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Mol Biol Cell. Nov 2002; 13(11): 3800–3810.
PMCID: PMC133593

The Intracellular pH-regulatory HCO3/Cl Exchanger in the Mouse Oocyte Is Inactivated during First Meiotic Metaphase and Reactivated after Egg Activation via the MAP Kinase Pathway

Guido Guidotti, Monitoring Editor

Abstract

The HCO3/Cl exchanger is quiescent in the unfertilized mouse egg but is highly active in regulating intracellular pH in the early embryo and required for normal development. We show here that the HCO3/Cl exchanger is active in first meiotic prophase (GV) oocyte but inactivated during meiotic metaphase before the MI to MII transition. Reactivation does not occur until the activated egg enters interphase. A quiescent HCO3/Cl exchanger is not simply a general feature of metaphase, because activity did not decrease during first mitotic metaphase. Inactivation of the HCO3/Cl exchanger during MI coincided with the activation of MAP kinase (MAPK), whereas its reactivation coincided with the loss of MAPK activity after egg activation. Maintaining high MAPK activity after egg activation prevented the normal reactivation of the HCO3/Cl exchanger. Inactivating MAPK in unfertilized MII eggs resulted in HCO3/Cl exchanger activation. Preventing MAPK activation during first meiotic metaphase prevented the inactivation of HCO3/Cl exchange. Conversely, activating MAPK in the GV oocyte resulted in inactivation of HCO3/Cl exchange. These results imply that the HCO3/Cl exchanger in mouse oocytes is negatively regulated by MAPK. Thus, suppression of pH-regulatory mechanisms during meiosis is a novel function of MAPK and cytostatic factor activity in the oocyte.

INTRODUCTION

Intracellular pH (pHi) in mammalian cells is maintained within a narrow range by transport mechanisms, including the Na+/H+ antiporter, which exports H+ and thus corrects low pHi, and the HCO3/Cl exchanger, which exports HCO3 and thus corrects increases in pHi (Roos and Boron, 1981 blue right-pointing triangle; Alper, 1994 blue right-pointing triangle; Orlowski and Grinstein, 1997 blue right-pointing triangle). The HCO3/Cl exchanger is highly active in preimplantation mammalian embryos from the pronuclear egg through the blastocyst stages (Baltz et al., 1991 blue right-pointing triangle; Zhao and Baltz, 1996 blue right-pointing triangle; Lane et al., 1999a blue right-pointing triangle; Phillips et al., 2000 blue right-pointing triangle; Phillips and Baltz, 1999 blue right-pointing triangle), and exchanger activity is required for mouse embryos to maintain pHi and for normal embryo development (Zhao et al., 1995 blue right-pointing triangle).

In a number of marine invertebrates and amphibians, activation of pHi-regulatory transporters is a fundamental event of egg activation (Johnson et al., 1976 blue right-pointing triangle; Webb and Nuccitelli, 1981 blue right-pointing triangle; Dube et al., 1985 blue right-pointing triangle; Dube, 1988 blue right-pointing triangle; Epel, 1988 blue right-pointing triangle; Freeman and Ridgway, 1993 blue right-pointing triangle; Dube and Eckberg, 1997 blue right-pointing triangle). In the sea urchin, the Na+/H+ antiporter is quiescent in the egg but becomes highly active within minutes of fertilization (Johnson et al., 1976 blue right-pointing triangle; Epel, 1988 blue right-pointing triangle), causing a permanent increase in pHi that is required for subsequent embryo development. In mammals, however, no increase in pHi follows fertilization (Kline and Zagray, 1995 blue right-pointing triangle; Ben Yosef et al., 1996 blue right-pointing triangle; Phillips and Baltz, 1996 blue right-pointing triangle; Dale et al., 1998 blue right-pointing triangle; Phillips et al., 2000 blue right-pointing triangle), and it had been assumed that activation of pHi-regulatory mechanisms is not a feature of mammalian fertilization.

We recently showed, however, that the HCO3/Cl exchanger is quiescent in ovulated mouse eggs and only becomes activated several hours after fertilization (Phillips and Baltz, 1999 blue right-pointing triangle). Similarly, both the HCO3/Cl exchanger and Na+/H+ antiporter are quiescent in hamster eggs and become activated after fertilization (Lane et al., 1999a blue right-pointing triangle, 1999b blue right-pointing triangle). Fertilization-induced activation of the HCO3/Cl exchanger in mouse or Na+/H+ antiporter in hamster did not require protein synthesis or rely on protein trafficking, indicating that preexisting exchanger proteins in the membrane likely become activated by fertilization (Lane et al., 1999b blue right-pointing triangle; Phillips and Baltz, 1999 blue right-pointing triangle). However, the mechanism of activation after fertilization was unknown.

Activation of Na+/H+ antiporter activity at fertilization in the sea urchin is controlled through intracellular calcium (Ca2+i)-dependent signaling via protein kinase C (Swann and Whitaker, 1985 blue right-pointing triangle; Epel, 1988 blue right-pointing triangle). In mouse and hamster, chelation of Ca2+i similarly inhibited the activation of pHi-regulatory mechanisms after fertilization (Lane et al., 1999b blue right-pointing triangle; Phillips and Baltz, 1999 blue right-pointing triangle). We thus proposed that the increase in Ca2+i and repetitive Ca2+i transients that follows fertilization in mammals (Jones et al., 1995 blue right-pointing triangle) might be involved in activating pHi-regulatory systems in mammalian eggs (Phillips and Baltz, 1999 blue right-pointing triangle). We show here, however, that activation of the HCO3/Cl exchanger in mouse eggs after fertilization is independent of Ca2+i transients. Instead, its activity is closely correlated with the meiotic cell cycle.

