The increase in intracellular pH associated with Xenopus egg activation is
a Ca2+-dependent wave
NATHALIE GRANDIN and MICHEL CHARBONNEAU*
Laboratoire de Biologie el Ginttique du Diveloppement, URA CNRS 256, University de Rennes I, Campus de Beaulieu, 35042 Rennes,France
•Present address: The Research Institute of Scripps Clinic, Department of Molecular Biology (MB7 and MB3), 10666 North Torrey PinesRoad, La Jolla, California 92037, U.S.A.
Summary
In Xenopus eggs, the transient increase in intracellularfree calcium ([Ca2+]0, or Ca2+ transient, which occurs1-3 min after egg activation, is likely to be partlyresponsible for the release of the cell cycle blockade. Inthe present study, we have used microinjection ofBAPTA or EGTA, two potent chelators of Ca2+, tobuffer [Ca2+]| at various steps during Xenopus eggactivation and evaluate the impact on some of theassociated events. Microinjection of either one of theCa2+ chelators into unactivated eggs prevented eggactivation without, however, lowering [Ca2+]|, sugges-ting that only physiological [Ca2+]| changes, but not[Ca2+]i levels, were affected by the Ca2+ buffer. WhenBAPTA was microinjected around the time of occur-rence of the Ca2+ transient, the egg activation-associatedincrease in intracellular pH (pH|) was clearly delayed.That delay was not due to a general slowing down of the
cell cycle, since under the same conditions of microinjec-tion of BAPTA the kinetics of MPF (a universal M-phasepromoting factor) inactivation were unaffected. Theseresults represent the first indication that the Ca2+
transient participates in determining the time of in-itiation of the pH| increase during Xenopus egg acti-vation. The present results also demonstrate that the eggactivation-associated pH| changes (a slight, transientdecrease in pH( followed by a permanent increase in pH,)proceed as a wave propagating from the site of triggeringof egg activation. Experiments of local microinjection ofBAPTA support the view that the pH wave is aconsequence of the Ca2+ wave, which it follows closely.
In Xenopus unfertilized eggs, the arrest in metaphase 2of meiotic maturation is under the control of MPF, auniversal M-phase promoting factor (recently reviewedby Hunt, 1989; Lohka, 1989; Dor6e, 1990; Mailer, 1990,1991; Nurse, 1990), first revealed in amphibian oocytes(Masui and Markert, 1971). In Xenopus oocytes andeggs, MPF has a high activity during metaphase and alow activity during interphase (Gerhart et al. 1984).Around 8 min after triggering of egg activation inXenopus, MPF activity drops, a reaction that permitsthe completion of meiotic maturation and drives thenewly activated or fertilized egg into the first mitoticinterphase (Gerhart et al. 1984). We have previouslydrawn attention to the finding that the increase inintracellular pH (pHj) associated with egg activationoccurred simultaneously with the inactivation of MPF,in both Xenopus and Pleurodeles, another amphibianthat has a naturally longer cell cycle than that ofXenopus (Grandin and Charbonneau, 1991a). The
close relationship between MPF activity and pHjchanges in amphibian eggs is attested by the finding thatboth activities fluctuate in phase during the embryoniccell cycle and that they are also functionally related toeach other (Grandin and Charbonneau, 1990a, 1991a).
Our interest in MPF activity and pH, variations isdirected by the fact that both activities representuniversal mechanisms of control of the cell cycle. Thep34cdc2 kinase and cyclins, the two components of MPF,have been found to operate in all eukaryotic systems sofar studied, from yeast to man (reviewed by Nurse,1990; Mailer, 1991). Similarly, an increase in pH; hasbeen recorded in response to cell activation or, moregenerally, in association with a change in the metabolicstate of the cell or at the onset of cell proliferation inmany cell types (reviewed by Busa and Nuccitelli, 1984;Boron, 1986; Busa, 1986; Moolenaar, 1986; Epel andDub6, 1987), including Xenopus eggs (Webb andNuccitelli, 1981). In many cell types, cell activation,which often corresponds to a reinitiation of the cellcycle, is triggered, or at least signaled, by a transient
56 N. Grandin and M. Charbonneau
increase in intracellular free calcium activity ([Ca2+],),a so-called Ca2+ transient (reviewed by Berridge andIrvine, 1989; Meyer, 1991). This is also the case inactivating Xenopus eggs (Busa and Nuccitelli, 1985).Following the initial observation that addition of Ca2+
to amphibian egg extracts inactivated MPF (Meyerhofand Masui, 1977; Masui, 1982), it has recently beendemonstrated that a Ca2+-calmodulin-dependent pro-cess was required to produce the degradation of cyclin,a component of MPF, in Xenopus egg extracts (Lorca etal. 1991). On the other hand, there is no indication inthe literature concerning the mechanisms producing theincrease in pHj in Xenopus eggs. Moreover, thereaction itself does not depend on classical plasmamembrane ion exchangers (Webb and Nuccitelli, 1982;Grandin and Charbonneau, 1990b) and has no knownionic or metabolic origin, besides the assumption that itis a consequence of MPF inactivation (Grandin andCharbonneau, 1991a).
In the present work, we report that microinjection ofBAPTA (l,2-bis(2-aminophenoxy)ethane-./V,./V,A'',./V'-tetraacetic acid), a highly selective calcium-chelatingreagent (Tsien, 1980; Pethig et al. 1989; Speksnijder etal. 1989) into Xenopus eggs during the Ca transient,2.5-3 min after triggering of egg activation, results in adelay in the occurrence of the physiological increase inpH; with respect to control eggs microinjected withBAPTA/CaCl2 buffers. In contrast, under the sameconditions, there was no delay in the inactivation ofMPF with respect to controls, suggesting that theBAPTA-induced delay in the increase in pH| was notdue to a general lengthening of the cell cycle. Theseresults suggest that (i) the Ca transient plays a role indetermining the time-lapse before the onset of the pHresponse, but may not be necessary for the responseitself and, (ii) MPF inactivation can proceed in theabsence of a propagating Ca2+ wave. Finally, we reportthat the transient cytoplasmic acidification and thefollowing permanent cytoplasmic alkalinization bothproceed as a wave starting around the site of triggeringof egg activation. This represents, to our knowledge,the first description of an intracellular pH wave.Experiments of local microinjection of limited amountsof BAPTA demonstrate that the pH wave necessitatesCa2+ for its propagation and closely follows the Ca2+
wave.
Materials and methods
Biological material and solutionsMature (metaphase 2-arrested) eggs were expressed fromfemales of Xenopus laevis (reared in the laboratory), inducedto ovulate following injection of 900 i.u. of human chorionicgonadotropin (Organon, Saint Denis, France), and immedi-ately dejellied in Fl solution (see below) containing 2%cysteine, pH 7.8. The physiological Fl solution in whichdejellied eggs were immersed, modified from Hollinger andCorton (1980), contained: 31.2 mM NaCl, 1.8 mM KC1, 1.0raM CaCl2, 0.1 mM MgCl2, 2.0 mM NaHCO3, 1.9 mMNaOH, buffered with 10.0 mM Hepes at pH 7.4-7.5.BAPTA (l,2-bis(2-aminophenoxy)ethane-Af,N,A'',A''-tetra-
acetic acid) and EGTA (ethylene glycol-bis(/J-aminoethylether)N,A',A'',Af'-tetraacetic acid), both purchased fromSigma Chemical Company (St Louis, MO, USA), wereprepared as stock solutions of 100 mM (in 10 mM Hepes,adjusted to pH 7.5 with NaOH) and used alone or mixed withvarious amounts of CaCl2 or MgCl2.
