55
Journal of Cell Science 101, 55-67 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
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 Pines
Road, La Jolla, California 92037, U.S.A.
Summary
In Xenopus eggs, the transient increase in intracellular
free calcium ([Ca 2+ ]0, or Ca 2+ transient, which occurs
1-3 min after egg activation, is likely to be partly
responsible for the release of the cell cycle blockade. In
the present study, we have used microinjection of
BAPTA or EGTA, two potent chelators of Ca 2 + , to
buffer [Ca 2+ ]| at various steps during Xenopus egg
activation and evaluate the impact on some of the
associated events. Microinjection of either one of the
Ca 2+ chelators into unactivated eggs prevented egg
activation without, however, lowering [Ca 2+ ]|, suggesting that only physiological [Ca 2+ ]| changes, but not
[Ca2+]i levels, were affected by the Ca 2+ buffer. When
BAPTA was microinjected around the time of occurrence of the Ca 2+ transient, the egg activation-associated
increase in intracellular pH (pH|) was clearly delayed.
That delay was not due to a general slowing down of the
Introduction
In Xenopus unfertilized eggs, the arrest in metaphase 2
of meiotic maturation is under the control of MPF, a
universal M-phase promoting factor (recently reviewed
by Hunt, 1989; Lohka, 1989; Dor6e, 1990; Mailer, 1990,
1991; Nurse, 1990), first revealed in amphibian oocytes
(Masui and Markert, 1971). In Xenopus oocytes and
eggs, MPF has a high activity during metaphase and a
low activity during interphase (Gerhart et al. 1984).
Around 8 min after triggering of egg activation in
Xenopus, MPF activity drops, a reaction that permits
the completion of meiotic maturation and drives the
newly activated or fertilized egg into the first mitotic
interphase (Gerhart et al. 1984). We have previously
drawn attention to the finding that the increase in
intracellular pH (pHj) associated with egg activation
occurred simultaneously with the inactivation of MPF,
in both Xenopus and Pleurodeles, another amphibian
that has a naturally longer cell cycle than that of
Xenopus (Grandin and Charbonneau, 1991a). The
cell cycle, since under the same conditions of microinjection of BAPTA the kinetics of MPF (a universal M-phase
promoting factor) inactivation were unaffected. These
results represent the first indication that the Ca 2+
transient participates in determining the time of initiation of the pH| increase during Xenopus egg activation. The present results also demonstrate that the egg
activation-associated pH| changes (a slight, transient
decrease in pH( followed by a permanent increase in pH,)
proceed as a wave propagating from the site of triggering
of egg activation. Experiments of local microinjection of
BAPTA support the view that the pH wave is a
consequence of the Ca 2+ wave, which it follows closely.
Key words: intracellular Ca2+ transient, intracellular pH
wave, M-phase promoting factor, egg activation, Xenopus.
close relationship between MPF activity and pHj
changes in amphibian eggs is attested by thefindingthat
both activities fluctuate in phase during the embryonic
cell cycle and that they are also functionally related to
each other (Grandin and Charbonneau, 1990a, 1991a).
Our interest in MPF activity and pH, variations is
directed by the fact that both activities represent
universal mechanisms of control of the cell cycle. The
p34cdc2 kinase and cyclins, the two components of MPF,
have been found to operate in all eukaryotic systems so
far studied, from yeast to man (reviewed by Nurse,
1990; Mailer, 1991). Similarly, an increase in pH; has
been recorded in response to cell activation or, more
generally, in association with a change in the metabolic
state of the cell or at the onset of cell proliferation in
many cell types (reviewed by Busa and Nuccitelli, 1984;
Boron, 1986; Busa, 1986; Moolenaar, 1986; Epel and
Dub6, 1987), including Xenopus eggs (Webb and
Nuccitelli, 1981). In many cell types, cell activation,
which often corresponds to a reinitiation of the cell
cycle, 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 Ca 2+ transient (reviewed by Berridge and
Irvine, 1989; Meyer, 1991). This is also the case in
activating Xenopus eggs (Busa and Nuccitelli, 1985).
Following the initial observation that addition of Ca 2+
to amphibian egg extracts inactivated MPF (Meyerhof
and Masui, 1977; Masui, 1982), it has recently been
demonstrated that a Ca2+-calmodulin-dependent process was required to produce the degradation of cyclin,
a component of MPF, in Xenopus egg extracts (Lorca et
al. 1991). On the other hand, there is no indication in
the literature concerning the mechanisms producing the
increase in pHj in Xenopus eggs. Moreover, the
reaction itself does not depend on classical plasma
membrane ion exchangers (Webb and Nuccitelli, 1982;
Grandin and Charbonneau, 1990b) and has no known
ionic or metabolic origin, besides the assumption that it
is a consequence of MPF inactivation (Grandin and
Charbonneau, 1991a).
In the present work, we report that microinjection of
BAPTA (l,2-bis(2-aminophenoxy)ethane-./V,./V,A'',./V'tetraacetic acid), a highly selective calcium-chelating
reagent (Tsien, 1980; Pethig et al. 1989; Speksnijder et
al. 1989) into Xenopus eggs during the Ca transient,
2.5-3 min after triggering of egg activation, results in a
delay in the occurrence of the physiological increase in
pH; with respect to control eggs microinjected with
BAPTA/CaCl2 buffers. In contrast, under the same
conditions, there was no delay in the inactivation of
MPF with respect to controls, suggesting that the
BAPTA-induced delay in the increase in pH| was not
due to a general lengthening of the cell cycle. These
results suggest that (i) the Ca transient plays a role in
determining the time-lapse before the onset of the pH
response, but may not be necessary for the response
itself and, (ii) MPF inactivation can proceed in the
absence of a propagating Ca 2+ wave. Finally, we report
that the transient cytoplasmic acidification and the
following permanent cytoplasmic alkalinization both
proceed as a wave starting around the site of triggering
of egg activation. This represents, to our knowledge,
the first description of an intracellular pH wave.
Experiments of local microinjection of limited amounts
of BAPTA demonstrate that the pH wave necessitates
Ca 2+ for its propagation and closely follows the Ca 2+
wave.