In most vertebrates, the fully grown oocyte is arrested in meiotic prophase, exhibiting a prominent nucleus or germinal vesicle (GV). On ovulation, meiosis resumes and the oocyte exits prophase I, as marked by the disappearance of the GV or GV breakdown (GVBD). Prophase I arrest appears to be maintained in mammalian eggs at least in part by an unknown inhibitory signal from the surrounding follicle cells, because most mammalian GV eggs spontaneously resume meiosis upon removal from the follicle. This is in contrast to many other animals, where resumption of meiosis requires a positive hormonal signal. In both cases, however, release from arrest appears to require a decrease in intracellular cAMP, whereas artificially maintaining elevated cAMP will prevent GVBD. After GVBD, the oocyte proceeds through first meiotic metaphase (MI), which ends with an unequal cytokinesis to emit the first polar body that removes one-half of the maternal chromosomes. The oocyte then reenters metaphase and becomes arrested in second meiotic metaphase (MII). On fertilization or egg activation, metaphase arrest is released, and the oocyte undergoes the second unequal cleavage to emit the second polar body and attain haploidy. The male and female genetic material form two separate pronuclei, which combine by the end of the first mitotic cell cycle to form the embryonic genome.

Two key players in regulating meiosis are maturation promoting factor (MPF), which induces metaphase, and cytostatic factor (CSF), which maintains arrest of ovulated oocytes (in MII in mammals) until after fertilization. MPF is a complex of cyclin B and cdk1 kinase and is the common trigger of both meiotic and mitotic metaphases. In contrast, CSF, which stabilizes MPF and maintains metaphase arrest, is specific to meiosis. The MOS/MEK/MAPK pathway has been shown to at least partly underlie CSF activity in oocytes (Colledge et al., 1994 blue right-pointing triangle; Hashimoto et al., 1994 blue right-pointing triangle; Gross et al., 2000 blue right-pointing triangle). Because, as we report here, HCO3/Cl exchanger activity is regulated by the meiotic cell cycle, we have investigated whether the signaling pathways known to control meiotic maturation in oocytes also regulate HCO3/Cl exchanger activity. Our results indicate, for the first time, that a pHi-regulatory mechanism in a mammalian oocyte is controlled by the developmental status of the oocyte and is cell cycle dependent during meiosis.

MATERIALS AND METHODS

Chemicals and Solutions

Cycloheximide, demecolcine, hyaluronidase, pregnant mare's serum gonadotropin (PMSG), human chorionic gonadotropin (hCG), EGTA, dibutyryl cyclic AMP (dbcAMP), okadaic acid (OA), and nigericin were obtained from Sigma (St. Louis, MO). 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), valinomycin, 4-bromo-A23187, and the acetoxymethyl esters of SNARF-1 and Fura-2 were obtained from Molecular Probes (Eugene, OR). U0126 was obtained from Calbiochem (La Jolla, CA). All stock solutions were prepared in DMSO, except for cycloheximide and dbcAMP (water), and demecolcine and nigericin (ethanol), and stored at −20°C.

All media were based on KSOM embryo culture medium (Lawitts and Biggers, 1993 blue right-pointing triangle; Phillips and Baltz, 1999 blue right-pointing triangle), equilibrated with 5% CO2/air except where noted. For pHi measurements, 9 of the 10 mM Na lactate was replaced with NaCl, and BSA was omitted. All solutions contained 110 mM Cl, except for Cl-free media, where all Cl salts were replaced with corresponding gluconate salts as described previously (Phillips and Baltz, 1999 blue right-pointing triangle).

Oocyte and Egg Collection and Egg Activation

HEPES-buffered medium (HEPES-KSOM, pH 7.4; Lawitts and Biggers, 1993 blue right-pointing triangle) was used for oocyte and egg collection. Female CF1 mice (6–8 week old; Charles River, Montreal, Quebec, Canada) were superovulated with 5 IU PMSG given intraperitoneally (IP). GV oocytes were collected from minced ovaries 45–48 h post-PMSG. dbcAMP (300 μM) was present during collection and culture to maintain GV arrest (Cho et al., 1974 blue right-pointing triangle) unless otherwise specified. For unfertilized eggs, ovulation was induced with 5 IU hCG IP 48 h post-PMSG, and eggs were collected 13.5–16 h post-hCG as previously described (Phillips and Baltz, 1999 blue right-pointing triangle). Eggs and oocytes were cultured in KSOM droplets under mineral oil in 5%CO2/air at 37°C (Lawitts and Biggers, 1993 blue right-pointing triangle).

For activation with Sr2+, eggs were incubated for 2 h in KSOM with CaCl2 omitted and 10 mM SrCl2 added (Fraser, 1987 blue right-pointing triangle; Bos-Mikich et al., 1995 blue right-pointing triangle). For activation with cycloheximide, eggs were incubated for 2 h with 50 μg/ml cycloheximide (Siracusa et al., 1978 blue right-pointing triangle; Moos et al., 1996a blue right-pointing triangle). After activation, eggs were washed and transferred to culture in KSOM.