Intracellular pH (pHj) and intracellular free calcium([Ca2+]i) measurements and microinjectionsIntracellular pH and Ca2+ microelectrodes were fabricatedand calibrated as described by Grandin and Charbonneau(1991b,c). The resins, contained in the microelectrode tips,used to detect intracellular ion activities, were hydrogen ionionophore I-cocktail A, designed by Ammann et al. (1981),and calcium ionophore I-cocktail A, designed by Lanter et al.(1982), both purchased from Fluka Chemical Corporation(Buchs, Switzerland). These ion-selective microelectrodespermit a very rapid (of the order of a few seconds), selectiveand sensitive detection of the ion activities concerned. It isimportant to note that it is necessary to use two microelec-trodes for each ion activity measured: a potential microelec-trode measuring only the membrane potential (Em) and anion-selective microelectrode measuring the ion activity plusthe membrane potential. The membrane potential recordedby the potential microelectrode was continuously subtractedfrom the total signal recorded by the ion-selective microelec-trode at the pen recorder input. Unactivated dejellied eggs,immersed in Fl solution in the recording chamber, wereimpaled with microelectrodes and remained unactivated afterachievement of impalement (no anesthetic was used). Foradditional details concerning the electrophysiological set-upand microelectrode impalement, see Grandin and Charbon-neau (1991b,c). Microinjections were performed as previouslydescribed (Grandin and Charbonneau, 1990b).
Egg activationActivation was triggered by pricking the egg cortex, aprocedure that allows Ca2+ to leak from the external mediuminto the cytoplasm (Wolf, 1974). In Xenopus eggs, artificialactivators, which all act by increasing intracellular free Ca2+,produce exactly the same events as those elicited by thesperm, with the exception of cell division. A major differencebetween prick-induced activation and activation induced byapplication of A23187, a calcium ionophore that activates theegg by releasing Ca2+ from intracellular stores even in theabsence of extracellular Ca2+ (Steinhardt et al. 1974), is thatpricking initiates the reaction from a single point, whereasA23187 initiates the activation reaction simultaneously fromseveral regions of the egg cortex (Charbonneau and Picheral,1983). In this respect, prick-induced egg activation moreclosely mimicks the physiological reaction induced by thesperm, which also proceeds as a wave starting from a singlepoint (Picheral and Charbonneau, 1982). Many of themetabolic reactions involved during anuran amphibian eggactivation proceed as propagating waves: the cortical reactionof exocytosis and the elongation of plasma membranemicrovilli (Picheral and Charbonneau, 1982), the opening ofCl~ and K+ channels participating in the initial plasmamembrane depolarization, the so-called activation potential(Jaffe et al. 1985; Kline and Nuccitelli, 1985), the Ca2+
transient (Busa and Nuccitelli, 1985) and the so-calledactivation waves, two successive waves of cortical movements(Hara and Tydeman, 1979; Takeichi and Kubota, 1984; Klineand Nuccitelli, 1985). It was therefore important, in thepresent study, to know exactly the spatial localization of thesite from which egg activation was initiated, that is the site ofpricking, with respect to the site of microinjection of the Ca2+
Ca2+ microelectrode
Animalhemisphere
pH microelectrode
Em microelectrodeT
Pricking
Egg activation
Em microelectrodeVegetal
Microinjection hemispherepipet
A Ca2+-dependent wave of intracellular pH change 57
forming interphasic nucleus, dejellied eggs were fixed for 24 hin Smith's fixative (Humason, 1972). After dehydration in aseries of ethanol and butyl alcohol, and embedding inparaffin, eggs were sectioned at 5 jim, stained with bisbenzi-mide (Hoechst 33258 or 33342, Sigma) to detect chromosomesand chromatin (Latt and Stetten, 1976; Critser and First,1986), and observed with a Leitz epifluorescence microscope.
Measurement of histone HI kinase activityHistone kinase activity in single Xenopus eggs, reflecting theirMPF (M-phase promoting factor) activity (see, for instance,Murray and Kirschner, 1989), was measured as described byFelix et al. (1989), using histone HI III-S from calf thymus(Sigma) and [}^2P]ATP (Amersham PB 218, Les Ulis,France). The filters were counted dry on the tritium channel.
Fig. 1. Schematic representation of the disposition ofmicroelectrodes and sites of pricking and microinjection, inXenopus eggs. This configuration was adopted in the wholestudy, with the exception of the experiments shown in Figs5 and 9. Dejellied mature eggs were immersed in therecording chamber and orientated, using forceps, animalpole (AP) up. Thus, the pigmented animal hemisphere(hatched zone) was always facing the experimentatorobserving from above, under a stereomicroscope. It shouldbe noted that this scheme is a perspective drawing in whichthe egg is viewed at an angle with respect to the vertical.Adopting such standard conditions was necessary, takinginto account the fact that many of the reactions associatedwith anuran egg activation proceed as waves propagatingfrom the site of triggering of egg activation (see referencesin Materials and methods). Unactivated eggs were eachimpaled with a pH microelectrode, a Ca2 microelectrodeand two potential microelectrodes (Em microelectrodes) asshown in the scheme. Once the electrical and ionicparameters had stabilized, the egg was prick-activated byrapidly withdrawing and re-impaling one of the Emmicroelectrodes (always the same, as shown in thescheme), which produced a local entry of external Ca2+,resulting in the triggering of egg activation (Wolf, 1974). Insome experiments, indicated in the text, a single egg wasimpaled with two pH microelectrodes or two Ca2+
microelectrodes and two Em microelectrodes.
chelator. This was particularly true when the time betweentriggering of egg activation and microinjection was short,because, for a given time, microinjecting into a region alreadyattained by the various waves of activation is not equivalent tomicroinjecting beyond these waves. Indeed, the Ca2+ tran-sient is not detected at the same time following egg activation,depending on the site of implantation of the Ca microelec-trode with respect to the site of pricking (see Busa andNuccitelli, 1985). It was therefore necessary for us tostandardize the conditions for microelectrode impalement,pricking and microinjection. These standard conditions aredescribed in Fig. 1. Criteria for Xenopus egg activationconsidered in the present study were: the activation potential(detected 2-5 seconds after pricking the egg cortex), elevationof the vitelline envelope (1-2 min), the Ca2+ transient (2-3min), the cortical contraction (3-4 min), the increase inintracellular pH (6-8 min) and the disappearance of thematuration spot (25-30 min).
Staining of the egg chromosomes and nucleusTo visualize the state of the chromosomes following release ofthe metaphase block during egg activation and that of the
Results
Microinjection of BAPT A or EGTA prevents eggactivation without affecting intracellular free Ccr+
levelsEGTA and BAPTA, two specific chelators of Ca2+,have already been used to prevent Xenopus eggactivation (Karsenti et al. 1984; Kline, 1988; Bementand Capco, 1990). However, none of these studiesreported measurement of the activity of intracellularfree Ca2+ ([Ca2+]0 in response to EGTA or BAPTAmicroinjection. It was particularly important to knowthat parameter in order to distinguish between twopossibilities: (1) the Ca2+ chelator lowers [Ca2+], levels,thus preventing [Ca2+], from reaching a threshold level(required for egg activation) upon stimulation with anactivating stimulus; (2) the Ca2+ chelator does notaffect [Ca2+], levels, but chelates Ca2+ as they arereleased from intraceUular stores (or enter the egg)upon stimulation with an activating stimulus. Ourresults demonstrate that BAPTA or EGTA (50 or 100mM in the microinjection pipet, around 5 or 10 mMfinal concentration in the egg) do not affect [Ca2+](
levels, although they prevent egg activation (Fig. 2), areaction involving rapid changes in [Ca2+];. In thisrespect, our results with Xenopus eggs are similar tothose reported in fibroblasts (Kao et al. 1990) and seaurchin eggs (Patel et al. 1990), but opposite to thoseobtained in plant cells in which Ca2 -free EGTA orBAPTA microinjection lowers the basal [Ca2+]i level(Zhang et al. 1990). Microinjection of 5 mM CaCl2 intoeggs previously microinjected (around 30 min before)with 50 mM BAPTA resulted in the immediatetriggering of egg activation (data not shown).