Materials and methods
Biological material and solutions
Mature (metaphase 2-arrested) eggs were expressed from
females of Xenopus laevis (reared in the laboratory), induced
to ovulate following injection of 900 i.u. of human chorionic
gonadotropin (Organon, Saint Denis, France), and immediately dejellied in Fl solution (see below) containing 2%
cysteine, pH 7.8. The physiological Fl solution in which
dejellied eggs were immersed, modified from Hollinger and
Corton (1980), contained: 31.2 mM NaCl, 1.8 mM KC1, 1.0
raM CaCl2, 0.1 mM MgCl2, 2.0 mM NaHCO3, 1.9 mM
NaOH, 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-aminoethyl
ether)N,A',A'',Af'-tetraacetic acid), both purchased from
Sigma Chemical Company (St Louis, MO, USA), were
prepared as stock solutions of 100 mM (in 10 mM Hepes,
adjusted to pH 7.5 with NaOH) and used alone or mixed with
various amounts of CaCl2 or MgCl2.
Intracellular pH (pHj) and intracellular free calcium
([Ca2+]i) measurements and microinjections
Intracellular pH and Ca2+ microelectrodes were fabricated
and calibrated as described by Grandin and Charbonneau
(1991b,c). The resins, contained in the microelectrode tips,
used to detect intracellular ion activities, were hydrogen ion
ionophore 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 microelectrodes
permit a very rapid (of the order of a few seconds), selective
and sensitive detection of the ion activities concerned. It is
important to note that it is necessary to use two microelectrodes for each ion activity measured: a potential microelectrode measuring only the membrane potential (Em) and an
ion-selective microelectrode measuring the ion activity plus
the membrane potential. The membrane potential recorded
by the potential microelectrode was continuously subtracted
from the total signal recorded by the ion-selective microelectrode at the pen recorder input. Unactivated dejellied eggs,
immersed in Fl solution in the recording chamber, were
impaled with microelectrodes and remained unactivated after
achievement of impalement (no anesthetic was used). For
additional details concerning the electrophysiological set-up
and microelectrode impalement, see Grandin and Charbonneau (1991b,c). Microinjections were performed as previously
described (Grandin and Charbonneau, 1990b).
Egg activation
Activation was triggered by pricking the egg cortex, a
procedure that allows Ca2+ to leak from the external medium
into the cytoplasm (Wolf, 1974). In Xenopus eggs, artificial
activators, which all act by increasing intracellular free Ca 2+ ,
produce exactly the same events as those elicited by the
sperm, with the exception of cell division. A major difference
between prick-induced activation and activation induced by
application of A23187, a calcium ionophore that activates the
egg by releasing Ca2+ from intracellular stores even in the
absence of extracellular Ca2+ (Steinhardt et al. 1974), is that
pricking initiates the reaction from a single point, whereas
A23187 initiates the activation reaction simultaneously from
several regions of the egg cortex (Charbonneau and Picheral,
1983). In this respect, prick-induced egg activation more
closely mimicks the physiological reaction induced by the
sperm, which also proceeds as a wave starting from a single
point (Picheral and Charbonneau, 1982). Many of the
metabolic reactions involved during anuran amphibian egg
activation proceed as propagating waves: the cortical reaction
of exocytosis and the elongation of plasma membrane
microvilli (Picheral and Charbonneau, 1982), the opening of
Cl~ and K+ channels participating in the initial plasma
membrane depolarization, the so-called activation potential
(Jaffe et al. 1985; Kline and Nuccitelli, 1985), the Ca2+
transient (Busa and Nuccitelli, 1985) and the so-called
activation waves, two successive waves of cortical movements
(Hara and Tydeman, 1979; Takeichi and Kubota, 1984; Kline
and Nuccitelli, 1985). It was therefore important, in the
present study, to know exactly the spatial localization of the
site from which egg activation was initiated, that is the site of
pricking, with respect to the site of microinjection of the Ca2+
A Ca2+-dependent wave of intracellular pH change
Ca 2+ microelectrode
Animal
hemisphere
Em microelectrode
T
Pricking
Egg activation
pH microelectrode
Em microelectrode
Vegetal
Microinjection hemisphere
pipet
Fig. 1. Schematic representation of the disposition of
microelectrodes and sites of pricking and microinjection, in
Xenopus eggs. This configuration was adopted in the whole
study, with the exception of the experiments shown in Figs
5 and 9. Dejellied mature eggs were immersed in the
recording chamber and orientated, using forceps, animal
pole (AP) up. Thus, the pigmented animal hemisphere
(hatched zone) was always facing the experimentator
observing from above, under a stereomicroscope. It should
be noted that this scheme is a perspective drawing in which
the egg is viewed at an angle with respect to the vertical.
Adopting such standard conditions was necessary, taking
into account the fact that many of the reactions associated
with anuran egg activation proceed as waves propagating
from the site of triggering of egg activation (see references
in Materials and methods). Unactivated eggs were each
impaled with a pH microelectrode, a Ca2 microelectrode
and two potential microelectrodes (Em microelectrodes) as
shown in the scheme. Once the electrical and ionic
parameters had stabilized, the egg was prick-activated by
rapidly withdrawing and re-impaling one of the Em
microelectrodes (always the same, as shown in the
scheme), which produced a local entry of external Ca 2+ ,
resulting in the triggering of egg activation (Wolf, 1974). In
some experiments, indicated in the text, a single egg was
impaled with two pH microelectrodes or two Ca2+
microelectrodes and two Em microelectrodes.
chelator. This was particularly true when the time between
triggering of egg activation and microinjection was short,
because, for a given time, microinjecting into a region already
attained by the various waves of activation is not equivalent to
microinjecting beyond these waves. Indeed, the Ca2+ transient is not detected at the same time following egg activation,
depending on the site of implantation of the Ca microelectrode with respect to the site of pricking (see Busa and
Nuccitelli, 1985). It was therefore necessary for us to
standardize the conditions for microelectrode impalement,
pricking and microinjection. These standard conditions are
described in Fig. 1. Criteria for Xenopus egg activation
considered in the present study were: the activation potential
(detected 2-5 seconds after pricking the egg cortex), elevation
of the vitelline envelope (1-2 min), the Ca2+ transient (2-3
min), the cortical contraction (3-4 min), the increase in
intracellular pH (6-8 min) and the disappearance of the
maturation spot (25-30 min).