Intracellular pH and Ca2+ Measurements

pHi and Ca2+i measurements using a quantitative fluorescence imaging microscopy system (Inovision, Durham, NC) have been described previously (Baltz et al., 1991 blue right-pointing triangle; Zhao et al., 1995 blue right-pointing triangle; Baltz and Phillips, 1999 blue right-pointing triangle). Briefly, pHi was determined in oocytes loaded intracellularly with the pH-sensitive fluorophore, SNARF-1 (Zhao et al., 1995 blue right-pointing triangle; Baltz and Phillips, 1999 blue right-pointing triangle). Calibration of ratio with pHi was done by the nigericin/high K+ method with valinomycin added to collapse the K+ gradient (Thomas et al., 1979 blue right-pointing triangle; Baltz and Phillips, 1999 blue right-pointing triangle). Ca2+i was determined with Fura-2 (Baltz and Phillips, 1999 blue right-pointing triangle; Phillips and Baltz, 1999 blue right-pointing triangle). Ca2+i was estimated from the intensity ratio as previously described (Grynkiewicz et al., 1985 blue right-pointing triangle; Baltz and Phillips, 1999 blue right-pointing triangle). Each replicate consisted of simultaneous measurements made upon groups of 5–20 eggs or oocytes. pHi was averaged for the group at each time point (Baltz and Phillips, 1999 blue right-pointing triangle; Phillips and Baltz, 1999 blue right-pointing triangle). All measurements were made with oocytes or eggs in a temperature- and atmosphere-controlled chamber (37°C, 5% CO2/air).

Cl Removal Assay for HCO3/Cl Exchange Activity

HCO3/Cl exchanger activity was quantified by the Cl removal method (Zhao and Baltz, 1996 blue right-pointing triangle; Baltz and Phillips, 1999 blue right-pointing triangle; Phillips et al., 2000 blue right-pointing triangle; Phillips and Baltz, 1999 blue right-pointing triangle). On exposure of cells to Cl-free solution, the HCO3/Cl exchanger will run in reverse, resulting in intracellular alkalinization due to HCO3 influx coupled to Cl efflux (Nord et al., 1988 blue right-pointing triangle). Increased pHi upon Cl removal thus indicates HCO3/Cl exchanger activity, and the initial rate of alkalinization provides a quantitative measure of activity (Nord et al., 1988 blue right-pointing triangle). Here, SNARF-1–loaded eggs or oocytes were placed in the chamber for 10 min, after which the solution was changed to Cl-free, low-lactate KSOM for 20 min. The initial rate of intracellular alkalinization upon Cl removal was determined using linear regression (SigmaPlot 5; SPSS, Chicago, IL), and exchanger activity is reported as the change in pHi per minute (pH U/min). This assay for HCO3/Cl exchanger activity has been extensively described and validated in mouse oocytes and embryos (Zhao et al., 1995 blue right-pointing triangle; Zhao and Baltz, 1996 blue right-pointing triangle; Baltz and Phillips, 1999 blue right-pointing triangle; Phillips and Baltz, 1999 blue right-pointing triangle; Phillips et al., 2000 blue right-pointing triangle). The change in pHi observed after external Cl removal was confirmed to be diagnostic of HCO3/Cl exchanger activity by its pharmacological profile and HCO3 requirement (Baltz et al., 1991 blue right-pointing triangle). In addition, the lack of HCO3/Cl exchanger activity in ovulated eggs indicated by this assay has been confirmed by demonstrating their inability to recover from ammonium-induced alkalosis (Phillips and Baltz, 1999 blue right-pointing triangle), ruling out the alternative possibility that unfertilized eggs instead have an abnormally low internal Cl concentration that would prevent HCO3/Cl exchanger reversal upon external Cl removal.

Simultaneous Measurement of Histone H1 Kinase and MBP Kinase Activities

Histone H1 kinase (MPF) and MBP kinase (MAPK) activities were measured simultaneously in a single assay as previously described (Phillips et al., 2002 blue right-pointing triangle). For each measurement, seven eggs along with ~1–1.5 μl medium were added to 3.5 μl lysis buffer and frozen at −80°C until the assay was performed. The lysis buffer used was identical to that described by Moos et al., except that 10 μg/ml leupeptin was added (Phillips et al., 2002 blue right-pointing triangle). The kinase assay reaction was initiated by thawing and addition of 5 μl kinase buffer, which contained 500 μCi/ml γ-32P-ATP (Amersham). The kinase buffer used was identical to that described by Moos et al. for the Histone H1 kinase assay, except that it contained 0.5 mg/ml MBP in addition to 2 mg/ml histone type III-S (Phillips et al., 2002 blue right-pointing triangle). Phosphorylation of histone and MBP (transfer of γ-32P) has been shown to increase linearly in this assay for at least 60 min (Phillips et al., 2002 blue right-pointing triangle), and the presence of phosphatase inhibitors prevented significant dephosphorylation during the assay period. After 30 min, the reaction was stopped by adding 10 μl 2× Laemmli sample buffer and boiling for 3 min. The extent of phosphorylation of MBP and histone was determined using SDS-PAGE (15% gels) quantified with a phosphorimager (Typhoon 8600 with ImageQuant software; Molecular Dynamics, Inc., Sunnyvale, CA). Background was determined from a reaction run without eggs. A sample of fresh unfertilized eggs was included in each gel, and H1 and MBP kinase activities were expressed relative to the values for eggs (set to 100%) within each gel. Histone H1 kinase and MBP phosphorylation were assumed to indicate MPF and MAPK activity, respectively, as previously demonstrated (Moos et al., 1996a blue right-pointing triangle; Phillips et al., 2002 blue right-pointing triangle).

