impaled with a pH microelectrode, a Ca2 microelectrode and two potential microelectrodes (Em microelectrodes) as shown in the scheme. Once the electrical ...
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
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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.
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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 • 10
5.
10 pH@
pH, 7.37pH, 7.37—
©
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
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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. References Ammann, D., Lanter, F., Steiner, R. A., Schulthess, P., Shijo, Y. and Simon, W. (1981). Neutral carrier-based hydrogen ion selective microelectrode for extra- and intracellular studies. Analyt. Chem. 53, 2267-2269. Bement, W. M. and Capco, D. G. (1990). Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis. Cell Regul. 1, 315-326. Berridge, M. J. and Irvine, R. F. (1989). Inositol phosphates and cell signalling. 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-mediated cell regulation. Annu. Rev. Physiol. 48, 389-402. Busa, W. B., Ferguson, J. E., Joseph, S. K., Williamson, J. R. and Nuccitelli, R. (1985). Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. I. Characterization of Ca 2 + release from intracellular stores. J. Cell Bwl. 101, 677-682. Busa, W. B. and Nuccitelli, R. (1984). Metabolic regulation via intracellular pH. Am. J. Physiol. 246, R4O9-R438. Busa, W. B. and Nuccitelli, R. (1985). An elevated free cytosolic Ca 2 + wave follows fertilization in eggs of the frog, Xenopus laevis. J. Cell Biol. 100, 1325-1329. Charbonneau, M. and Picheral, B. (1983). Early events in anuran amphibian fertilization: an ultrastrucural study of changes occurring in the course of monospermic fertilization and artificial activation. Dev. Growth Differ. 25, 23-37. Critser, E. S. and First, N. L. (1986). Use of a fluorescent stain for visualization of nuclear material in living oocytes and early embryos. Stain Technol. 61, 1-5. Doree, M. (1990). Control of M-phase by maturation-promoting factor. 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. Boynton and H. M. Leffert), pp. 363-393. Academic Press, New York. Felix, M. A., Pines, J., Hunt, T. and Karsenti, E. (1989). A postribosomal supernatant from activated Xenopus eggs that displays post-translationally regulated oscillation of its cdc2+ mitotic kinase activity. EM BO J. 8, 3059-3069. Gerhart, J. C , Wu, M. and Klrschner, M. (1984). Cell cycle dynamics of an M-phase-specific cytoplasmic factor in Xenopus laevis oocytes and eggs. /. Cell Biol. 98, 1247-1255.
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(Received 29 July 1991 - Accepted 25 September 1991)
