilized eggs, as illustrated in Fig. 4 A. All supplemental material is available online at http://www.jcb.org/cgi/content/full/jcb.200311023/DC1.
![]() |
Acknowledgments |
---|
This work was supported by a Medical Research Council Career establishment grant to J. Carroll and by the Wellcome Trust.
Submitted: 5 November 2003
Accepted: 20 February 2004
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berridge, M.J. 1993. Inositol trisphosphate and calcium signalling. Nature. 361:315325.[CrossRef][Medline]
Berridge, M.J. 1995. Capacitative calcium entry. Biochem. J. 312:111.[Medline]
Berridge, M.J., M.D. Bootman, and H.L. Roderick. 2003. Calcium signalling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4:517529.[CrossRef][Medline]
Chauhan, V.P., and H. Brockerhoff. 1988. Phosphatidylinositol-4,5-bisphosphate may antecede diacylglycerol as activator of protein kinase C. Biochem. Biophys. Res. Commun. 155:1823.[Medline]
Colonna, R., and C. Tatone. 1993. Protein kinase C-dependent and independent events in mouse egg activation. Zygote. 1:243256.[Medline]
Corbalán-García, S., J. García-García, J.A. Rodríguez-Alfaro, and J.C. Gómez-Fernández. 2003. A new phosphatidylinositol 4,5-bisphosphate-binding site located in the C2 domain of protein kinase C. J. Biol. Chem. 278:49724980.
Cuthbertson, K.S., and P.H. Cobbold. 1985. Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca2+. Nature. 316:541542.[Medline]
Deguchi, R., H. Shirakawa, S. Oda, T. Mohri, and S. Miyazaki. 2000. Spatiotemporal analysis of Ca2+ waves in relation to the sperm entry site and animal-vegetal axis during Ca2+ oscillations in fertilized mouse eggs. Dev. Biol. 218:299313.[CrossRef][Medline]
Ducibella, T., and L. LeFevre. 1997. Study of protein kinase C antagonists on cortical granule exocytosis and cell-cycle resumption in fertilized mouse eggs. Mol. Reprod. Dev. 46:216226.[CrossRef][Medline]
Ducibella, T., D. Huneau, E. Angelichio, Z. Xu, R.M. Schultz, G.S. Kopf, R. Fissore, S. Madoux, and J.P. Ozil. 2002. Egg-to-embryo transition is driven by differential responses to Ca2+ oscillation number. Dev. Biol. 250:280291.[CrossRef][Medline]
Eliyahu, E., and R. Shalgi. 2002. A role for protein kinase C during rat egg activation. Biol. Reprod. 67:189195.
Fan, H.Y., C. Tong, M.Y. Li, L. Lian, D.Y. Chen, H. Schatten, and Q.Y. Sun. 2002. Translocation of the classic protein kinase C isoforms in porcine oocytes: implications of protein kinase C involvement in the regulation of nuclear activity and cortical granule exocytosis. Exp. Cell Res. 277:183191.[CrossRef][Medline]
Gallicano, G.I., R.W. McGaughey, and D.G. Capco. 1995. Protein kinase M, the cytosolic counterpart of protein kinase C remodels the internal cytoskeleton of the mammalian egg during activation. Dev. Biol. 167:482501.[CrossRef][Medline]
Gallicano, G.I., R.W. McGaughey, and D.G. Capco. 1997. Activation of protein kinase C after fertilization is required for remodeling the mouse egg into the zygote. Mol. Reprod. Dev. 46:587601.[CrossRef][Medline]
Gordo, A.C., H. Wu, C.L. He, and R.A. Fissore. 2000. Injection of sperm cytosolic factor into mouse metaphase II oocytes induces different developmental fates according to the frequency of [Ca2+]i oscillations and oocyte age. Biol. Reprod. 62:13701379.
Grynkiewicz, G., M. Poenie, and R.Y. Tsien. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:34403450.[Abstract]
Halet, G., R. Tunwell, T. Balla, K. Swann, and J. Carroll. 2002. The dynamics of plasma membrane PtdIns(4,5)P2 at fertilization of mouse eggs. J. Cell Sci. 115:21392149.