Effects of microinjection of BAPTA on the Ca2*transientSince BAPTA or EGTA block egg activation bypreventing [Ca2+]j changes, as seen above, they can beused to determine the period of time during which anintracellular release of Ca2+ is needed to accomplishthe various events of egg activation. BAPTA waspreferred over EGTA, because the capacity of the latterto bind Ca2+ is known to depend on pH, a parameterthat varies in the cytoplasm of the activating egg. In our
58 N. Grandin and M. Charbonneau
£-10 L
CD
-20L
6.0p
"J-lOmin
^
-10
-20
L
6.o
6.5 iBAPTA
Fig. 2. Prevention of egg activation followingmicroinjection of BAPTA into unactivated Xenopus eggs.Two Ca2+ microelectrodes and two potentialmicroelectrodes were implanted in single unactivated eggsas shown in Fig. 1 (the pH microelectrode represented inFig. 1 was replaced by a second Ca2+ microelectrode).BAPTA (30-40 nl of a 100 raM solution, pH 7.5, preparedin 10 mM Hepes, pH 7.4-7.5) was microinjected(arrowhead) at the site indicated in Fig. 1. Although theegg was pricked upon microinjection, there was nosubsequent activation of the egg, as indicated by theabsence of an activation potential on the first and thirdtraces (Em, membrane potential) and of any otherreactions normally associated with egg activation (seeMaterials and methods). BAPTA did not change the[Ca2+]| level (see text), as indicated on the second andfourth traces (pCa traces, pCa is the negative logarithm ofintracellular free Ca2+ activity). In the whole study, themean value of the [Ca2+]i level in unactivated eggs impaledwith four microelectrodes was 0.49 ± 0.21 /*M (SD, n=42).
hands, and according to the location of the microelec-trodes (implanted as shown in Fig. 1), the beginning ofthe Ca transient was found to occur 2.7 ± 1.1 min(mean value ± standard deviation, n=29 eggs) after thebeginning of the activation potential, a CP-dependentplasma membrane depolarization, which is the earliestknown event of egg activation. The main goal of theexperiments using microinjection of BAPTA was todetermine whether or not the increase in pHj wasdependent on the increase in [Ca2+]j (see below).However, analysis of the relationships between thesetwo events should not be complicated by possibleinterference between BAPTA and the triggering of eggactivation, independently of the [Ca2+],-pHi relations.In other words, the effects of BAPTA on the pHjincrease due to a perturbation of the triggering of eggactivation itself, which is upstream of the Catransient, were undesirable. We therefore decided thatin the experiments looking at the effects of BAPTA onthe increase in pH,, microinjection would always be
•6.6
B
E.
uf
+10
-10tillOmin
6.0r
6.4L ABAPTA
D
aOQ.
+10-
0 -
-10-
6.0p
6.4L
+10
10 mm 10min
iBAPTA
kBAPTA
Fig. 3. Effects of microinjection of BAPTA on the Ca2+
transient. Single eggs were impaled with fourmicroelectrodes: two potential microelectrodes plus eithertwo Ca2+ microelectrodes (see Fig. 2) or one Ca2+ and onepH microelectrode, implanted according to the configurationshown in Fig. 1. For each egg, only the pCa trace or one ofthe two pCa traces, and the corresponding membranepotential trace are represented. Pricking (triggering of eggactivation) and microinjection were realized at the sitesindicated in Fig. 1. (A) Control non-microinjected egg,activated by pricking, displaying a normal activation potential(top trace) followed by a transient increase in [Ca2+];(bottom trace). (B, C) Two examples of eggs microinjectedwith 50 mM BAPTA (arrowheads) 3.3 and 2.7 min,respectively, after the onset of the activation potential. TheCa transient, which had already started at the time ofmicroinjection, was abruptly reduced by BAPTA. In some(C), but not all (B), cases, the [Ca2+], level decreased as aresult of BAPTA microinjection. (D, E) Two examples ofeggs microinjected with 50 mM BAPTA (arrowheads) 1.5min after the onset of the activation potential. In both cases,microinjection took place before the onset of the Ca2+
transient. In some cases BAPTA totally blocked the Ca2+
transient (D), while in other cases a diminished Ca2+
transient still took place (E). In all cases, the egg activation-associated reactions considered (with the exception of theincrease in pH,, see text) normally took place.
performed 2.5-3 min after the onset of the activationpotential. Under such conditions, the Ca2+ transientwas strongly reduced (Fig. 3B,C). On the other hand,
A Ca2*-dependent wave of intracellular pH change 59
when BAPTA was microinjected earlier (1.5 min afterthe activation potential), the Ca2+ transient wasreduced still more (Fig. 3E) or even suppressed (Fig.3D). The Ca2+ response of eggs microinjected withBAPTA varied slightly from one egg to the other,probably due to some egg-to-egg variability and to thefact that the Ca2+ microelectrode recording the Ca2+
wave could not always be inserted exactly at the sameplace with respect to the site of pricking, at least notwith a precision greater than a few tens of /an.However, it is important, at this point, to note that thedifferences in the ability of BAPTA to affect the Ca2+
transient illustrated in Fig. 3 are not due to somebiological variability, but to differences in the times ofmicroinjection: 1.5 min after the activation potential inFig. 3D and E versus 3.3 and 2.7 min after the activationpotential, respectively, in Fig. 3B and C. For reasonsexplained above, all subsequent microinjections wereperformed 2.5-3 min after the activation potential,which is the case in all following figures.
The Co2* transient is needed for the normaloccurrence of the subsequent increase in pHt
Egg activation in Xenopus is accompanied by a slowincrease in intracellular pH (pHj) that starts 6-8 minafter egg activation. When BAPTA was microinjected
(30-40 nl of a 50 or 100 mM solution) 2.5-3 min after eggactivation, this resulted in a delay, in the occurrence ofthe physiological increase in pH;, and, sometimes, in areduction of its amplitude (Fig. 4, Table 1). Microinjec-tion by itself was not responsible for that delay, sincemicroinjection of 10 mM Hepes had no effect on thekinetics of the pHj increase (Table 1). In addition, thedelay in the initiation of the pHj increase produced byBAPTA appeared to be specifically due to intracellularCa2+ chelation, since solutions containing 100 mMBAPTA/lOO mM MgCl2, but not solutions containing100 mM BAPTA/lOO mM CaCl2, caused a delay in theoccurrence of the pHj increase (Fig. 5, Table 1). It isimportant to note that the period between the begin-ning of the increase in pH, and the time at which theplateau level was attained was not affected by BAPTA,or slightly lengthened in some cases (Table 1). Thismeans that BAPTA produced a delay in the initiation ofthe increase in pH|, but that, once started, the reactionproceeded almost as rapidly as in control eggs.Likewise, the amplitude of the delayed increase in pHjwas only slightly affected following microinjection ofBAPTA (Table 1). In fact, in most cases that amplitudewas unaffected by BAPTA (Fig. 4B, Table 1), while inother cases it was clearly diminished (Figs 4C, 5B; TableI)-
To know whether the BAPTA-induced delay in the
Table 1. Effects of microinjection of BAPTA on the pHt response to Xenopus egg activation
Controls§
Non-injected
10 mM Hepes
50 mM BAPTA/50 mM CaCl2
100 mM BAPTA/lOO mM CaCl2
BAPTA1
50 mM BAPTA
100 mM BAPTA
100 mM BAPTA/lOO mM MgCl2
Beginning ofpH] increase*
(min)
6.2±1.4("=21)5.4±0.9("=7)
6.0±1.8("=5)
6.1±1.3(n=4)
7.011.8("=5)
20.5±7.4(*=26)
17.5±5.6(" = 12)
22.8±4.9("=9)
23.7±12.5("=5)
Time of elevatedpH[ plateaut
(min)
29.115.5("=19)
25.912.6(n=l)
26.412.1("=3)
26.313.5("=4)
34.516.6("=5)
46.8112.5(" = H)
34.017.4(71 = 4)
50.118.1(n=4)
59.513.4("=3)
Amplitude ofpH, increased
(pH unit)
0.2810.05(* = 19)
0.2610.06(«=7)
0.2610.04("=3)
0.2810.07("=4)
0.2810.05(n=5)
0.2310.09(" = 12)
0.2410.11("=5)
0.2810.09(n=4)
0.1710.03("=3)
•Measured with respect to the onset of the activation potential, a Cl -dependent plasma membrane depolarization, which is the earliestknown event of egg activation, following pricking, by 2-5 seconds.
tTime between the onset of the activation potential and the stabilized elevated pH( value (plateau level).tin the whole study, pHi in unactivated eggs impaled with four microelectrodes was 7.4010.09 pH unit (SD, n=58). MicToinjection of
BAPTA sometimes produced a small change in the pH| level. Therefore, the amplitude of the egg activation-associated pH, increase wasmeasured as the difference between the pH| level existing just before the beginning of the pH| increase and the stable elevated value(plateau level). Changes in pH] following microinjection of BAPTA, 2.5-3 min after the triggering of egg activation (but before the eggactivation-associated pHi increase) were as follows. 50 mM BAPTA: +0.0810.04 pH unit (alkalinization) in 2 eggs, -0.1310.06 pH unit(acidification) in 7 eggs, no effect in 8 eggs; 50 mM BAPTA/50 mM CaCl2: no effect (n=4); 100 mM BAPTA: +0.07 pH unit (n = l),-0.1010.05 pH unit (n=5), no effect (/i=3); 100 mM BAPTA/lOO mM CaCl2: -0.09 pH unit (n = l), no effect (n=4); 100 mMBAPTA/lOO mM MgCl2: -0.0610.01 pH unit (n=3), no effect (/i=3). Microinjection of 10 mM Hepes, pH 7.4-7.5, had no effect on thepH| level. Experiments in which the pH| level of BAPTA-microinjected eggs had not stabilized at the time of the beginning of the eggactivation-associated pH| increase were discarded.