Staining of the egg chromosomes and nucleus
To visualize the state of the chromosomes following release of
the metaphase block during egg activation and that of the
57
forming interphasic nucleus, dejellied eggs were fixed for 24 h
in Smith's fixative (Humason, 1972). After dehydration in a
series of ethanol and butyl alcohol, and embedding in
paraffin, eggs were sectioned at 5 jim, stained with bisbenzimide (Hoechst 33258 or 33342, Sigma) to detect chromosomes
and chromatin (Latt and Stetten, 1976; Critser and First,
1986), and observed with a Leitz epifluorescence microscope.
Measurement of histone HI kinase activity
Histone kinase activity in single Xenopus eggs, reflecting their
MPF (M-phase promoting factor) activity (see, for instance,
Murray and Kirschner, 1989), was measured as described by
Felix 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.
Results
Microinjection of BAPT A or EGTA prevents egg
activation without affecting intracellular free Ccr+
levels
EGTA and BAPTA, two specific chelators of Ca 2+ ,
have already been used to prevent Xenopus egg
activation (Karsenti et al. 1984; Kline, 1988; Bement
and Capco, 1990). However, none of these studies
reported measurement of the activity of intracellular
free Ca 2+ ([Ca2+]0 in response to EGTA or BAPTA
microinjection. It was particularly important to know
that parameter in order to distinguish between two
possibilities: (1) the Ca 2+ chelator lowers [Ca2+], levels,
thus preventing [Ca2+], from reaching a threshold level
(required for egg activation) upon stimulation with an
activating stimulus; (2) the Ca 2+ chelator does not
affect [Ca2+], levels, but chelates Ca 2+ as they are
released from intraceUular stores (or enter the egg)
upon stimulation with an activating stimulus. Our
results demonstrate that BAPTA or EGTA (50 or 100
mM in the microinjection pipet, around 5 or 10 mM
final concentration in the egg) do not affect [Ca2+](
levels, although they prevent egg activation (Fig. 2), a
reaction involving rapid changes in [Ca 2+ ] ; . In this
respect, our results with Xenopus eggs are similar to
those reported in fibroblasts (Kao et al. 1990) and sea
urchin eggs (Patel et al. 1990), but opposite to those
obtained in plant cells in which Ca2 -free EGTA or
BAPTA microinjection lowers the basal [Ca2+]i level
(Zhang et al. 1990). Microinjection of 5 mM CaCl2 into
eggs previously microinjected (around 30 min before)
with 50 mM BAPTA resulted in the immediate
triggering of egg activation (data not shown).
Effects of microinjection of BAPTA on the Ca2*
transient
Since BAPTA or EGTA block egg activation by
preventing [Ca2+]j changes, as seen above, they can be
used to determine the period of time during which an
intracellular release of Ca 2+ is needed to accomplish
the various events of egg activation. BAPTA was
preferred over EGTA, because the capacity of the latter
to bind Ca 2+ is known to depend on pH, a parameter
that varies in the cytoplasm of the activating egg. In our
58
N. Grandin and M. Charbonneau
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Fig. 2. Prevention of egg activation following
microinjection of BAPTA into unactivated Xenopus eggs.
Two Ca 2 + microelectrodes and two potential
microelectrodes were implanted in single unactivated eggs
as shown in Fig. 1 (the pH microelectrode represented in
Fig. 1 was replaced by a second Ca 2 + microelectrode).
BAPTA (30-40 nl of a 100 raM solution, pH 7.5, prepared
in 10 mM Hepes, pH 7.4-7.5) was microinjected
(arrowhead) at the site indicated in Fig. 1. Although the
egg was pricked upon microinjection, there was no
subsequent activation of the egg, as indicated by the
absence of an activation potential on the first and third
traces (Em, membrane potential) and of any other
reactions normally associated with egg activation (see
Materials and methods). BAPTA did not change the
[Ca 2 + ]| level (see text), as indicated on the second and
fourth traces (pCa traces, pCa is the negative logarithm of
intracellular free Ca 2 + activity). In the whole study, the
mean value of the [Ca 2+ ]i level in unactivated eggs impaled
with four microelectrodes was 0.49 ± 0.21 /*M (SD, n=42).
hands, and according to the location of the microelectrodes (implanted as shown in Fig. 1), the beginning of
the Ca transient was found to occur 2.7 ± 1.1 min
(mean value ± standard deviation, n=29 eggs) after the
beginning of the activation potential, a CP-dependent
plasma membrane depolarization, which is the earliest
known event of egg activation. The main goal of the
experiments using microinjection of BAPTA was to
determine whether or not the increase in pHj was
dependent on the increase in [Ca2+]j (see below).
However, analysis of the relationships between these
two events should not be complicated by possible
interference between BAPTA and the triggering of egg
activation, independently of the [Ca2+],-pHi relations.
In other words, the effects of BAPTA on the pHj
increase due to a perturbation of the triggering of egg
activation itself, which is upstream of the Ca
transient, were undesirable. We therefore decided that
in the experiments looking at the effects of BAPTA on
the increase in pH,, microinjection would always be
A
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Fig. 3. Effects of microinjection of BAPTA on the Ca 2 +
transient. Single eggs were impaled with four
microelectrodes: two potential microelectrodes plus either
two Ca 2 + microelectrodes (see Fig. 2) or one Ca 2 + and one
pH microelectrode, implanted according to the configuration
shown in Fig. 1. For each egg, only the pCa trace or one of
the two pCa traces, and the corresponding membrane
potential trace are represented. Pricking (triggering of egg
activation) and microinjection were realized at the sites
indicated in Fig. 1. (A) Control non-microinjected egg,
activated by pricking, displaying a normal activation potential
(top trace) followed by a transient increase in [Ca 2+ ];
(bottom trace). (B, C) Two examples of eggs microinjected
with 50 mM BAPTA (arrowheads) 3.3 and 2.7 min,
respectively, after the onset of the activation potential. The
Ca transient, which had already started at the time of
microinjection, was abruptly reduced by BAPTA. In some
(C), but not all (B), cases, the [Ca 2+ ], level decreased as a
result of BAPTA microinjection. (D, E) Two examples of
eggs microinjected with 50 mM BAPTA (arrowheads) 1.5
min after the onset of the activation potential. In both cases,
microinjection took place before the onset of the Ca 2 +
transient. In some cases BAPTA totally blocked the Ca 2 +
transient (D), while in other cases a diminished Ca 2 +
transient still took place (E). In all cases, the egg activationassociated reactions considered (with the exception of the
increase in pH,, see text) normally took place.
performed 2.5-3 min after the onset of the activation
potential. Under such conditions, the Ca 2+ transient
was strongly reduced (Fig. 3B,C). On the other hand,
A Ca2*-dependent wave of intracellular pH change
when BAPTA was microinjected earlier (1.5 min after
the activation potential), the Ca2+ transient was
reduced still more (Fig. 3E) or even suppressed (Fig.