U0126, a specific inhibitor of the MAPK kinase, MEK, was used to decrease MAPK activity. We have extensively validated the use of U0126 to inhibit MEK and hence MAPK in eggs, including demonstrating, by Western blot, a complete shift from the slower-migrating (active) forms of ERK1 and -2 to the faster-migrating (inactive) forms after U0126 treatment of eggs, and the complete loss of immunoreactivity with antiphospho-MAPK antibody after U0126 treatment (Phillips et al., 2002 blue right-pointing triangle). We previously found that 50 μM U0126 reduced MAPK activity to background in unfertilized eggs (Phillips et al., 2002 blue right-pointing triangle). U0126 is somewhat more effective at preventing MEK activation than in inhibiting active MEK, and thus we found here that 20 μM U0126 completely prevented MAPK activation during meiotic maturation (below). Therefore, these concentrations of U0126 were used here.

Okadaic acid was used in some experiments to maintain high MAPK activity. The induction of high MAPK activity in activated eggs and GV oocytes, where MAPK is normally inactive, by OA treatment was previously shown by both the kinase assay described here and by the expected mobility shifts of ERK by Western blot (Moos et al., 1995 blue right-pointing triangle; Lu et al., 2002 blue right-pointing triangle).

Statistics

Means of replicates were compared by t test (2 groups) or ANOVA (≥3 groups) using InStat (GraphPad, San Diego, CA). A parametric t test (Student's) or ANOVA (pANOVA) was used if variances were not significantly different by F-test or Bartlett's test, respectively, or else a nonparametric t test (Welch's) or ANOVA (nANOVA) was used. For ANOVAs, post hoc tests (Tukey-Kramer Multiple Comparisons test for pANOVA; Dunn's test for nANOVA) were performed to compare treatment groups. Plots and curve fits were done using SigmaPlot 5 (SPSS, Chicago, IL).

RESULTS

Development of HCO3/Cl Exchanger Activity after Sr2+Activation of Eggs

To allow precise timing and to permit manipulation, we parthenogenetically activated eggs with Sr2+. These activated eggs exhibited repetitive Ca2+i transients (Figure (Figure1A),1A), similar to those after IVF (Phillips and Baltz, 1999 blue right-pointing triangle). The time courses of polar body emission and pronuclear development in Sr2+ activated eggs (Figure (Figure1B)1B) and in eggs after IVF (Phillips and Baltz, 1999 blue right-pointing triangle) were similar. Furthermore, MPF (Histone H1 kinase) and MAPK activity (MBP kinase) decreased after Sr2+ activation (Figure (Figure1C)1C) with nearly identical time courses as after IVF (Phillips et al., 2002 blue right-pointing triangle). Sr2+-activated eggs developed in culture past the two-cell stage (96%) to morulae (58%; n = 60, N = 4), but development to blastocysts was lower (15%), as is typical for parthenogenotes.

Figure 1
Sr2+activation of mouse eggs and activation of HCO3/Cl exchanger. (A) Ca2+i transients induced in individual eggs (n = 5) upon exposure to 10 mM Sr2+. (B) Timing of emission of second polar body (PB2; 65 eggs ...

The HCO3/Cl exchanger became activated in eggs that were parthenogenetically activated with Sr2+ (Figure (Figure1D).1D). Half-maximal activation occurred at ~5 h, and maximal activation at 7 h post-Sr2+ activation. This time course is nearly identical to that obtained for HCO3/Cl exchanger activation after IVF, where HCO3/Cl exchanger activity was half-maximal at ~6 h after sperm-egg incubation and maximal by ~8 h (Phillips and Baltz, 1999 blue right-pointing triangle). Activation occurred just after pronuclear formation and the decrease in MAPK activity that follows egg activation.

Upregulation of HCO3/Cl Exchanger Does Not Depend on Ca2+i Transients

Exposing unfertilized eggs to a 5-min SrCl2 (10 mM) pulse produced an immediate, single Ca2+i transient of ~7 min duration (unpublished data). This was sufficient to activate eggs (100%), which emitted a second polar body and developed pronuclei within 6 h (unpublished data). Activated eggs were found to develop HCO3/Cl exchanger activity after a single Ca2+ transient, with a mean activity of 0.050 ± 0.017 pH U/min (n = 69, N = 5) at 7–9 h after activation, as determined by the Cl-removal assay.

To determine whether HCO3/Cl exchanger activity would develop in the absence of Ca2+i transients, eggs were activated by exposure to cycloheximide, a protein synthesis inhibitor that activates eggs by disrupting the continuous cyclin synthesis required for MII arrest (Moos et al., 1996a blue right-pointing triangle). Pronuclei developed after cycloheximide exposure with essentially the same time course as with Sr2+ activation (Figure (Figure2A).2A). Cycloheximide-induced egg activation occurs without Ca2+i transients (Jones et al., 1995 blue right-pointing triangle; Moos et al., 1996a blue right-pointing triangle), which we confirmed (Figure (Figure2B).2B). Cycloheximide-activated eggs developed HCO3/Cl exchanger activity to the same level (Figure (Figure2C)2C) as after Sr2+ activation (above) or IVF (Phillips and Baltz, 1999 blue right-pointing triangle) at 7–9 h postactivation. This indicates that Ca2+i transients are unnecessary for activation of HCO3/Cl exchange. Inhibition of HCO3/Cl exchanger activation after fertilization by chelation of Ca2+i with BAPTA (Phillips and Baltz, 1999 blue right-pointing triangle), must therefore reflect an effect of abnormally low Ca2+i rather than any requirement for Ca2+i transients. MPF inactivation after egg activation requires an intact metaphase spindle in mouse eggs (Kubiak et al., 1993 blue right-pointing triangle; Winston et al., 1995 blue right-pointing triangle). We arrested eggs in metaphase by disrupting the spindle (1 μg/ml demecolcine for 1 h) and then induced Ca2+i transients with Sr2+ (2 h, as above). This prevented emission of the second polar body or pronuclear formation (Figure (Figure3A)3A) and maintained high MPF and MAPK activities (Figure (Figure3B),3B), whereas Ca2+i transients occurred normally (unpublished data). HCO3/Cl exchanger activity did not develop despite the presence of Ca2+i transients (Figure (Figure3C).3C). This indicates that Ca2+i transients are not sufficient to induce activation of HCO3/Cl exchange.