§Mean values (SD, number of eggs) for all control eggs, shown in the four lines below for each of the four categories of controls.UMean values for Ca2+-free-BAPTA-micToinjected eggs, shown in the three lines below for each of the three categories.
60 N. Grandin and M. Charbonneau
l ° [E-10L
7.2r
lOmin
Q.
7.7
B
1uf -10 Jl
lOmin
7.2
Q.
7.7
uf JllOmin
7.4
a.
7.8
initiation of the pHj increase was due to a concomitantslowing down of the main events controlling the cellcycle, the kinetics of MPF inactivation were measuredunder conditions exactly identical to those in which adelay in the increase in pH, had been observed. When100 mM BAPTAwas microinjected into Xenopus eggs,2.5 min after artificial activation, MPF activity(measured as its histone HI kinase activity) rapidlydropped within 5 min, with identical kinetics to those incontrol eggs microinjected with 100 mM BAPTA/lOOmM CaCl2 (Fig. 6). This was observed in three otherexperiments (2 using 50 mM BAPTA, 1 using 100 mMBAPTA; controls microinjected with either 50 mMBAPTA/50 mM CaCl2 or 100 mM BAPTA/100 mMCaCl2). The absence of interference between micro-injection of BAPTA and the timing of the cell cycleevents was confirmed morphologically by the fact thatmeiosis resumption normally took place in eggs micro-injected with 50 or 100 mM BAPTA, 2.5-3 min after eggactivation (Fig. 7).
Fig. 4. Effects of microinjection of BAPTA on the kineticsof the egg activation-associated increase in intracellular pH(pHj). Single unactivated eggs were each impaled with apH microelectrode, a Ca2+ microelectrode and twopotential microelectrodes, implanted as indicated in Fig. 1.The respective sites of triggering of egg activation(pricking) and microinjection were as shown in Fig. 1.Only the pH, trace and its corresponding membranepotential (Em) trace are represented. (A) Control non-microinjected egg, activated by pricking. The activationpotential was followed by a typical increase in pH, (0.34pH unit) occurring 5.4 min after egg activation (see meanvalues in Table 1). Note the transient cytoplasmicacidification occurring just before the beginning of thealkalinization. (B) Typical effect of 100 mM BAPTA,microinjected 3 min after triggering of egg activation(arrowhead). The physiological increase in pHj was clearlydelayed, since it occurred 19.2 min after egg activation (seemean values in Table 1). The amplitude of the increase,0.31 pH unit, was not affected by BAPTA (see Table 1).Note that the transient cytoplasmic acidification, which hadbeen initiated a few seconds before microinjection, was notmodified. (C) Example of a reduction in the amplitude ofthe physiological increase in pH, following microinjectionof 50 mM BAPTA (arrowhead) 2.6 min after triggering ofegg activation. That amplitude, 0.21 pH unit, was slightlyreduced with respect to controls (see Table 1). As in B,the increase in pHj was clearly delayed by BAPTA,occurring 15.6 min after egg activation (see mean values inTable 1). Although BAPTAwas microinjected before theonset of the transient cytoplasmic acidification, the latterwas not delayed with respect to the onset of the activationpotential, contrary to the subsequent increase in pH,.
A wave of intracellular pH changes in Xenopus eggsThe Ca2+ transient in Xenopus eggs proceeds as a wavestarting from the site of triggering of egg activation(Busa and Nuccitelli, 1985). This can be seen when twoCa2+ microelectrodes are impaled in a single egg (Fig.8A). Ca2+ waves represent cell-signaling second mess-engers widely used by various cell types (Meyer, 1991).However, the existence of pH waves has never beenconsidered or, at least, demonstrated, either inXenopus eggs or in any other system. When two pHmicroelectrodes were inserted into a single egg accord-ing to the configuration shown in Fig. 1, the differencebetween the distances of each of the pH microelec-trodes to the pricking site was too small to allow us todecide whether the increase in pHj proceeded as apropagating wave (data not shown). Therefore, thedistances between the site of pricking and each of thetwo pH microelectrodes were chosen so as to be verydifferent from each other. Under such conditions, wecould clearly demonstrate the existence of a pH wavetravelling from the site of pricking over the entire cortexof the egg (Fig. 8B,C). Both the initial transientdecrease and the permanent increase in pH; were foundto propagate as a wave. The mean value of thedifference in time between the onset of the pH, increasemeasured by the pH microelectrode located near thesite of pricking and that measured by the pH microelec-trode located on the opposite side of the egg (see Fig.8B,C) was 1.3 ± 0.5 min (SD, n=5 eggs).
A Ca2+-dependent wave of intracellular pH change
8r •
61
5•10
-10
7.3
7.8
lOmin
A. BAPTA/CaClj
B
lOmin7.3
IQ.
7.6 BAPTA/MgClj
Fig. 5. Control experiments showing that BAPTA retardsthe onset of the egg activation-associated increase in pH,by specifically chelating intracellular Ca2+. In this series ofexperiments, single unactivated eggs were each impaledwith two microelectrodes (a pH microelectrode and apotential microelectrode), which were implanted onopposite sides of the egg, placed animal pole up. (A) Amixture of 100 mM BAPTA/lOO mM CaCl2, pH 7.5, wasmicroinjected (at the arrowhead) 3 min after the onset ofthe activation potential. There was no effect on thephysiological increase in pHj that started 6.9 min after eggactivation and had an amplitude of 0.37 pH unit. (B) Amixture of 100 mM BAPTA/lOO mM MgCl2, pH 7.5, wasmicroinjected (at the arrowhead) 3 min after the onset ofthe activation potential. The physiological increase in pH;was clearly delayed, occurring 25.8 min after eggactivation, and had a reduced amplitude (0.20 pH unit).Mean values corresponding to the experiments ofmicroinjection of mixtures of BAPTA and CaCl2 or MgCl2are given in Table 1.
Because of the influence of [Ca2+]j levels on the onsetof the egg activation-associated pHt changes (see Figs 4,5), we could reasonably suppose that the pH wave was aconsequence of the Ca wave. To confirm thisassumption, eggs were impaled with two pH microelec-trodes each and two potential microelectrodes, prick-activated and locally microinjected, very near one ofthe two pH microelectrodes, with a small amount ofBAPTA (5-10 nl of a 100 mM solution), 2.5-3 min afterthe onset of the activation potential. Under theseconditions, the pH wave was considerably slowed downin the microinjected region of the egg (Fig. 9).Meanwhile, physiological pH; changes proceeded morerapidly at the opposite end of the egg, away from the
cE£ 5-Si
1CO
o
10 15 20Time (min)
25 30
Fig. 6. Microinjection of BAPTA, under conditions thataffected the kinetics of the egg activation-associatedincrease in pHi; did not modify the kinetics of theinactivation of histone HI kinase activity (see Materialsand methods). Each point corresponds to the activity of asingle egg, microinjected 2.5 min after triggering of eggactivation (pricking) with 30-40 nl of either 50 mM BAPTA(•) or 50 mM BAPTA/50 mM CaCl2 (controls, • ) . Time 0corresponds to unactivated eggs. Both kinetics of histoneHI kinase activity decrease, a reaction associated with cellcycle reinitiation triggered by egg activation, were exactlyparallel. This demonstrates that the BAPTA-induced delayin the triggering of the physiological increase in pHi (seeFigs 4, 5) is not due to a general lengthening of the cellcycle.
site of microinjection of BAPTA (Fig. 9). In that non-microinjected region, the kinetics of pHj changes wereslightly modified with respect to those in control non-microinjected eggs, probably due to some diffusion ofBAPTA from near the site of microinjection (Fig. 9).The mean value of the difference of time between theonset of the pHi increase measured in the non-microinjected region of the egg and that measured bythe pH microelectrode located in the region locallymicroinjected with BAPTA (as shown in Fig. 9) was10.1 ± 6.8 min (SD, n=A eggs). These results confirmthe view that pH( changes proceed as a wave, thenormal delay (1.3 min) in the kinetics of pHj changes attwo distinct sites of the egg cortex being accentuated(10.1 min) following local microinjection of BAPTA.They also confirm that the normal time-lag between eggactivation and pH, changes is partly determined by theCa2+ transient, the effect on the kinetics of pHj changesbeing restricted, or at least more pronounced, in theregion of the egg that has been previously microinjectedwith BAPTA.