3D). The Ca2+ response of eggs microinjected with
BAPTA varied slightly from one egg to the other,
probably due to some egg-to-egg variability and to the
fact that the Ca2+ microelectrode recording the Ca2+
wave could not always be inserted exactly at the same
place with respect to the site of pricking, at least not
with a precision greater than a few tens of /an.
However, it is important, at this point, to note that the
differences in the ability of BAPTA to affect the Ca2+
transient illustrated in Fig. 3 are not due to some
biological variability, but to differences in the times of
microinjection: 1.5 min after the activation potential in
Fig. 3D and E versus 3.3 and 2.7 min after the activation
potential, respectively, in Fig. 3B and C. For reasons
explained above, all subsequent microinjections were
performed 2.5-3 min after the activation potential,
which is the case in all following figures.
The Co2* transient is needed for the normal
occurrence of the subsequent increase in pHt
Egg activation in Xenopus is accompanied by a slow
increase in intracellular pH (pHj) that starts 6-8 min
after egg activation. When BAPTA was microinjected
(30-40 nl of a 50 or 100 mM solution) 2.5-3 min after egg
activation, this resulted in a delay, in the occurrence of
the physiological increase in pH;, and, sometimes, in a
reduction of its amplitude (Fig. 4, Table 1). Microinjection by itself was not responsible for that delay, since
microinjection of 10 mM Hepes had no effect on the
kinetics of the pHj increase (Table 1). In addition, the
delay in the initiation of the pHj increase produced by
BAPTA appeared to be specifically due to intracellular
Ca2+ chelation, since solutions containing 100 mM
BAPTA/lOO mM MgCl2, but not solutions containing
100 mM BAPTA/lOO mM CaCl2, caused a delay in the
occurrence of the pHj increase (Fig. 5, Table 1). It is
important to note that the period between the beginning of the increase in pH, and the time at which the
plateau level was attained was not affected by BAPTA,
or slightly lengthened in some cases (Table 1). This
means that BAPTA produced a delay in the initiation of
the increase in pH|, but that, once started, the reaction
proceeded almost as rapidly as in control eggs.
Likewise, the amplitude of the delayed increase in pHj
was only slightly affected following microinjection of
BAPTA (Table 1). In fact, in most cases that amplitude
was unaffected by BAPTA (Fig. 4B, Table 1), while in
other cases it was clearly diminished (Figs 4C, 5B; Table
I)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
59
Beginning of
pH] increase*
(min)
Time of elevated
pH[ plateaut
(min)
Amplitude of
pH, increased
(pH unit)
6.2±1.4
("=21)
5.4±0.9
29.115.5
("=19)
25.912.6
0.2810.05
(* = 19)
0.2610.06
(«=7)
0.2610.04
("=3)
0.2810.07
("=4)
0.2810.05
(n=5)
("=7)
(n=l)
6.0±1.8
("=5)
6.1±1.3
(n=4)
7.011.8
("=5)
26.412.1
("=3)
26.313.5
("=4)
34.516.6
("=5)
20.5±7.4
(*=26)
17.5±5.6
(" = 12)
22.8±4.9
("=9)
23.7±12.5
("=5)
46.8112.5
50.118.1
(n=4)
59.513.4
0.2310.09
(" = 12)
0.2410.11
("=5)
0.2810.09
(n=4)
0.1710.03
("=3)
("=3)
(" = H)
34.017.4
(71 = 4)
•Measured with respect to the onset of the activation potential, a Cl -dependent plasma membrane depolarization, which is the earliest
known 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 was
measured 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 egg
activation-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 mM
BAPTA/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 the
pH| level. Experiments in which the pH| level of BAPTA-microinjected eggs had not stabilized at the time of the beginning of the egg
activation-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
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initiation of the pHj increase was due to a concomitant
slowing down of the main events controlling the cell
cycle, the kinetics of MPF inactivation were measured
under conditions exactly identical to those in which a
delay in the increase in pH, had been observed. When
100 mM BAPTAwas microinjected into Xenopus eggs,
2.5 min after artificial activation, MPF activity
(measured as its histone H I kinase activity) rapidly
dropped within 5 min, with identical kinetics to those in
control eggs microinjected with 100 mM BAPTA/lOO
mM CaCl 2 (Fig. 6). This was observed in three other
experiments (2 using 50 mM BAPTA, 1 using 100 mM
BAPTA; controls microinjected with either 50 mM
BAPTA/50 mM CaCl 2 or 100 mM BAPTA/100 mM
CaCl 2 ). The absence of interference between microinjection of BAPTA and the timing of the cell cycle
events was confirmed morphologically by the fact that
meiosis resumption normally took place in eggs microinjected with 50 or 100 mM BAPTA, 2.5-3 min after egg
activation (Fig. 7).