Figure 2
Activation of HCO3/Cl exchange after egg activation by cycloheximide. (A) Timing of pronuclear development after cycloheximide exposure (109 eggs in 8 replicates, combined; normalized to total number of eggs activated). Eggs were activated ...
Figure 3
Effect of arrest with demecolcine on activation of HCO3/Cl exchanger. (A) Emission of second polar body (PB2, [filled square]) and pronuclear development (PN, ●) assessed as a function of time after activation of eggs with Sr2+ ...

HCO3/Cl Exchanger Is Inactivated during Meiotic Maturation

Under our conditions, release of GV oocytes from prophase I arrest (GVBD) was complete by ~3 h after removal from dbcAMP, and the transition from MI to MII, marked by emission of the first polar body, occurred at around 14 h (Figure (Figure4A).4A). Freshly obtained GV oocytes exhibited a high level of HCO3/Cl exchanger activity, which could be maintained for more than 12 h in GV oocytes arrested with dbcAMP (Figure (Figure4B).4B). The alkalinization measured upon Cl removal in GV oocytes was confirmed to indicate HCO3/Cl exchanger activity, because it was blocked by the anion transport inhibitor DIDS (100 μM; unpublished data). dbcAMP itself has no effect on HCO3/Cl exchanger activity in eggs (Phillips and Baltz, 1999 blue right-pointing triangle).

Figure 4
HCO3/Cl exchanger activity during meiotic maturation. Oocytes were induced to undergo spontaneous GVBD and meiotic maturation by removal from dbcAMP (t = 0) or were maintained in GV arrest by the continuous presence of dbcAMP. (A) Proportion ...

To determine whether the HCO3/Cl exchanger was deactivated during meiosis, HCO3/Cl exchanger activity was measured in oocytes as a function of time after they were released from prophase I (GV) arrest. After removal of dbcAMP, HCO3/Cl exchanger activity remained high in oocytes that still possessed a GV and in early MI eggs after GVBD (Figure (Figure4B).4B). Activity then decreased between 6 and 8 h after removal from dbcAMP, reached a minimum several hours before the transition to MII, and then remained low (Figure (Figure4B).4B). Thus, the HCO3/Cl exchanger is active in prophase I oocytes and is inactivated during first meiosis in the mouse oocyte.

Inactivation of the HCO3/Cl Exchanger Is Not a General Feature of Metaphase

Inactivation of HCO3/Cl exchange during meiotic metaphase might indicate that such inactivation is a feature of metaphase in general, at least during early development. Therefore, we examined HCO3/Cl exchanger activity during the subsequent metaphase—the first mitotic metaphase at the 1- to 2-cell transition. HCO3/Cl exchanger activity was measured in Sr2+-activated eggs from just before pronuclear envelope breakdown (late G2 and prophase) through cytokinesis. Pronuclear envelope breakdown was complete in most parthenogenotes by ~18.5 h after egg activation, with cytokinesis ~3 h later (Figure (Figure5A).5A). We found no decrease in the very high HCO3/Cl exchanger activity evident during this period (Figure (Figure5B),5B), indicating that activity does not decrease during first mitotic metaphase.

Figure 5
HCO3/Cl exchanger activity during first mitotic metaphase. (A) Timing of pronuclear envelope breakdown (NEBD) and cytokinesis (to 2-cell stage) in Sr2+-activated eggs as a function of time after activation (60 eggs in 3 replicates). ...

Metaphase-induced inactivation of the exchanger may require a longer period in metaphase than normally occurs during mitosis but that is typical of meiotic metaphase and MII arrest. We thus arrested parthenogenotes in first mitotic metaphase with demecolcine added at 12 h post-Sr2+ activation and then maintained them in demecolcine for 8 h. We found that HCO3/Cl exchanger activity remained as high in activated eggs arrested in first mitotic metaphase as in activated eggs exposed to vehicle alone, which had progressed through metaphase and cleaved to the two-cell stage (Figure (Figure5C).5C). Thus, an inactive HCO3/Cl exchanger appears to be a specific feature of meiotic metaphase and is not a general consequence of metaphase.

Maintenance of MAPK Activity in Activated Eggs Prevents Activation of HCO3/Cl Exchanger

Both MPF and MAPK become activated during meiotic maturation, are active in MII eggs, and are inactivated after egg activation (Verlhac et al., 1994 blue right-pointing triangle, 1996 blue right-pointing triangle; Moos et al., 1995 blue right-pointing triangle; Verlhac et al., 1996 blue right-pointing triangle), which is the converse of HCO3/Cl exchanger activity. However, HCO3/Cl exchanger activity did not decrease again during first mitotic metaphase (Figure (Figure5),5), where MPF is reactivated but MAPK activity is reported to remain low (Haraguchi et al., 1998 blue right-pointing triangle). HCO3/Cl exchanger activity also appeared to more closely mirror MAPK activity. Therefore, we investigated whether MAPK activity affects HCO3/Cl exchanger activity in mouse oocytes.