Discussion
Three main findings emerge from the present study.
62 N. Grandin and M. Charbonneau
Fig. 7. Microinjection of BAPTA, under conditions thataffected the kinetics of the egg activation-associatedincrease in pH,, did not delay the nuclear events followingegg activation. Eggs were microinjected 2.5-3 min aftertriggering of egg activation (pricking) with 30-40 nl of 50mM BAPTA/50 raM CaCl2 (B, D: controls), or 50 mMBAPTA (C) or 100 mM BAPTA (E) and fixed at 5-minintervals from between the time of pricking and 30 minlater. Paraffin sections were stained with bisbenzimide (seeMaterials and methods). (A) Control unactivated (non-microinjected) egg showing a typical metaphase 2 spindle.(B, C) Eggs microinjected with 50 mM BAPTA/50 mMCaCl2 (B, control) or 50 mM BAPTA (C) and fixed 10 minafter egg activation. BAPTA alone did not delay thepassage into anaphase, indicated by the presence of twosets of chromosomes, which were at the same stage asthose in controls (B). (D, E) Eggs microinjected with 50mM BAPTA/50 mM CaCl2 (D, control) or 100 mMBAPTA (E) and fixed 30 min after egg activation. Re-formation of an interphasic nucleus occurred at the sametime in the two cases. Therefore, exit from mitosis cannotbe blocked by chelating intracellular Ca2+ under conditionsthat delayed the physiological increase in pH;.
The first one is that the normal time-lag between eggactivation and the increase in intracellular pH (pH,) inXenopus is partly controlled by the transient increase inintracellular free calcium ([Ca2+];), as shown bymicroinjection of BAPTA, a chelator of Ca2+. Mostimportantly, the BAPTA-induced delay in the initiationof the pH response to egg activation was not the resultof a slowing down of the events controlling the cell
cycle. The second finding is that the increase in pHjassociated with Xenopus egg activation proceeds as awave, which represents, to our knowledge, the firstreported case of an intracellular pH wave. Since the pHwave closely follows the Ca2+ wave and is locallyslowed down following local microinjection of BAPTA,this suggests that the pH wave needs Ca2+ for itspropagation. The third main finding of the presentstudy is that inactivation of MPF and, hence, the entryinto the first mitotic cell cycle, can proceed in theabsence of a propagating Ca wave.
The Ca2+ transient determines the normal time-lagbetween egg activation and the pHt increaseThe present report is the first to provide direct evidenceof a relationship between the increases in [Ca2+]j andpH,, both associated with Xenopus egg activation.Because the Ca2+ transient represents a ubiquitoussignal for triggering of cell activation (recently reviewedby Berridge and Irvine, 1989; Meyer, 1991) andprecedes the increase in pH; in Xenopus eggs, it hasbeen frequently proposed that, in this system at least,the increase in pHj resulted from the increase in[Ca2+]j. This, however, was not found experimentally,although it must be admitted that attempts to under-stand the problem better may have been discouraged bythe complexity of the technical approaches needed. Itshould be borne in mind that the technical difficulty ofimpaling a single egg of Xenopus with four microelec-trodes, without activating it, was increased in thepresent study by the necessity to microinject BAPTAfurther without artefactually perturbing the measuredionic activities. A straightforward explanation of thepresent observation that microinjection of BAPTAresults in a delay in the initiation of the increase in pH;(without retarding reinitiation of the cell cycle; see Figs6, 7) is that a certain amount of the Ca2+ releasedintracellularly is needed to determine the time-lag (6-8min) that is normally present between the onset of eggactivation (the activation potential) and the pH re-sponse. Control experiments show that microinjectionby itself is not responsible for that delay and that Ca2+
represents the intracellular ion specifically chelated byBAPTA (Fig. 5). The increase in pHj during Xenopusegg activation has been previously reported to takeplace in the absence of extracellular Ca2+ (nominallyCa2+-free solution supplemented with 1 mM EGTA)(Grandin and Charbonneau, 1990b). Therefore, theCa2+ that is necessary for a correct initiation of theincrease in pHj has an intracellular origin. An alterna-tive hypothesis to explain the BAPTA-induced delay inthe pH response relies on the effect of BAPTA on theegg membrane potential (Em) repeatedly observed inthis study (compare Fig. 3A with B or C, for instance).Owing to the possible influence of the Em level on thefunctioning of plasma membrane ion transportersinvolved in pH; regulation, the effect of BAPTA on theXenopus egg Em might be responsible for the observeddelay in the pH response. However, this is unlikely,since that pH response has been shown to be indepen-dent of any of the known plasma membrane ion
A Ca2+-dependent wave of intracellular pH change 63
+10
0
-10
pCa 6.27—
pCa 6.27—
10min
ik
pricking
Fig. 8. An intracellular pH wave that followsthe intracelllular Ca2+ wave in Xenopus eggs.(A) A very impressive illustration of the factthat the increase in [Ca2+], taking placeduring Xenopus egg activation proceeds as awave. A single egg was impaled with twoCa2+ microelectrodes and two potentialmicroelectrodes, according to theconfiguration shown in Fig. 1 (the pHmicroelectrode represented in Fig. 1 wasreplaced by a second Ca2+ microelectrode).Only one of the two membrane potential(Em) traces is represented (in A, B and C),since the membrane potential is always thesame at any place within the egg. The Ca2+
transient, a propagating front initiated fromaround the site of pricking, had very differentproperties in these two distinct regions of theegg cortex, both concerning the amount ofQ r + released and the Ca gradient of theCa2+ wave. The Ca2+ gradient was muchsteeper in the region corresponding to thepCa bottom trace than in the regioncorresponding to the pCa top trace, whichmight explain the larger amplitude of theCa transient in the former (pCa bottomtrace). The distance between the twoarrowheads represents the delay between theonset of the Ca2+ transient recorded by thetwo microelectrodes. The existence of such adelay provided the first demonstration of theexistence of a Ca2+ wave in Xenopus eggs(Busa and Nuccitelli, 1985). (B, C) Twoexamples illustrating the existence of anintracellular pH wave propagating fromaround the site of triggering of egg activation.Eggs were each impaled with two pHmicroelectrodes and two potentialmicroelectrodes as represented on theaccompanying schemes B' and C , which alsoindicate the site of pricking (by one of thetwo Em microelectrodes). The distancebetween the two arrowheads, in B and C,indicates the difference of time (delay)between the onset of the physiologicalincrease in pHj recorded by the twomicroelectrodes at the sites represented on
the corresponding schemes. This clearly demonstrates the existence of a pH wave traveling throughout the egg cortex fromthe site of pricking. The mean value of the velocity of the pH wave under the conditions represented here is given in thetext.
transporters (Webb and Nuccitelli, 1982; Grandin andCharbonneau, 1990b).