Fig. 4. Effects of microinjection of BAPTA on the kinetics
of the egg activation-associated increase in intracellular pH
(pHj). Single unactivated eggs were each impaled with a
pH microelectrode, a Ca2+ microelectrode and two
potential 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 membrane
potential (Em) trace are represented. (A) Control nonmicroinjected egg, activated by pricking. The activation
potential was followed by a typical increase in pH, (0.34
pH unit) occurring 5.4 min after egg activation (see mean
values in Table 1). Note the transient cytoplasmic
acidification occurring just before the beginning of the
alkalinization. (B) Typical effect of 100 mM BAPTA,
microinjected 3 min after triggering of egg activation
(arrowhead). The physiological increase in pHj was clearly
delayed, since it occurred 19.2 min after egg activation (see
mean 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 had
been initiated a few seconds before microinjection, was not
modified. (C) Example of a reduction in the amplitude of
the physiological increase in pH, following microinjection
of 50 mM BAPTA (arrowhead) 2.6 min after triggering of
egg activation. That amplitude, 0.21 pH unit, was slightly
reduced 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 in
Table 1). Although BAPTAwas microinjected before the
onset of the transient cytoplasmic acidification, the latter
was not delayed with respect to the onset of the activation
potential, contrary to the subsequent increase in pH,.
A wave of intracellular pH changes in Xenopus eggs
The Ca 2+ transient in Xenopus eggs proceeds as a wave
starting from the site of triggering of egg activation
(Busa and Nuccitelli, 1985). This can be seen when two
Ca 2+ microelectrodes are impaled in a single egg (Fig.
8A). Ca 2+ waves represent cell-signaling second messengers widely used by various cell types (Meyer, 1991).
However, the existence of pH waves has never been
considered or, at least, demonstrated, either in
Xenopus eggs or in any other system. When two pH
microelectrodes were inserted into a single egg according to the configuration shown in Fig. 1, the difference
between the distances of each of the pH microelectrodes to the pricking site was too small to allow us to
decide whether the increase in pHj proceeded as a
propagating wave (data not shown). Therefore, the
distances between the site of pricking and each of the
two pH microelectrodes were chosen so as to be very
different from each other. Under such conditions, we
could clearly demonstrate the existence of a pH wave
travelling from the site of pricking over the entire cortex
of the egg (Fig. 8B,C). Both the initial transient
decrease and the permanent increase in pH; were found
to propagate as a wave. The mean value of the
difference in time between the onset of the pH, increase
measured by the pH microelectrode located near the
site of pricking and that measured by the pH microelectrode 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
61
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Fig. 5. Control experiments showing that BAPTA retards
the onset of the egg activation-associated increase in pH,
by specifically chelating intracellular Ca 2+ . In this series of
experiments, single unactivated eggs were each impaled
with two microelectrodes (a pH microelectrode and a
potential microelectrode), which were implanted on
opposite sides of the egg, placed animal pole up. (A) A
mixture of 100 mM BAPTA/lOO mM CaCl2, pH 7.5, was
microinjected (at the arrowhead) 3 min after the onset of
the activation potential. There was no effect on the
physiological increase in pHj that started 6.9 min after egg
activation and had an amplitude of 0.37 pH unit. (B) A
mixture of 100 mM BAPTA/lOO mM MgCl2, pH 7.5, was
microinjected (at the arrowhead) 3 min after the onset of
the activation potential. The physiological increase in pH;
was clearly delayed, occurring 25.8 min after egg
activation, and had a reduced amplitude (0.20 pH unit).
Mean values corresponding to the experiments of
microinjection of mixtures of BAPTA and CaCl2 or MgCl2
are given in Table 1.
Because of the influence of [Ca2+]j levels on the onset
of the egg activation-associated pHt changes (see Figs 4,
5), we could reasonably suppose that the pH wave was a
consequence of the Ca
wave. To confirm this
assumption, eggs were impaled with two pH microelectrodes each and two potential microelectrodes, prickactivated and locally microinjected, very near one of
the two pH microelectrodes, with a small amount of
BAPTA (5-10 nl of a 100 mM solution), 2.5-3 min after
the onset of the activation potential. Under these
conditions, the pH wave was considerably slowed down
in the microinjected region of the egg (Fig. 9).
Meanwhile, physiological pH; changes proceeded more
rapidly at the opposite end of the egg, away from the
15
20
Time (min)
25
30
Fig. 6. Microinjection of BAPTA, under conditions that
affected the kinetics of the egg activation-associated
increase in pHi; did not modify the kinetics of the
inactivation of histone HI kinase activity (see Materials
and methods). Each point corresponds to the activity of a
single egg, microinjected 2.5 min after triggering of egg
activation (pricking) with 30-40 nl of either 50 mM BAPTA
(•) or 50 mM BAPTA/50 mM CaCl2 (controls, • ) . Time 0
corresponds to unactivated eggs. Both kinetics of histone
HI kinase activity decrease, a reaction associated with cell
cycle reinitiation triggered by egg activation, were exactly
parallel. This demonstrates that the BAPTA-induced delay
in the triggering of the physiological increase in pHi (see
Figs 4, 5) is not due to a general lengthening of the cell
cycle.
site of microinjection of BAPTA (Fig. 9). In that nonmicroinjected region, the kinetics of pHj changes were
slightly modified with respect to those in control nonmicroinjected eggs, probably due to some diffusion of
BAPTA from near the site of microinjection (Fig. 9).
The mean value of the difference of time between the
onset of the pHi increase measured in the nonmicroinjected region of the egg and that measured by
the pH microelectrode located in the region locally
microinjected with BAPTA (as shown in Fig. 9) was
10.1 ± 6.8 min (SD, n=A eggs). These results confirm
the view that pH( changes proceed as a wave, the
normal delay (1.3 min) in the kinetics of pHj changes at
two 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 egg
activation and pH, changes is partly determined by the
Ca2+ transient, the effect on the kinetics of pHj changes
being restricted, or at least more pronounced, in the
region of the egg that has been previously microinjected
with BAPTA.
Discussion
Three main findings emerge from the present study.
62
N. Grandin and M. Charbonneau
cycle. The second finding is that the increase in pHj
associated with Xenopus egg activation proceeds as a
wave, which represents, to our knowledge, the first
reported case of an intracellular pH wave. Since the pH
wave closely follows the Ca 2+ wave and is locally
slowed down following local microinjection of BAPTA,
this suggests that the pH wave needs Ca 2+ for its
propagation. The third main finding of the present
study is that inactivation of MPF and, hence, the entry
into the first mitotic cell cycle, can proceed in the
absence of a propagating Ca wave.