OA, an inhibitor of protein phosphatases PP1 and PP2A (Cohen et al., 1990 blue right-pointing triangle), has been used as a tool to maintain high MAPK activity in fertilized eggs (Schwartz and Schultz, 1991 blue right-pointing triangle; Moos et al., 1995 blue right-pointing triangle). When OA (2.5 μM) was added 15 min before (Figure (Figure6A)6A) or coincident with (unpublished data) Sr2+, it prevented the development of pronuclei. This is consistent with previous reports that the decrease in MAPK activity after fertilization regulates the formation of pronuclei (Moos et al., 1995 blue right-pointing triangle, 1996b blue right-pointing triangle), and OA thus prevents formation of pronuclei (Schwartz and Schultz, 1991 blue right-pointing triangle; Moos et al., 1995 blue right-pointing triangle). Introduction of OA immediately after the 2-h period of Sr2+ exposure permitted only a transient (~2 h) appearance of pronuclei (unpublished data), whereas OA introduced to the pronucleate stage or two-cell stage parthenogenotes (Sr2+-activated eggs after cleavage to the 2-cell stage) caused the pronuclei to disappear after ~4 h (Figure (Figure6A).6A). We confirmed that OA, added 15 min before Sr2+, maintained high MAPK activity in activated eggs (Figure (Figure6B).6B). In contrast, OA had no effect on MPF activity, which was similarly low in OA-treated and control eggs 8 h after Sr2+ activation (Figure (Figure6B).6B). Therefore, OA can be used to produce activated eggs with low MPF activity and inappropriately high MAPK activity.

Figure 6
HCO3/Cl exchanger activity after okadaic acid (OA) treatment of activated eggs. (A) Proportion with pronuclei or nuclei assessed as a function of time after OA addition (OA addition at t = 0 in each case), with OA added either just before ...

An additional strategy for maintaining high MAPK activity by expressing exogenous constitutively activated MEK in eggs has been reported previously (Moos et al., 1995 blue right-pointing triangle). However, we have been unable to obtain sufficient expression to prevent pronuclear formation after activation by Sr2+ (unpublished data; constitutively-activated MEK was a gift of S. Gutkind, NIH). Thus, we have relied on OA here to maintain high MAPK activity after egg activation.

We thus determined whether HCO3/Cl exchanger activity developed in Sr2+-activated parthenogenotes where elevated MAPK activity was maintained by OA. OA, added 15 min before Sr2+, completely prevented the appearance of HCO3/Cl exchanger activity at 7–9 h after egg activation, by which time maximal HCO3/Cl exchanger activity had developed in control groups of Sr2+-activated eggs (Figure (Figure6C).6C). Similarly, no HCO3/Cl exchanger activity developed in Sr2+-activated eggs at 7–9 h after egg activation when OA was added immediately after the 2-h period of Sr2+ exposure, 1.5 h later or 3 h later (unpublished data).

To determine if OA was capable of inactivating the fully active HCO3/Cl exchanger in pronucleate stage eggs, we added OA to eggs 10 h after Sr2+ activation, several hours after full HCO3/Cl exchanger activity had developed. There was no significant decrease in HCO3/Cl exchanger activity 4 h after OA addition (unpublished data). However, 8 h after OA addition, HCO3/Cl exchanger activity had been reduced to background levels (Figure (Figure6C).6C). Similarly, 8 h of OA exposure was able to eliminate the robust HCO3/Cl exchanger activity in two-cell parthenogenotes (Figure (Figure66C).

Inactivation of MAPK in Unfertilized Eggs Activates HCO3/Cl Exchange

We used U0126, a specific inhibitor of the MAPK kinase MEK, to decrease MAPK activity in unfertilized eggs. We previously showed (Phillips et al., 2002 blue right-pointing triangle) that U0126 (50 μM) rapidly and completely inactivates MAPK in unfertilized eggs (<1 h versus 5-h post-IVF or -Sr2+ activation). The loss of CSF activity in the presence of U0126 in turn caused MPF activity to decrease, with a time course similar to that after IVF or Sr2+ activation (1–2 h; Phillips et al., 2002 blue right-pointing triangle). We found here that exposure of eggs to U0126 (50 μM) caused them to develop HCO3/Cl exchanger activity (Figure (Figure7).7). Activity appeared to begin to develop immediately, without the 3–4-h lag time as seen with Sr2+ (Figure (Figure1D,1D, D,7)7) or after IVF (Phillips and Baltz, 1999 blue right-pointing triangle), and was maximal by 5 h after U0126 was added.

Figure 7
Effect of U0126 on HCO3/Cl exchanger activity in eggs. Mean (±SEM) HCO3/Cl exchanger activity in unfertilized eggs treated with the MEK inhibitor U0126 (50 μM; ●) or in control eggs (vehicle ...

HCO3/Cl Exchanger Inactivation during Meiotic Maturation Requires MAPK Activity

MI oocytes cultured with U0126 (20 μM) after GVBD developed normally high MPF (H1 kinase) activity. However, unlike normal MI oocytes, MAPK activity remained minimal and was still not detectable at 8 h after release from prophase arrest (Figure (Figure8A).8A). MI oocytes treated with U0126 exhibited high HCO3/Cl exchanger activity at 8 h, which was not significantly different from that of GV oocytes maintained in prophase arrest (Figure (Figure8B).8B). In contrast, control oocytes treated in parallel had the low HCO3/Cl exchanger activity expected for late MI oocytes (Figure (Figure8B).8B).