Neither the kinetics of the increase in pH, (timebetween the beginning of the increase and the elevatedpHj plateau) nor its amplitude was dramatically affec-ted by BAPTA (Table 1). This would tend to suggestthat intracellular Ca2 + is principally needed for theinitiation of the pH response, but less for its unfolding.There are two possible explanations. The first one isthat, besides Ca 2 + , there exists a second triggeringsignal for the increase in pHj itself, and that [Ca2+],might just play a role in ensuring that a normal time-lagis established between egg activation and the pHresponse. There is no major problem with such an
interpretation. However, the nature of that possiblesecond triggering signal, which might also depend on[Ca2+]i but on a sensitivity basis different from that ofthe initiation of the pH response, is still unknown. Thesecond possibility is that Ca2 + alone determines boththe time to the initiation of the pH response and theresponse itself, but that the failure of BAPTA to diffusethroughout the egg may result in failure to abolish thepH response completely. On first analysis, this appearsto be unlikely, since several of our experiments showthat BAPTA can diffuse relatively large distances fromthe site of microinjection (see, for instance, Figs 3, 9).However, Fig. 9 also shows that the amount of BAPTAthat diffused far from the site of microinjection was
64 N. Grandin and M. Charbonneau
AC^10
uf0
-101
pH, 7.48-
pH, 7.48-
B
pricking
BAPTA
Fig. 9. The propagation of the pH wave depends on thepreceding Ca2+ wave. Unactivated eggs were each impaledwith two pH microlectrodes and two potentialmicroelectrodes as described in scheme C. (A and B)represent two examples of the same phenomenon. SchemeC also shows the sites of pricking and microinjection. Eggswere microinjected with 5-10 nl of a 100 mM BAPTAsolution, pH 7.5 (arrowheads), 2.5-3 min after triggering ofegg activation (pricking), indicated by the occurrence ofthe activation potential on the membrane potential (£m)trace. Microinjection was done on purpose very near oneof the two pH microelectrodes (the pH microelectrodenoted pH-c in scheme C, recording the pHj bottom trace inboth A and B). The pH microelectrode noted pH-d inscheme C corresponds to the pHj top trace in both A andB. It is important to note that the amount of microinjectedBAPTA in this series of experiments (5-10 nl) was smallerthan in the rest of this work (30-40 nl). When comparedwith the normal situation in which there was nomicroinjection (Fig. 8), local microinjection of smallamounts of BAPTA, as here, caused a very dramaticslowing down of the pH wave. This was seen as anincrease in the difference of time (delay) between detectionof the onset of the pHj increase at the two distinctlocations (pH-c and pH-d), with respect to the equivalentdelay in non-microinjected eggs (Fig. 8). That difference intime corresponds to the distance between the twoarrowheads in both A and B. In other words, thepropagation of the pH wave detected with two pHmicroelectrodes was slowed down in the region in whichBAPTA was microinjected. The mean value of the velocityof the pH wave under the conditions presented here isgiven in the text. These experiments confirm that thephysiological increase in pH, in Xenopus eggs proceeds as awave. They also demonstrate that the propagation of thepH wave depends on preceding variations in [Ca2+],.
probably much less (had much less effect on the pHresponse) than near the site of microinjection, althoughit is true that in this particular example the total amountof injected BAPTA was limited with respect to thestandard conditions. Incidentally, it should be notedthat recording an effect of BAPTA on the Em (theabrupt hyperpolarization) at a given place within theegg is not indicative of the fact that BAPTA has reallyreached the region located around that particular Emmicroelectrode. Indeed, even if there were a local Emchange, this could not be detected with intracellular Emmicroelectrodes, because cells are equipotential (due tothe resistance of the cytoplasm being much smaller thanthat of the plasma membrane), a situation bestillustrated by the fact that the sperm, which interactswith only a tiny portion of the egg plasma membrane,nevertheless produces an Em change that can besimultaneously recorded at any place within the egg. Infact, in Xenopus eggs, the demonstration of theexistence of local Em changes or propagating conduc-tance changes necessitated the use of patch electrodes(Jaffe et al. 1985) or of an extracellular vibrating probe(Kline and Nuccitelli, 1985). Like the problems regard-ing diffusion of BAPTA, the time of BAPTA micro-injection is of importance in evaluating the role of[Ca2+]; in determining the pH response. Indeed, in allexperiments in which the pH response was delayed butnot suppressed, BAPTA had been microinjected 2.5-3min after the activation potential, a standard conditiondenned in this study (see corresponding text to Fig.3B,C in Results). One may wonder if the pH responsewould have been both delayed and suppressed follow-ing a much earlier microinjection of BAPTA, forinstance 1.5 min after the activation potential as shownin Fig. 3D,E. We decided to apply a strict rule todetermine our standard conditions and avoid anysituation in which we would not be totally sure thatBAPTA had not interfered with the triggering of eggactivation itself. Situations with such interferencesmight be difficult to analyze, because BAPTA mightblock many early events of egg activation, most ofwhich might be only distantly related to the pHresponse. In conclusion, it is not possible yet to decidewith certainty whether the Ca2+ transient is the onlyevent that controls the pH response to egg activation inXenopus.
Relationships between [Ca2+]i, pH, and MPFSea urchin eggs and various types of cultured mam-malian cells certainly represent the best-known systemsconcerning the relationships of the [Ca2+]j and pHjchanges to the cell cycle, although, to our knowledge,no experiments aiming at buffering the Ca2+ transienthave been performed in these systems. In thesesystems, the increase in pH, associated with cellactivation involves the activation of a Na+-H+
exchange (reviewed by Epel and Dube", 1987). In thesesystems, the transduction of the activating signalinvolves stimulation of the inositol phospholipid metab-olism leading to two independent pathways, oneresponsible for the [Ca2+], increase via production of
A Co2*-dependent wave of intracellular pH change 65
inositol 1,4,5-trisphosphate (IP3), the other for the pH,increase via production of diacylglycerol (DAG) andactivation of protein kinase C (PKC) (see referencesand schemes; Pouyss6gur, 1985; Houslay, 1987). Itshould be noted that these two pathways are probablynever totally independent of each other, since [Ca2+]ilevels are known to modulate the activity of PKC byacting on its translocation to the plasma membranewhere it associates with DAG (reviewed by Huang,1989). In sea urchin eggs, it is possible to produce the[Ca2+], increase in the absence of the subsequentincrease in pH( when egg activation is triggered in Na+-free sea water (Whitaker and Patel, 1990). However,this does not provide additional information on the[Ca2+],-pHi relations, since it is the reaction itself, theNa+-H+ exchange, that is blocked.
The situation in Xenopus eggs is quite different fromthat in sea urchin eggs and cultured mammalian cells.Indeed, Xenopus eggs do not possess a Na+-H+
exchange system or any other of the classical ionic pHrregulating systems in their plasma membrane (Webband Nuccitelli, 1982; Grandin and Charbonneau,1990b). In addition, PKC is not included in the pH,response to Xenopus egg activation (Grandin andCharbonneau, 1991c). On the other hand, IP3 (Busa etal. 1985) and a G protein (Kline et al. 1988) appear to beinvolved during Xenopus egg activation. The originalityof the situation in Xenopus eggs also resides in the factthat the increase in pHj appears to have a metabolicorigin, most probably associated with the inactivationof MPF (see Grandin and Charbonneau, 1991a). Theassumption that the increase in pHj might be a directconsequence of MPF inactivation is based on theexistence of temporal relationships between the twoevents, in both Xenopus laevis and Pleurodeles waltlii,another amphibian (Grandin and Charbonneau,1991a), as well as functional relationships between thepH, oscillations and the oscillations in the activity ofMPF accompanying the mitotic cell cycle (Grandin andCharbonneau, 1990a). MPF activity, measured as abiological activity inducing meiosis resumption inXenopus oocytes arrested in prophase 1 of meioticmaturation, was found to start decreasing 8 min afteregg activation in Xenopus (Gerhart et al. 1984).Meanwhile, the pH; level, stable during the last part ofmeiotic maturation until the arrest in second meta-phase, starts elevating around 10 min after eggactivation (Webb and Nuccitelli, 1981). In our hands,pH, in Xenopus eggs starts increasing 6-7 min after eggactivation, at 22°C. Although both the timing of MPFinactivation and that of the pH, increase appear to beclosely coincident, it should be noted that the kinetics ofMPF inactivation previously reported were measured at19°C (Gerhart et al. 1984). In our hands, at 22°C, MPFactivity starts decreasing around 5 min after eggactivation, whether measured as its histone HI kinaseactivity (Fig. 6) or as its oocyte maturation-inducingactivity (Grandin and Charbonneau, unpublished re-sults). The present observation that MPF inactivationoccurs slightly before the increase in pH, supports ourprevious assumption that the increase in pHj is a
consequence of MPF inactivation rather than theconverse (Grandin and Charbonneau, 1991a). Ourexperiments using microinjection of BAPTA alsosuggest that the intermediate reactions between MPFinactivation and the initiation of the pH response, ifthey exist, are probably Ca2+-dependent.
