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Differences in Intracellular pH Regulation by Na⁺/H⁺ Antiporter among Two-Cell Mouse Embryos Derived from Females of Different Strains¹

^a Loeb Research Institute, Ottawa Hospital, and ^b Departments of Obstetrics and Gynecology, ^c Division of Reproductive Medicine, and Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9 ^d Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT

Regulation of intracellular pH (pH_i) by two-cell-stage embryos derived from female mice of three different strains (CF-1, Balb/c, and BDF) was investigated. Embryos recovered at a slow rate from intracellular acidosis produced by a pulse of NH₄Cl; the rate did not differ significantly among strains. Recovery was reversibly inhibited by amiloride or the absence of Na⁺, implicating Na⁺/H⁺ antiporter activity. The threshold pH_i (setpoint) below which Na⁺/H⁺ antiporter activity was elicited was approximately 7.15 for each strain. No recovery from induced acidosis occurred in the absence of external Na⁺ in any strain, and thus embryos could be maintained in acidosis for an extended period. Upon reintroduction of Na⁺, embryos derived from either CF-1 or BDF females recovered at a slow rate comparable to that measured in embryos not maintained for a period in Na⁺-free medium, but embryos derived from Balb/c females consistently recovered at a highly accelerated rate. This accelerated recovery appeared to be due, in part, to an activation of the Na⁺/H⁺ antiporter in Balb/c-derived embryos, which did not occur in CF-1- or BDF-derived embryos. Thus, embryos derived from different strains of female mice differ in their control of mechanisms for pH_i regulation.

conceptus, embryo

INTRODUCTION

Intracellular pH (pH_i) regulation is an important homeostatic function of cells. Mammalian cells maintain pH_i within a narrow range by means of several nearly ubiquitous mechanisms including HCO₃^-/Cl^- exchangers belonging to the AE gene family, which regulate against alkalosis, and Na⁺/H⁺ antiporters belonging to the NHE gene family, which oppose acidosis [1–3]. The NHE family has five known members thought to localize to the plasma membrane (NHE1–5) and a sixth (NHE6) that may be intracellular [3, 4]. The Na⁺/H⁺ antiporters examined to date are activated below a pH_i threshold or setpoint, with the antiporter exhibiting a steep increase in activity as pH_i decreases below this level [5]. NHE1 is the ubiquitous, growth factor-activatable isoform [6]. Activation of NHE1 by growth factors generally causes a shift in the setpoint to a higher value, thus increasing activity at physiological pH_i. NHE1 activation is mediated by a number of intracellular signaling pathways, including protein kinase C and the p42/44 mitogen-activated protein kinase pathway, which either directly phosphorylate NHE1 or work via intermediary proteins that bind NHE1 [3, 4]. In contrast, NHE3 appears to be activated via an increase in maximal transport rate rather than setpoint, and to be regulated by signaling pathways including cAMP-dependent protein kinase A [4]. In addition to pH_i regulation, NHE isoforms mediate cell volume regulation and transepithelial ion transport.

Mammalian preimplantation embryos can regulate pH_i. Regulation against alkalosis by HCO₃^-/Cl^- exchanger activity was first shown in two-cell-stage mouse embryos [7] and was subsequently found throughout preimplantation development, with the most robust activity at the one- and two-cell stages [8, 9]. Similar regulation of pH_i by HCO₃^-/Cl^- exchange has been demonstrated in preimplantation hamster [10] and human [11] embryos, although in contrast this exchange appears to be essentially nonfunctional in bovine embryos [12]. Unlike embryos from the late one-cell stage onwards, unfertilized ovulated mouse and hamster eggs are nearly devoid of HCO₃^-/Cl^- exchanger activity. Activity appears slowly, over the course of several hours following egg activation, and is only fully activated at around the time pronuclei form [10, 13].