Jones, K.T., J. Carroll, and D.G. Whittingham. 1995. Ionomycin, thapsigargin, ryanodine, and sperm induced Ca2+ release increase during meiotic maturation of mouse oocytes. J. Biol. Chem. 270:66716677.
Kline, D., and J.T. Kline. 1992a. Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. Dev. Biol. 149:8089.[Medline]
Kline, D., and J.T. Kline. 1992b. Thapsigargin activates a calcium influx pathway in the unfertilized mouse egg and suppresses repetitive calcium transients in the fertilized egg. J. Biol. Chem. 267:1762417630.
Kochs, G., R. Hummel, B. Fiebich, T.F. Sarre, D. Marme, and H. Hug. 1993. Activation of purified human protein kinase C and ß I isoenzymes in vitro by Ca2+, phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate. Biochem. J. 291:627633.[Medline]
Lee, M.H., and R.M. Bell. 1991. Mechanism of protein kinase C activation by phosphatidylinositol 4,5-bisphosphate. Biochemistry. 30:10411049.[Medline]
Louzao, M.C., C.M.P. Ribeiro, G.S.J. Bird, and J.W. Putney, Jr. 1996. Cell type-specific modes of feedback regulation of capacitative calcium entry. J. Biol. Chem. 271:1480714813.
Luria, A., T. Tennenbaum, Q.Y. Sun, S. Rubinstein, and H. Breitbart. 2000. Differential localization of conventional protein kinase C isoforms during mouse oocyte development. Biol. Reprod. 62:15641570.
Maasch, C., S. Wagner, C. Lindschau, G. Alexander, K. Buchner, M. Gollasch, F.C. Luft, and H. Haller. 2000. Protein kinase C targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca2+]i. FASEB J. 14:16531663.
Marangos, P., G. FitzHarris, and J. Carroll. 2003. Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei. Development. 130:14611472.
McGuinness, O.M., R.B. Moreton, M.H. Johnson, and M.J. Berridge. 1996. A direct measurement of increased divalent cation influx in fertilised mouse oocytes. Development. 122:21992206.
Mellor, H., and P.J. Parker. 1998. The extended protein kinase C superfamily. Biochem. J. 332:281292.[Medline]
Miyazaki, S., H. Shirakawa, K. Nakada, and Y. Honda. 1993. Essential role of the inositol 1,4,5-trisphosphate receptor/Ca2+ release channel in Ca2+ waves and Ca2+ oscillations at fertilization of mammalian eggs. Dev. Biol. 158:6278.[CrossRef][Medline]
Mochly-Rosen, D., and A.S. Gordon. 1998. Anchoring proteins for protein kinase C: a means for isozyme selectivity. FASEB J. 12:3542.
Mostafavi-Pour, Z., J.A. Askari, S.J. Parkinson, P.J. Parker, T.T. Ng, and M.J. Humphries. 2003. Integrin-specific signaling pathways controlling focal adhesion formation and cell migration. J. Cell Biol. 161:155167.
Newton, A.C. 2001. Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem. Rev. 101:23532364.[CrossRef][Medline]
Newton, A.C. 2003. Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm. Biochem. J. 370:361371.[CrossRef][Medline]
Oancea, E., and T. Meyer. 1998. Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals. Cell. 95:307318.[Medline]
Oancea, E., M.N. Teruel, A.F.G. Quest, and T. Meyer. 1998. Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells. J. Cell Biol. 140:485498.
Ogden, D., K. Khodakhah, T. Carter, M. Thomas, and T. Capiod. 1995. Analogue computation of transient changes of intracellular free Ca2+ concentration with the low affinity Ca2+ indicator furaptra during whole-cell patch-clamp recording. Pflugers Arch. 429:587591.[Medline]
Pap, E.H.W., P.I.H. Bastiaens, J.W. Borst, P.A.W. van den Berg, A. van Hoek, G.T. Snoek, K.W.A. Wirtz, and A.J.W.G. Visser. 1993. Quantitation of the interaction of protein kinase C with diacylglycerol and phosphoinositides by time-resolved detection of resonance energy transfer. Biochemistry. 32:1331013317.[Medline]
Pauken, C.M., and D.G. Capco. 2000. The expression and stage-specific localization of protein kinase C isotypes during mouse preimplantation development. Dev. Biol. 223:411421.[CrossRef][Medline]
Petersen, C.C.H., and M.J. Berridge. 1994. The regulation of capacitative calcium entry by calcium and protein kinase C in Xenopus oocytes. J. Biol. Chem. 269:3224632253.