Cell-free extracts prepared with metaphase-blockedXenopus or Rana eggs have MPF activities that aresensitive to Ca2+ (Meyerhof and Masui, 1977; Masui,1982; Lohka and Mailer, 1985). Similarly, in a cell-freesystem from clam embryos, added Ca2+ leads to a rapiddestruction of endogenous cyclin, one of the com-ponents of MPF (Luca and Ruderman, 1989). In thepresent study, the question of the role of intracellularCa2+ release in MPF inactivation was not addressed.However, our results do show that microinjectingBAPTA 2.5-3 min after the onset of egg activation didnot change the normal time-lag to the onset of MPFinactivation or the reaction itself. These experiments,as well as those showing an absence of effect of BAPTAon meiosis resumption under the same conditions,suggest that MPF inactivation can proceed normallyeven when the Ca2+ transient is strongly reduced, thatis under conditions that prevent a Ca2+ wave frompropagating throughout the egg cortex. This confirmsrecent experiments in which increasing Ca2+ to 1-1.5JJM in a Xenopus metaphase extract for only 30 s wasfound to be sufficient to trigger cyclin degradation(Lorca et al. 1991).
A wave of intracellular pH changes during Xenopusegg activationTo our knowledge, the present results are the first todemonstrate the existence of a wave of intracellular pHchange (Figs 8, 9). The delay between egg activationand pHj changes was clearly dependent on the distancebetween the site of pricking and the pH microelectrode(Fig. 8). The finding that the pH wave was slowed downonly in the region that had been microinjected withBAPTA (Fig. 9) confirms the view that the pH wave is aconsequence of the Ca2+ wave. Since the increase in[Ca2+]i also proceeds as a wave starting from the site oftriggering of egg activation (Busa and Nuccitelli, 1985)and given the relationships between pH, changes and[Ca ], levels uncovered in the present study, it is highlyprobable that the pH wave is initiated by the precedingCa2+ wave.
The existence of a pH wave might representimportant information to be used in the comprehensionof still poorly understood mechanisms, not only inXenopus eggs. A pH wave, even if it existed in othersystems, might be undetectable using the availabletechniques, principally because most cells are muchsmaller than Xenopus eggs. The mechanisms underly-ing the propagating pHj changes in Xenopus eggs mightbe related to those responsible for the generation ofCa2+ waves. Most cells have multiple calcium pools,sensitive or not to IP3, which in most cases areconstituted of endoplasmic reticulum (ER) or havecharacteristics related to ER (reviewed by Berridge andIrvine, 1989). The egg of Xenopus possesses both an
66 N. Grandin and M. Charbonneau
IP3-sensitive and an IP3-insensitive calcium pools (Busaand Nuccitelli, 1985), and the propagating wavetriggered by IP3, which is indistinguishable from thatobserved at fertilization, originates from an ER-enriched layer in stratified eggs (Han and Nuccitelli,1990). Given that the pH wave demonstrated heredepends on [Ca2+], levels and closely follows the Ca2+
wave, and that the mechanisms and location of the pHjchanges in Xenopus eggs are still unknown, one canpostulate the existence of some cortically localizedintracellular compartment that might start pumpingprotons as it is reached by the Ca wave. Alterna-tively, such a compartment might be the same as theER-calcium pool supposed to be at the origin of theCa2+ wave, and contain a Ca2+-H+ exchange system inits membrane. However, these hypotheses need to beexperimentally challenged.
We thank Mrs M. Manceau for cutting paraffin sections,and Mr L. Communier for help in preparing the photographicillustration. This work was supported by grants from theLigue contre le Cancer (Comit6 D6partemental d'llle-et-Vilaine), the Association pour la Recherche sur le Cancer,and the Fondation pour la Recherche M6dicale.
References
Ammann, D., Lanter, F., Steiner, R. A., Schulthess, P., Shijo, Y. andSimon, W. (1981). Neutral carrier-based hydrogen ion selectivemicroelectrode for extra- and intracellular studies. Analyt. Chem.53, 2267-2269.
Bement, W. M. and Capco, D. G. (1990). Protein kinase C actsdownstream of calcium at entry into the first mitotic interphase ofXenopus laevis. Cell Regul. 1, 315-326.
Berridge, M. J. and Irvine, R. F. (1989). Inositol phosphates and cellsignalling. Nature 341, 197-205.
Boron, W. F. (1986). Intracellular pH regulation in epithelial cells.Annu. Rev. Physio!. 48, 377-388.
Busa, W. B. (1986). Mechanisms and consequences of pH-mediatedcell regulation. Annu. Rev. Physiol. 48, 389-402.
Busa, W. B., Ferguson, J. E., Joseph, S. K., Williamson, J. R. andNuccitelli, R. (1985). Activation of frog (Xenopus laevis) eggs byinositol trisphosphate. I. Characterization of Ca2+ release fromintracellular stores. J. Cell Bwl. 101, 677-682.
Busa, W. B. and Nuccitelli, R. (1984). Metabolic regulation viaintracellular pH. Am. J. Physiol. 246, R4O9-R438.
Busa, W. B. and Nuccitelli, R. (1985). An elevated free cytosolic Ca2+
wave follows fertilization in eggs of the frog, Xenopus laevis. J. CellBiol. 100, 1325-1329.
Charbonneau, M. and Picheral, B. (1983). Early events in anuranamphibian fertilization: an ultrastrucural study of changesoccurring in the course of monospermic fertilization and artificialactivation. Dev. Growth Differ. 25, 23-37.
Critser, E. S. and First, N. L. (1986). Use of a fluorescent stain forvisualization of nuclear material in living oocytes and earlyembryos. Stain Technol. 61, 1-5.
Doree, M. (1990). Control of M-phase by maturation-promotingfactor. Curr. Opin. Cell Biol. 2, 269-273.
Epel, D. and Dube", F. (1987). Intracellular pH and cell proliferation.In Control of Animal Cell Proliferation, vol. II (ed A. L. Boyntonand H. M. Leffert), pp. 363-393. Academic Press, New York.
Felix, M. A., Pines, J., Hunt, T. and Karsenti, E. (1989). A post-ribosomal supernatant from activated Xenopus eggs that displayspost-translationally regulated oscillation of its cdc2+ mitotic kinaseactivity. EM BO J. 8, 3059-3069.
Gerhart, J. C , Wu, M. and Klrschner, M. (1984). Cell cycledynamics of an M-phase-specific cytoplasmic factor in Xenopuslaevis oocytes and eggs. / . Cell Biol. 98, 1247-1255.
Grandin, N. and Charbonneau, M. (1990a). Cycling of intracellularpH during cell division of Xenopus embryos is a cytoplasmicactivity depending on protein synthesis and phosphorylation. / .Cell Biol. I l l , 523-532.
Grandin, N. and Charbonneau, M. (1990b). Is the egg activation-induced intracellular pH increase necessary for the embryonicdevelopment of Xenopus laevis (anuran amphibian)? InMechanism of Fertilization: Plants to Humans (ed. B. Dale), pp.503-517. Springer-Verlag, Berlin.
Grandin, N. and Charbonneau, M. (1991a). Cycling of intracellularfree calcium and intracellular pH in Xenopus embryos: a possiblerole in the control of the cell cycle. / . Cell Sci. 99, 5-11.
Grandin, N. and Charbonneau, M. (1991b). Changes in intracellularfree calcium activity in Xenopus eggs following imposedintracellular pH changes using weak acids and weak bases.Biochim. Biophys. Ada 1091, 242-250.
Grandin, N. and Charbonneau, M. (1991c). Intracellular pH andintracellular free calcium responses to protein kinase C activatorsand inhibitors in Xenopus eggs. Development 112, 461-470.