In contrast to the clear data indicating regulation against alkalosis via HCO₃^-/Cl^- exchanger activity in embryos, it is not clear whether preimplantation embryos can regulate pH_i against acidosis using a Na⁺/H⁺ antiporter. It was first reported that mouse two-cell embryos derived from CF-1 females lacked demonstrable Na⁺/H⁺ antiporter activity [14]. These embryos were able to recover from experimentally imposed acidosis, but the recovery was not slowed either by absence of external Na⁺ or by the presence of amiloride, either of which should have inhibited recovery from acidosis mediated by Na⁺/H⁺ antiporter activity. Recently, however, Gibb et al. [15] reported a completely different result, showing extremely robust recovery from acidosis in two-cell embryos derived from Quackenbush (QS) strain female mice. This recovery was completely inhibited by amiloride derivatives or absence of external Na⁺, indicating that pH_i was regulated by Na⁺/H⁺ antiporter activity in these embryos. In addition, mouse eggs and embryos derived from CF-1 strain females have been shown to express mRNA encoding two members of the Na⁺/H⁺ antiporter family, NHE1 and NHE3 [16], also consistent with the presence of Na⁺/H⁺ antiporter activity in mouse embryos. However, it is not clear whether either or both isoforms contribute to pH_i regulation in embryos before they become differentially localized in the trophectoderm at the blastocyst stage, where NHE3 appears to mediate vectorial fluid transport into the blastocoel [16]. Hamster [17], human [11], and bovine [12] embryos also exhibit Na⁺/H⁺ antiporter activity. In hamsters, Na⁺/H⁺ antiporter activity followed a pattern similar to that of HCO₃^-/Cl^- exchanger activity, with no detectable activity in the unfertilized egg and robust activity slowly appearing over the course of several hours after egg activation [18]. Thus, it is now clear that preimplantation embryos of several species and at least some strains of mice regulate pH_i via Na⁺/H⁺ antiporter activity.

Why robust Na⁺/H⁺ antiporter activity in two-cell-stage mouse embryos was detected in some studies but not in others has been an unresolved question. One possibility is that differences in culture medium composition, specifically the presence of lactate, can mask Na⁺/H⁺ antiporter activity [15]. Alternatively, there might be some variation among strains of mice in their level of Na⁺/H⁺ antiporter activity and ability to regulate pH_i. To begin to address these issues, we assayed for Na⁺/H⁺ antiporter activity in embryos derived from different strains of female mice under identical conditions and in identical media. Although the most useful comparison would also involve QS-derived embryos, this strain was not available. Nonetheless, a preliminary screening of embryos derived from a number of available strains of female mice identified several that exhibited potential variations in pH_i regulation. Here, we report the results of studies in which the ability to regulate pH_i against an induced acidosis was compared among two-cell-stage embryos derived from three different common strains of female mice: CF-1, BDF, and Balb/c.

MATERIALS AND METHODS

Embryos

Female mice of the CF-1 outbred strain, the Balb/c inbred strain, and the BDF hybrid strain (B6D2F₁, a cross between C57Bl/6 females and DBA/2 males) were obtained from Charles River (St. Hyacinth, PQ, Canada) at 6–8 wk of age. Mice were maintained on a 12-h light:dark cycle. They were superovulated using 5 IU eCG (Sigma, St. Louis, MO) followed 47.5 h later by 5 IU hCG (Sigma), both administered i.p. These females were then mated with BDF strain males (regardless of the strain of female) by individually caging the females overnight with males, beginning immediately after the hCG injection. Two-cell embryos were obtained approximately 42 h after administration of hCG. Preliminary experiments were done with females of these three strains and with females of the CB6F₁ hybrid, Swiss outbred, and C57Bl/6 inbred strains, which were treated similarly. Embryos were retrieved by flushing them from freshly excised oviducts into culture medium. Embryos were either used immediately for pH_i measurements or were cultured.

Chemicals and Solutions

All chemicals were obtained from Sigma unless otherwise noted.

Media used for pH_i measurements were based on KSOM mouse embryo culture medium [19]. KSOM contains 95 mM NaCl, 2.5 mM KCl, 0.35 mM KH₂PO₄, 0.2 mM MgSO₄, 10.0 mM Na⁺ lactate, 0.2 mM glucose, 0.2 mM Na⁺ pyruvate, 25.0 mM NaHCO₃, 1.7 mM CaCl₂, 1.0 mM glutamine, 0.01 mM tetrasodium EDTA, 0.16 mM K penicillin G, and 0.03 mM streptomycin SO₄. Hepes-buffered KSOM (H-KSOM) [19] was used for flushing embryos from the oviduct and for embryo handling. This medium was produced by replacing 21 mM (of 25 mM) NaHCO₃ with equimolar Hepes (pH adjusted to 7.4 with NaOH).