Fig. 7. Microinjection of BAPTA, under conditions that
affected the kinetics of the egg activation-associated
increase in pH,, did not delay the nuclear events following
egg activation. Eggs were microinjected 2.5-3 min after
triggering of egg activation (pricking) with 30-40 nl of 50
mM BAPTA/50 raM CaCl2 (B, D: controls), or 50 mM
BAPTA (C) or 100 mM BAPTA (E) and fixed at 5-min
intervals from between the time of pricking and 30 min
later. Paraffin sections were stained with bisbenzimide (see
Materials and methods). (A) Control unactivated (nonmicroinjected) egg showing a typical metaphase 2 spindle.
(B, C) Eggs microinjected with 50 mM BAPTA/50 mM
CaCl2 (B, control) or 50 mM BAPTA (C) and fixed 10 min
after egg activation. BAPTA alone did not delay the
passage into anaphase, indicated by the presence of two
sets of chromosomes, which were at the same stage as
those in controls (B). (D, E) Eggs microinjected with 50
mM BAPTA/50 mM CaCl2 (D, control) or 100 mM
BAPTA (E) and fixed 30 min after egg activation. Reformation of an interphasic nucleus occurred at the same
time in the two cases. Therefore, exit from mitosis cannot
be blocked by chelating intracellular Ca2+ under conditions
that delayed the physiological increase in pH;.
The first one is that the normal time-lag between egg
activation and the increase in intracellular pH (pH,) in
Xenopus is partly controlled by the transient increase in
intracellular free calcium ([Ca2+];), as shown by
microinjection of BAPTA, a chelator of Ca 2+ . Most
importantly, the BAPTA-induced delay in the initiation
of the pH response to egg activation was not the result
of a slowing down of the events controlling the cell
The Ca2+ transient determines the normal time-lag
between egg activation and the pHt increase
The present report is the first to provide direct evidence
of a relationship between the increases in [Ca2+]j and
pH,, both associated with Xenopus egg activation.
Because the Ca 2+ transient represents a ubiquitous
signal for triggering of cell activation (recently reviewed
by Berridge and Irvine, 1989; Meyer, 1991) and
precedes the increase in pH; in Xenopus eggs, it has
been 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 understand the problem better may have been discouraged by
the complexity of the technical approaches needed. It
should be borne in mind that the technical difficulty of
impaling a single egg of Xenopus with four microelectrodes, without activating it, was increased in the
present study by the necessity to microinject BAPTA
further without artefactually perturbing the measured
ionic activities. A straightforward explanation of the
present observation that microinjection of BAPTA
results in a delay in the initiation of the increase in pH ;
(without retarding reinitiation of the cell cycle; see Figs
6, 7) is that a certain amount of the Ca 2+ released
intracellularly is needed to determine the time-lag (6-8
min) that is normally present between the onset of egg
activation (the activation potential) and the pH response. Control experiments show that microinjection
by itself is not responsible for that delay and that Ca 2+
represents the intracellular ion specifically chelated by
BAPTA (Fig. 5). The increase in pHj during Xenopus
egg activation has been previously reported to take
place in the absence of extracellular Ca 2+ (nominally
Ca2+-free solution supplemented with 1 mM EGTA)
(Grandin and Charbonneau, 1990b). Therefore, the
Ca 2+ that is necessary for a correct initiation of the
increase in pHj has an intracellular origin. An alternative hypothesis to explain the BAPTA-induced delay in
the pH response relies on the effect of BAPTA on the
egg membrane potential (Em) repeatedly observed in
this study (compare Fig. 3A with B or C, for instance).
Owing to the possible influence of the Em level on the
functioning of plasma membrane ion transporters
involved in pH; regulation, the effect of BAPTA on the
Xenopus egg Em might be responsible for the observed
delay in the pH response. However, this is unlikely,
since that pH response has been shown to be independent of any of the known plasma membrane ion
A Ca2+-dependent wave of intracellular pH change
+10
0
-10
10min
pCa 6.27—
pCa 6.27—
ik
pricking
C
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pH, 7.37pH, 7.37—
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63
Fig. 8. An intracellular pH wave that follows
the intracelllular Ca 2+ wave in Xenopus eggs.
(A) A very impressive illustration of the fact
that the increase in [Ca2+], taking place
during Xenopus egg activation proceeds as a
wave. A single egg was impaled with two
Ca 2+ microelectrodes and two potential
microelectrodes, according to the
configuration shown in Fig. 1 (the pH
microelectrode represented in Fig. 1 was
replaced by a second Ca 2+ microelectrode).
Only one of the two membrane potential
(Em) traces is represented (in A, B and C),
since the membrane potential is always the
same at any place within the egg. The Ca 2+
transient, a propagating front initiated from
around the site of pricking, had very different
properties in these two distinct regions of the
egg cortex, both concerning the amount of
Q r + released and the Ca gradient of the
Ca 2+ wave. The Ca 2+ gradient was much
steeper in the region corresponding to the
pCa bottom trace than in the region
corresponding to the pCa top trace, which
might explain the larger amplitude of the
Ca transient in the former (pCa bottom
trace). The distance between the two
arrowheads represents the delay between the
onset of the Ca 2+ transient recorded by the
two microelectrodes. The existence of such a
delay provided the first demonstration of the
existence of a Ca 2+ wave in Xenopus eggs
(Busa and Nuccitelli, 1985). (B, C) Two
examples illustrating the existence of an
intracellular pH wave propagating from
around the site of triggering of egg activation.
Eggs were each impaled with two pH
microelectrodes and two potential
microelectrodes as represented on the
accompanying schemes B' and C , which also
indicate the site of pricking (by one of the
two Em microelectrodes). The distance
between the two arrowheads, in B and C,
indicates the difference of time (delay)
between the onset of the physiological
increase in pHj recorded by the two
microelectrodes at the sites represented on
the corresponding schemes. This clearly demonstrates the existence of a pH wave traveling throughout the egg cortex from
the site of pricking. The mean value of the velocity of the pH wave under the conditions represented here is given in the
text.
transporters (Webb and Nuccitelli, 1982; Grandin and
Charbonneau, 1990b).
Neither the kinetics of the increase in pH, (time
between the beginning of the increase and the elevated
pHj plateau) nor its amplitude was dramatically affected by BAPTA (Table 1). This would tend to suggest
that intracellular Ca 2 + is principally needed for the
initiation of the pH response, but less for its unfolding.