Figure 8
Effect of U0126 on HCO3/Cl exchanger activity in first meiotic metaphase oocytes. (A) Mean (±SEM) MPF (H1 kinase) and MAPK (MBP kinase) in GV oocytes maintained in GV arrest with dbcAMP, or in MI oocytes that had been allowed ...

HCO3/Cl Exchanger in GV Oocytes Can Be Inactivated by MAPK Activation in the Absence of MPF Activation

It has recently been shown that the treatment of dbcAMP-arrested GV oocytes with a brief pulse of OA induces the irreversible activation of MAPK and breakdown of the GV, but MPF does not become activated (de Vantery Arrighi et al., 2000 blue right-pointing triangle; Lu et al., 2002 blue right-pointing triangle). When we treated GV oocytes with an OA pulse (2.5 μM, 1 h), they exhibited the characteristic “ruffled” appearance (Schwartz and Schultz, 1991 blue right-pointing triangle; Moos et al., 1995 blue right-pointing triangle; Zernicka-Goetz et al., 1997 blue right-pointing triangle) and underwent breakdown of the GV despite the presence of dbcAMP. We confirmed that this treatment produced oocytes with low MPF activity and high MAPK activity at 10 h post-GVBD (Figure (Figure9A).9A). As expected, at the same time post-GVBD, MPF, and MAPK were still inactive in GV oocytes maintained in arrest with dbcAMP and were both maximally active in MI oocytes exposed to a pulse of vehicle (DMSO) alone (Figure (Figure9A).9A). After an OA pulse, oocytes maintained in dbcAMP exhibited very low HCO3/Cl exchanger activity at 10 h, which was not significantly different from that in MI oocytes and was significantly lower than that in oocytes maintained in GV arrest for the same period (Figure (Figure9B).9B).

Figure 9
Effect of activation of MAPK by OA pulse on HCO3/Cl exchanger activity in GV oocytes. (A) Mean (±SEM) MPF (H1 kinase) and MAPK (MBP kinase) activities in oocytes. Oocytes were maintained in GV arrest with dbcAMP (GV), maintained ...

To show that the decrease in HCO3/Cl exchanger activity was due specifically to MAPK activity induced by the OA pulse, we blocked activity of the MAPK kinase, MEK, in GV oocytes before the OA pulse by pretreating them with 50 μM U0126 for 30 min in the continuous presence of dbcAMP. Consistent with previous reports (de Vantery Arrighi et al., 2000 blue right-pointing triangle), U0126 inhibited GVBD in OA-treated oocytes by ~50% (unpublished data), and we assumed here that the continued presence of a GV indicated successful reversal of the effects of the OA pulse by U0126. As before (Figure (Figure9A),9A), MAPK activity was high in OA pulse-treated oocytes, whereas MPF activity was low (Figure (Figure9C),9C), although a minor but statistically significant activation of MPF was evident. Treatment with U0126 eliminated MAPK activity, thereby producing an OA-treated oocyte with low MPF and MAPK activities. In these oocytes, HCO3/Cl exchanger activity was significantly higher compared with oocytes treated with an OA pulse alone (Figure (Figure99D).

DISCUSSION

We report here that the HCO3/Cl exchanger in mouse oocytes is inactivated during meiotic metaphase. Activity decreases from maximal in the prophase I–arrested GV oocyte as the oocyte proceeds through first meiotic metaphase and reaches a minimum approximately 2 h before the emission of the first polar body and entry into second meiotic metaphase. Reactivation does not occur until the end of meiosis, after the second polar body is emitted and pronuclei have developed, as the embryo enters interphase.

In the mouse, MAPK activation and the development of CSF activity occurs about 2 h after MPF activation and GVBD (Verlhac et al., 1994 blue right-pointing triangle). High MAPK activity persists through meiosis, remaining high in the MII-arrested egg. Inactivation of MAPK does not occur until several hours after egg activation, at the time of pronuclear formation (Moos et al., 1995 blue right-pointing triangle; Zernicka-Goetz et al., 1995 blue right-pointing triangle). In contrast, MPF deactivation occurs quickly after egg activation, preceding second polar body formation. Thus, MAPK activity appeared to be inversely correlated with HCO3/Cl exchanger activity in the mouse oocyte, whereas changes in MPF activity preceded changes in HCO3/Cl exchanger activity (Figure (Figure10).10). We therefore hypothesized that MAPK negatively regulates HCO3/Cl exchanger activity in mouse oocytes.

Figure 10
Schematic diagram of HCO3/Cl exchanger, MAPK, and MPF activities in oocytes from GV to pronuclear stage. MPF and MAPK activities are taken from data of Verlhac et al. (1994) blue right-pointing triangle for meiotic maturation (0–10 h), Kubiac et al. (1992) ...

Several pieces of evidence support this hypothesis (summarized in Table Table1).1). First, HCO3/Cl exchanger activity did not develop in eggs when high MAPK activity was maintained with OA for an extended period after egg activation, even although the eggs were activated and MPF decreased. In addition, HCO3/Cl exchange could be inactivated by OA in pronuclear stage or two-cell stage parthenogenotes. Second, the rapid inactivation of MAPK using the MEK inhibitor U0126 in unfertilized eggs (Phillips et al., 2002 blue right-pointing triangle) resulted in accelerated HCO3/Cl exchanger activation. Third, oocytes in which the normal activation of MAPK after GVBD was prevented with U0126 did not exhibit decreased HCO3/Cl exchanger activity, even although GVBD occurred and MPF was activated. Fourth, activation of MAPK in GV oocytes with an OA pulse induced GVBD and suppressed HCO3/Cl exchanger activity without activating MPF, and HCO3/Cl exchanger activity could be at least partially restored when MAPK activity was inhibited by U0126 in oocytes treated with an OA pulse. Thus, in each case where MAPK activity was low, HCO3/Cl exchange was activated, and vice versa (Table (Table1),1), consistent with negative regulation of HCO3/Cl exchanger by MAPK in the mouse oocyte.