Putney, J.W., Jr. 1990. Capacitative calcium entry revisited. Cell Calcium. 11:611624.[Medline]
Rhee, J.S., A. Betz, S. Pyott, K. Reim, F. Varoqueaux, I. Augustin, D. Hesse, T.C. Südhof, M. Takahashi, C. Rosenmund, and N. Brose. 2002. ß Phorbol ester- and diacylglycerol-induced augmentation of transmitter release is mediated by Munc13s and not by PKCs. Cell. 108:121133.[Medline]
Sakai, N., K. Sasaki, N. Ikegaki, Y. Shirai, Y. Ono, and N. Saito. 1997. Direct visualization of the translocation of the -subspecies of protein kinase C in living cells using fusion proteins with green fluorescent protein. J. Cell Biol. 139:14651476.
Saunders, C.M., M.G. Larman, J. Parrington, L.J. Cox, J. Royse, L.M. Blayney, K. Swann, and F.A. Lai. 2002. PLC: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development. Development. 129:35333544.
Schaefer, M., N. Albrecht, T. Hofmann, T. Gudermann, and G. Schultz. 2001. Diffusion-limited translocation mechanism of protein kinase C isotypes. FASEB J. 15:16341636.
Stahelin, R.V., J.D. Rafter, S. Das, and W. Cho. 2003. The molecular basis of differential subcellular localization of C2 domains of protein kinase C- and group IVa cytosolic phospholipase A2. J. Biol. Chem. 278:1245212460.
Stricker, S.A. 1999. Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev. Biol. 211:157176.[CrossRef][Medline]
Takahashi, A., P. Camacho, J.D. Lechleiter, and B. Herman. 1999. Measurement of intracellular calcium. Physiol. Rev. 79:10891125.
Tanimura, A., A. Nezu, T. Morita, N. Hashimoto, and Y. Tojyo. 2002. Interplay between calcium, diacylglycerol, and phosphorylation in the spatial and temporal regulation of PKC-GFP. J. Biol. Chem. 277:2905429062.
Tatone, C., S. Della Monache, A. Francione, L. Gioia, B. Barboni, and R. Colonna. 2003. Ca2+-independent protein kinase C signalling in mouse eggs during the early phases of fertilization. Int. J. Dev. Biol. 47:327333.[CrossRef][Medline]
Teruel, M.N., and T. Meyer. 2000. Translocation and reversible localization of signaling proteins: a dynamic future for signal transduction. Cell. 103:181184.[Medline]
Toullec, D., P. Pianetti, H. Coste, P. Bellevergue, T. Grand-Perret, M. Ajakane, V. Baudet, P. Boissin, E. Boursier, F. Loriolle, et al. 1991. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266:1577115781.
Várnai, P., and T. Balla. 1998. Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools. J. Cell Biol. 143:501510.
Verdaguer, N., S. Corbalán-García, W.F. Ochoa, I. Fita, and J.C. Gómez-Fernández. 1999. Ca2+ bridges the C2 membrane-binding domain of protein kinase C directly to phosphatidylserine. EMBO J. 18:63296338.
Violin, J.D., J. Zhang, R.Y. Tsien, and A.C. Newton. 2003. A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J. Cell Biol. 161:899909.
Viveiros, M.M., M. O'Brien, K. Wigglesworth, and J.J. Eppig. 2003. Characterization of protein kinase C- in mouse oocytes throughout meiotic maturation and following egg activation. Biol. Reprod. 69:14941499.
Xu, Z., G.S. Kopf, and R.M. Schultz. 1994. Involvement of inositol 1,4,5-trisphosphate-mediated Ca2+ release in early and late events of mouse egg activation. Development. 120:18511859.