There are two possible explanations. The first one is
that, besides C a 2 + , there exists a second triggering
signal for the increase in pHj itself, and that [Ca 2 + ],
might just play a role in ensuring that a normal time-lag
is established between egg activation and the pH
response. There is no major problem with such an
interpretation. However, the nature of that possible
second triggering signal, which might also depend on
[Ca 2+ ]i but on a sensitivity basis different from that of
the initiation of the pH response, is still unknown. The
second possibility is that Ca 2 + alone determines both
the time to the initiation of the pH response and the
response itself, but that the failure of BAPTA to diffuse
throughout the egg may result in failure to abolish the
pH response completely. On first analysis, this appears
to be unlikely, since several of our experiments show
that BAPTA can diffuse relatively large distances from
the site of microinjection (see, for instance, Figs 3, 9).
However, Fig. 9 also shows that the amount of BAPTA
that diffused far from the site of microinjection was
N. Grandin and M. Charbonneau
64
A
C^10
0
uf -101
pH, 7.48-
pH, 7.48-
B
pricking
BAPTA
Fig. 9. The propagation of the pH wave depends on the
preceding Ca2+ wave. Unactivated eggs were each impaled
with two pH microlectrodes and two potential
microelectrodes as described in scheme C. (A and B)
represent two examples of the same phenomenon. Scheme
C also shows the sites of pricking and microinjection. Eggs
were microinjected with 5-10 nl of a 100 mM BAPTA
solution, pH 7.5 (arrowheads), 2.5-3 min after triggering of
egg activation (pricking), indicated by the occurrence of
the activation potential on the membrane potential (£ m )
trace. Microinjection was done on purpose very near one
of the two pH microelectrodes (the pH microelectrode
noted pH-c in scheme C, recording the pHj bottom trace in
both A and B). The pH microelectrode noted pH-d in
scheme C corresponds to the pHj top trace in both A and
B. It is important to note that the amount of microinjected
BAPTA in this series of experiments (5-10 nl) was smaller
than in the rest of this work (30-40 nl). When compared
with the normal situation in which there was no
microinjection (Fig. 8), local microinjection of small
amounts of BAPTA, as here, caused a very dramatic
slowing down of the pH wave. This was seen as an
increase in the difference of time (delay) between detection
of the onset of the pHj increase at the two distinct
locations (pH-c and pH-d), with respect to the equivalent
delay in non-microinjected eggs (Fig. 8). That difference in
time corresponds to the distance between the two
arrowheads in both A and B. In other words, the
propagation of the pH wave detected with two pH
microelectrodes was slowed down in the region in which
BAPTA was microinjected. The mean value of the velocity
of the pH wave under the conditions presented here is
given in the text. These experiments confirm that the
physiological increase in pH, in Xenopus eggs proceeds as a
wave. They also demonstrate that the propagation of the
pH wave depends on preceding variations in [Ca2+],.
probably much less (had much less effect on the pH
response) than near the site of microinjection, although
it is true that in this particular example the total amount
of injected BAPTA was limited with respect to the
standard conditions. Incidentally, it should be noted
that recording an effect of BAPTA on the Em (the
abrupt hyperpolarization) at a given place within the
egg is not indicative of the fact that BAPTA has really
reached the region located around that particular Em
microelectrode. Indeed, even if there were a local Em
change, this could not be detected with intracellular Em
microelectrodes, because cells are equipotential (due to
the resistance of the cytoplasm being much smaller than
that of the plasma membrane), a situation best
illustrated by the fact that the sperm, which interacts
with only a tiny portion of the egg plasma membrane,
nevertheless produces an Em change that can be
simultaneously recorded at any place within the egg. In
fact, in Xenopus eggs, the demonstration of the
existence of local Em changes or propagating conductance changes necessitated the use of patch electrodes
(Jaffe et al. 1985) or of an extracellular vibrating probe
(Kline and Nuccitelli, 1985). Like the problems regarding diffusion of BAPTA, the time of BAPTA microinjection is of importance in evaluating the role of
[Ca2+]; in determining the pH response. Indeed, in all
experiments in which the pH response was delayed but
not suppressed, BAPTA had been microinjected 2.5-3
min after the activation potential, a standard condition
denned in this study (see corresponding text to Fig.
3B,C in Results). One may wonder if the pH response
would have been both delayed and suppressed following a much earlier microinjection of BAPTA, for
instance 1.5 min after the activation potential as shown
in Fig. 3D,E. We decided to apply a strict rule to
determine our standard conditions and avoid any
situation in which we would not be totally sure that
BAPTA had not interfered with the triggering of egg
activation itself. Situations with such interferences
might be difficult to analyze, because BAPTA might
block many early events of egg activation, most of
which might be only distantly related to the pH
response. In conclusion, it is not possible yet to decide
with certainty whether the Ca2+ transient is the only
event that controls the pH response to egg activation in
Xenopus.
Relationships between [Ca2+]i, pH, and MPF
Sea urchin eggs and various types of cultured mammalian cells certainly represent the best-known systems
concerning the relationships of the [Ca2+]j and pHj
changes to the cell cycle, although, to our knowledge,
no experiments aiming at buffering the Ca 2+ transient
have been performed in these systems. In these
systems, the increase in pH, associated with cell
activation involves the activation of a Na + -H +
exchange (reviewed by Epel and Dube", 1987). In these
systems, the transduction of the activating signal
involves stimulation of the inositol phospholipid metabolism leading to two independent pathways, one
responsible for the [Ca2+], increase via production of
A Co2*-dependent wave of intracellular pH change
inositol 1,4,5-trisphosphate (IP3), the other for the pH,
increase via production of diacylglycerol (DAG) and
activation of protein kinase C (PKC) (see references
and schemes; Pouyss6gur, 1985; Houslay, 1987). It
should be noted that these two pathways are probably
never totally independent of each other, since [Ca2+]i
levels are known to modulate the activity of PKC by
acting on its translocation to the plasma membrane
where 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 subsequent
increase 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, the
Na + -H + exchange, that is blocked.