Table 1
Regulation of HCO3/CI exchanger

The data do not support regulation of the HCO3/Cl exchanger by MPF. OA treatment of activated eggs or OA pulse treatment of GV oocytes did not activate MPF but did suppress HCO3/Cl exchanger activity (Table (Table1).1). Conversely, oocytes undergoing meiotic maturation in the presence of U0126 and parthenogenotes in mitotic metaphase possess high MPF activity, whereas the HCO3/Cl exchanger was not inactivated (Table (Table11).

There is some precedent for a role for MAPK in the control of pHi-regulatory transporters. MAPK activity mediates growth factor and arginine vasopressin activation of Na+/H+ antiporter activity in mammalian cells (Sardet et al., 1991 blue right-pointing triangle; Aharonovitz and Granot, 1996 blue right-pointing triangle; Bianchini et al., 1997 blue right-pointing triangle), whereas in Xenopus oocytes upregulation of Na+/H+ antiporter activity during meiotic maturation is dependent on RAF (Kang et al., 1998 blue right-pointing triangle) and can be induced by MOS (Rezai et al., 1994 blue right-pointing triangle). These examples, however, involve positive regulation of pHi-regulatory mechanisms by the MAPK pathway. In contrast, the HCO3/Cl exchanger appears to be negatively regulated by MAPK or downstream effectors in mouse oocytes during meiosis.

Similar to the HCO3/Cl exchanger, the Na+/H+ antiporter is quiescent in MII eggs and only becomes activated a number of hours after fertilization (Lane et al., 1999b blue right-pointing triangle). Thus, pHi-regulatory mechanisms in general may prove to be inactive during meiotic metaphase. The physiological reason for specific inactivation of pHi-regulatory mechanisms during meiosis and reactivation after fertilization remains unclear, since pHi in mouse oocytes does not change upon fertilization (Kline and Zagray, 1995 blue right-pointing triangle) in contrast to, e.g., sea urchin. Reactivation could be explained by a requirement to robustly regulate pHi as the egg becomes more metabolically active after fertilization, as has been found for activation of somatic cells (Ganz et al., 1989 blue right-pointing triangle). However, this would not explain the initial inactivation of pHi regulation during meiosis. At present, we can only speculate that it may reflect a need to restrict transmembrane ion transport during meiosis or during fertilization, but further work is clearly needed.

More generally, a picture is emerging of regulation of transport and homeostatic mechanisms in oocytes, eggs, and early embryos by the cell cycle interacting with developmental clocks, to which pHi-regulatory mechanisms conform. The cell swelling–activated anion channel, which functions in cell volume regulation, was shown to become inactivated during prophase and inactive in metaphase after the two-cell stage in mouse embryos but is not affected by previous mitotic or meiotic metaphases (Kolajova et al., 2001 blue right-pointing triangle). In addition, a large conductance K+ channel in mouse oocytes of unknown function is regulated by a cytoplasmic cell cycle, becoming activated during meiotic metaphase and each mitotic metaphase during early embryo development (Day et al., 1998a blue right-pointing triangle). The T-type Ca2+ channel in mouse embryos is activated upon exit from metaphase at the end of the one-cell stage and then deactivated before entry into the next metaphase, maintaining high activity during interphase of the two-cell stage (Day et al., 1998b blue right-pointing triangle).

Like these ion channels, the HCO3/Cl exchanger is under cell cycle control in the mouse oocyte. Inactivation during metaphase is restricted to meiosis, demonstrating an interaction with a developmental clock, similar to such control of the K+ and T-type Ca2+ channels in oocytes and embryos. Unlike these channels, however, a possible regulatory pathway for the HCO3/Cl exchanger has been identified, with MAPK or its downstream effectors implicated. Given the similar behavior of the Na+/H+ antiporter in hamster eggs, suppression of pH-regulatory mechanisms during meiosis may prove to be a novel function of MAPK, and hence CSF activity, in the mammalian oocyte.

ACKNOWLEDGMENTS

We acknowledge Mary-Anne Hammer for excellent technical support. This work was supported by Canadian Institutes of Health Research (CIHR) operating grant MOP12040. J.M.B. is the recipient of a Premier's Research Excellence Award (Government of Ontario). K.P.P. was supported by a Bombardier Foundation for Higher Education Studentship, an Ontario Graduate Science and Technology Studentship, and an Ontario Graduate Scholarship. M.A.F.P. was supported by an Ontario Graduate Scholarship.

Abbreviations used:

Ca2+i
intracellular calcium
CSF
cytostatic factor
GV
germinal vesicle (prominent nucleus of prophase I oocytes)
GVBD
germinal vesicle breakdown (release from prophase I arrest)
IVF
in vitro fertilization
MAPK
mitogen-activated protein kinase (ERK1,2)
MBP
myelin basic protein
MI
first meiotic metaphase
MII
second meiotic metaphase
MPF
maturation- (or mitosis-) promoting factor
OA
okadaic acid
pHi
intracellular pH

Footnotes

Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E02–04–0242. Article and publication date are at www.molbiolcell.org/cgi/doi/10.1091/mbc.E02–04–0242.

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