Han, J. K. and Nuccitelli, R. (1990). Inositol 1,4,5-trisphosphate-induced calcium release in the organelle layers of the stratified,intact egg of Xenopus laevis. J. Cell Biol. 110, 1103-1110.
Hara, K. and Tydeman, P. (1979). Cinematographic observation ofan "activation wave" (AW) on the locally inseminated egg ofXenopus laevis. Wilhem Roux's Arch. Dev. Biol. 186, 91-94.
Holllnger, T. J. and Corton, G. L. (1980). Artificial fertilization ofgametes from the south african clawed frog, Xenopus laevis. Gam.Res. 3, 45-57.
Houslay, M. D. (1987). Egg activation unscrambles a potential rolefor IP4. Trends Biochem. Sci. 12, 1-2.
Huang, K. (1989). The mechanism of protein kinase C activation.Trends Neurosci. 12, 425-432.
Humason, G. L. (1972). Animal Tissue Techniques. FreemanPublications, San Francisco, CA.
Hunt, T. (1989). Maturation promoting factor, cyclin and the controlof M-phase. Curr. Opin. Cell Biol. 1, 268-274.
JafTe, L. A., Kado, R. T. and Muncy, L. (1985). Propagatingpotassium and chloride conductances during activation andfertilization of the egg of the frog, Rana pipiens. J. Physiol.(Lond.). 368, 227-242.
Kao, J. P. Y., Alderton, J. M., Tslen, R. Y. and Steinhardt, R. A.(1990). Active involvement of Ca2+ in mitotic progression of Swiss3T3 fibroblasts. J. Cell Biol. I l l , 183-196.
Karsenti, E., Newport, J., Hubble, R. and Kirschner, M. (1984).Interconversion of metaphase and interphase microtubule arrays,as studied by the injection of centrosomes and nuclei into Xenopuseggs. J. Cell Biol. 98, 1730-1745.
Kline, D. (1988). Calcium-dependent events at fertilization of the frogegg: injection of a calcium buffer blocks ion channel opening,exocytosis, and formation of pronuclei. Dev. Biol. 126, 346-361.
Kline, D. and Nuccitelli, R. (1985). The wave of activation current inthe Xenopus egg. Dev. Biol. I l l , 471-487.
Kline, D., Simondni, L., Mandel, G., Maue, R. A., Kado, R. T. andJaffe, L. A. (1988). Fertilization events induced byneurotransmitters after injection of mRNA in Xenopus eggs.Science 241, 464-467.
Lanter, F., Stelner, R. A., Ammann, D. and Simon, W. (1982).Critical evaluation of the applicability of neutral carrier-basedcalcium selective microelectrodes. Analytica. Chim. Acta 135, 51-59.
Latt, S. A. and Stetten, G. (1976). Spectral studies on 33258 Hoechst andrelated bisbenzimidazole dyes useful for fluorescent detection ofdeoxyribonucleic acid synthesis. J. Histochem. Cytochem. 24, 24-33.
Lohka, M. J. (1989). Mitotic control by metaphase promoting factorand ede proteins. J. Cell Sci. 92, 131-135.
Lohka, M. J. and Mailer, J. L. (1985). Induction of nuclear envelopebreakdown, chromosome condensation, and spindle formation incell-free extracts. J. Cell Biol. 101, 518-523.
Lorca, T., Galas, S., Fesquet, D., Dcvault, A., Cavadore, J. C. andDoree, M. (1991). Degradation of the proto-oncogene productp39mo* is not necessary for cyclin proteolysis and exit from meioticmetaphase: requirement for a Ca -calmodulin dependent event."EM BO J. 10, 2087-2093.
A Ca2+-dependent wave of intracellular pH change 67
Luca, F. C. and Ruder man, J. V. (1989). Control of programmedcyclin destruction in a cell-free system. / . Cell Biol. 109,1895-1909.
Mailer, J. L. (1990). Xenopus oocytes and the biochemistry of celldivision. Biochemistry 29, 3157-3166.
Mailer, J. L. (1991). Mitotic control. Curr. Opin. Cell Biol. 3, 269-275.
Masul, Y. (1982). Oscillatory activity of Maturation Promoting Factor(MPF) in extracts of Ranapip'tens eggs. J. Exp. Zool. 224,389-399.
Masul, Y. and Markert, C. L. (1971). Cytoplasmic control of nuclearbehavior during meiotic maturation of frog oocytes. / . Exp. Zool.177, 129-146.
Meyer, T. (1991). Cell signaling by second messenger waves. Cell 64,675-678.
Meyerhof, P. G. and Masul, Y. (1977). Ca and Mg control ofcytostatic factors from Rana pipiens oocytes which causemetaphase and cleavage arrest. Dev. Biol. 61, 214-229.
Moolenaar, W. H. (1986). Effects of growth factors on intracellularpH regulation. Ann. Rev. Physiol. 48, 363-376.
Murray, A. W. and Kirschner, M. (1989). Cyclin synthesis drives theeaily embryonic cell cycle. Nature 339, 275-280.
Nurse, P. (1990). Universal control mechanism regulating onset of M-phase. Nature 344, 503-508.
Patel, R., Twigg, J., Sheppard, B. and Whltaker, M. (1990). Calcium,cyclin and cell cycle control in sea urchin embryos. InDevelopmental Biology, UCLA Symposia on Molecular andCellular Biology, New Series, vol. 125 (ed. E. Davidson, J.Ruderman and J. Posakony), pp. 21-35. Wiley-Liss, Inc., NewYork.
Petnlg, R. R., Kuhn, M., Payne, R., Adler, E., Chen, T. H. and Jaffa,L. F. (1989). On the dissociation constants of BAPTA-typc calciumbuffers. Cell Calcium 10, 491-498.
Plcheral, B. and Charbonneau, M. (1982). Anuran fertilization: amorphological reinvestigation of some early events. J. Ultrastruct.Res. 81, 306-321.
Pouyssegur, J. (1985). The growth factor-activatable Na + -H +
exchange system: a genetic approach. Trends Biochem. Sci. 10,453-455.
Speksnjjder, J. E., Miller, A. L., Weisenseel, M. H., Chen, T. H. andJaffe, L. F. (1989). Calcium buffer injections block fucoid eggdevelopment by facilitating calcium diffusion. Proc. Nat. Acad. Sci.U.S.A. 86, 6607-6611.
Steinhardt, R. A., Epel, D., Carroll, E. J. and Yanaglmachl, R.(1974). Is calcium ionophore a universal activator for unfertilisedeggs? Nature 252, 41-43.
Takeichi, T. and Kubota, H. Y. (1984). Structural basis of theactivation wave in the egg of Xenopus laevis. J. Embryol. Exp.Morphol. 81, 1-16.
Tsien, R. Y. (1980). New calcium indicators and buffers with highselectivity against magnesium and protons: Design, synthesis, andproperties of prototype structures. Biochemistry 19, 2396-2404.
Webb, D. J. and NucdteUl, R. (1981). Direct measurement ofintracellular pH changes in Xenopus eggs at fertilization andcleavage. / . Cell Biol. 91, 562-567.
Webb, D. J. and Nucdtelli, R. (1982). Intracellular pH changesaccompanying the activation of development in frog eggs:comparison of pH microelectrodes and 31P-NMR measurements.In Intracellular pH: its Measurement, Regulation, and Utilization inCellular Functions (ed. R. Nucdtelli and D. W. Deamer), pp. 293-324. Alan Liss, New York.
Whltaker, M. and Patel, R. (1990). Calcium and cell cycle control.Development 108, 525-542.
Wolf, D. P. (1974). The cortical response in Xenopus laevis ova. Dev.Biol. 40, 102-115.
Zhang, D. H., Callaham, D. A. and Hepler, P. K. (1990). Regulationof anaphase chromosome motion in Tradescentia stamen hair cellsby calcium and related signaling agents. / . Cell Biol. I l l , 171-182.
(Received 29 July 1991 - Accepted 25 September 1991)
![Regulation of the intracellular Ca2+. Regulation of intracellular [H]:](https://static.documents.pub/doc/80x56/5a4d1b717f8b9ab0599b56a5/regulation-of-the-intracellular-ca2-regulation-of-intracellular-h.jpg)