The situation in Xenopus eggs is quite different from
that 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 pH r
regulating systems in their plasma membrane (Webb
and Nuccitelli, 1982; Grandin and Charbonneau,
1990b). In addition, PKC is not included in the pH,
response to Xenopus egg activation (Grandin and
Charbonneau, 1991c). On the other hand, IP3 (Busa et
al. 1985) and a G protein (Kline et al. 1988) appear to be
involved during Xenopus egg activation. The originality
of the situation in Xenopus eggs also resides in the fact
that the increase in pHj appears to have a metabolic
origin, most probably associated with the inactivation
of MPF (see Grandin and Charbonneau, 1991a). The
assumption that the increase in pHj might be a direct
consequence of MPF inactivation is based on the
existence of temporal relationships between the two
events, in both Xenopus laevis and Pleurodeles waltlii,
another amphibian (Grandin and Charbonneau,
1991a), as well as functional relationships between the
pH, oscillations and the oscillations in the activity of
MPF accompanying the mitotic cell cycle (Grandin and
Charbonneau, 1990a). MPF activity, measured as a
biological activity inducing meiosis resumption in
Xenopus oocytes arrested in prophase 1 of meiotic
maturation, was found to start decreasing 8 min after
egg activation in Xenopus (Gerhart et al. 1984).
Meanwhile, the pH; level, stable during the last part of
meiotic maturation until the arrest in second metaphase, starts elevating around 10 min after egg
activation (Webb and Nuccitelli, 1981). In our hands,
pH, in Xenopus eggs starts increasing 6-7 min after egg
activation, at 22°C. Although both the timing of MPF
inactivation and that of the pH, increase appear to be
closely coincident, it should be noted that the kinetics of
MPF inactivation previously reported were measured at
19°C (Gerhart et al. 1984). In our hands, at 22°C, MPF
activity starts decreasing around 5 min after egg
activation, whether measured as its histone HI kinase
activity (Fig. 6) or as its oocyte maturation-inducing
activity (Grandin and Charbonneau, unpublished results). The present observation that MPF inactivation
occurs slightly before the increase in pH, supports our
previous assumption that the increase in pHj is a
65
consequence of MPF inactivation rather than the
converse (Grandin and Charbonneau, 1991a). Our
experiments using microinjection of BAPTA also
suggest that the intermediate reactions between MPF
inactivation and the initiation of the pH response, if
they exist, are probably Ca2+-dependent.
Cell-free extracts prepared with metaphase-blocked
Xenopus or Rana eggs have MPF activities that are
sensitive to Ca2+ (Meyerhof and Masui, 1977; Masui,
1982; Lohka and Mailer, 1985). Similarly, in a cell-free
system from clam embryos, added Ca 2+ leads to a rapid
destruction of endogenous cyclin, one of the components of MPF (Luca and Ruderman, 1989). In the
present study, the question of the role of intracellular
Ca2+ release in MPF inactivation was not addressed.
However, our results do show that microinjecting
BAPTA 2.5-3 min after the onset of egg activation did
not change the normal time-lag to the onset of MPF
inactivation or the reaction itself. These experiments,
as well as those showing an absence of effect of BAPTA
on meiosis resumption under the same conditions,
suggest that MPF inactivation can proceed normally
even when the Ca2+ transient is strongly reduced, that
is under conditions that prevent a Ca 2+ wave from
propagating throughout the egg cortex. This confirms
recent experiments in which increasing Ca2+ to 1-1.5
JJM in a Xenopus metaphase extract for only 30 s was
found to be sufficient to trigger cyclin degradation
(Lorca et al. 1991).
A wave of intracellular pH changes during Xenopus
egg activation
To our knowledge, the present results are the first to
demonstrate the existence of a wave of intracellular pH
change (Figs 8, 9). The delay between egg activation
and pHj changes was clearly dependent on the distance
between the site of pricking and the pH microelectrode
(Fig. 8). Thefindingthat the pH wave was slowed down
only in the region that had been microinjected with
BAPTA (Fig. 9) confirms the view that the pH wave is a
consequence of the Ca 2+ wave. Since the increase in
[Ca2+]i also proceeds as a wave starting from the site of
triggering of egg activation (Busa and Nuccitelli, 1985)
and given the relationships between pH, changes and
[Ca ], levels uncovered in the present study, it is highly
probable that the pH wave is initiated by the preceding
Ca2+ wave.
The existence of a pH wave might represent
important information to be used in the comprehension
of still poorly understood mechanisms, not only in
Xenopus eggs. A pH wave, even if it existed in other
systems, might be undetectable using the available
techniques, principally because most cells are much
smaller than Xenopus eggs. The mechanisms underlying the propagating pHj changes in Xenopus eggs might
be related to those responsible for the generation of
Ca2+ waves. Most cells have multiple calcium pools,
sensitive or not to IP3, which in most cases are
constituted of endoplasmic reticulum (ER) or have
characteristics related to ER (reviewed by Berridge and
Irvine, 1989). The egg of Xenopus possesses both an
66
N. Grandin and M. Charbonneau
IP3-sensitive and an IP3-insensitive calcium pools (Busa
and Nuccitelli, 1985), and the propagating wave
triggered by IP3, which is indistinguishable from that
observed at fertilization, originates from an ERenriched layer in stratified eggs (Han and Nuccitelli,
1990). Given that the pH wave demonstrated here
depends on [Ca2+], levels and closely follows the Ca 2+
wave, and that the mechanisms and location of the pHj
changes in Xenopus eggs are still unknown, one can
postulate the existence of some cortically localized
intracellular compartment that might start pumping
protons as it is reached by the Ca wave. Alternatively, such a compartment might be the same as the
ER-calcium pool supposed to be at the origin of the
Ca2+ wave, and contain a Ca 2+ -H + exchange system in
its membrane. However, these hypotheses need to be
experimentally challenged.
We thank Mrs M. Manceau for cutting paraffin sections,
and Mr L. Communier for help in preparing the photographic
illustration. This work was supported by grants from the
Ligue contre le Cancer (Comit6 D6partemental d'llle-etVilaine), the Association pour la Recherche sur le Cancer,
and the Fondation pour la Recherche M6dicale.
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(Received 29 July 1991 - Accepted 25 September 1991)