Other media used for pH_i measurements were based on a modified form of H-KSOM in which lactate was reduced to 1.0 mM and from which HCO₃^- was omitted. This medium is designated here as pH-KSOM. Lactate was reduced by replacing 9.0 mM (of 10.0 mM) Na⁺ lactate and all NaHCO₃ with equimolar NaCl. For Na⁺-free (i.e., Na⁺ < 1 mM) pH-KSOM, NaCl was replaced with equimolar choline Cl^-, and Na⁺ lactate and Na⁺ pyruvate were replaced by lactic acid and K⁺ pyruvate, respectively. The pH was adjusted with KOH.

To induce intracellular acidosis, a pulse of medium containing 25 mM NH₄Cl was used wherein 25 mM NaCl had been replaced by NH₄Cl. Embryos were exposed to medium containing NH₄Cl for 10 min, after which the medium was replaced with an appropriate NH₄Cl-free medium, resulting in a net intracellular acidosis [1]. In some experiments, a Na⁺-free version of NH₄Cl-containing KSOM was used, with Na⁺ replaced as described above. Following the NH₄Cl pulse, the medium used was either pH-KSOM or Na⁺-free pH-KSOM. In some experiments, the pH-KSOM contained 1 mM amiloride. Calibration solutions used in pH_i measurements contained 100 mM KCl, 25 mM NaCl, 21 mM Hepes, and 75 mM sucrose. Solutions were calibrated to pH 6.7, 7.0, 7.4, and 7.8 with NaOH or KOH.

KSOM was used for embryo culture. It was equilibrated with 5% CO₂/air at 37°C and 100% humidity before use. Embryos were cultured in drops of medium (approximately 50 µl) in 35-mm culture dishes (Falcon 3001; Fisher, Pittsburgh, PA) with a KSOM-washed mineral oil overlayer (embryo tested; Sigma). The dishes were preequilibrated with 5% CO₂/air at 37°C and 100% humidity before use.

pH_i Measurements

pH_i was measured using the pH-sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1), loaded into embryos by incubation with 5.0 µM SNARF-1-acetoxymethyl ester (SNARF-1-AM; Molecular Probes, Eugene, OR) at 37°C for 30 min in pH-KSOM [13, 20]. After SNARF-1 loading, embryos were washed several times with pH-KSOM and placed in a temperature-controlled chamber (Biophysica, Baltimore, MD), which was modified to allow solution changes. During measurements, embryos were maintained at 37°C (±0.5°C).

The methods used for pH_i measurements have been described in detail previously [8, 13, 14, 20–22]. Simultaneous measurements of groups of embryos were made, and data were recorded for individual blastomeres. An intensified charged-coupled device camera with output to an image storage and quantification system (Inovision, Durham, NC) detected fluorescence at two emission wavelengths, 640 nm (pH sensitive) and 600 nm (pH insensitive), using an excitation wavelength of 535 nm. The ratio 640/600, which is dependent only on pH, was calculated by dividing the images after background subtraction. This ratio was calibrated to pH_i using calibration solutions with 10 µg/ml nigericin and 5 µg/ml valinomycin added [14, 23]. pH_i calibration curves were generated regularly and were not different among strains (data not shown). A small number of pilot experiments were performed on different equipment and using 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein as the pH-sensitive fluorophore. These pilot experiments were done in Madison, WI, as previously described for hamster embryos [17], and the rest of the experiments were performed in Ottawa, ON.

Baseline pH_i

Steady state pH_i was measured under two conditions. First, pH_i of two-cell embryos was determined in pH-KSOM in the temperature-controlled chamber as part of the experiments in which recovery from acidosis was assessed. Second, pH_i was measured in two-cell embryos in standard microdrop cultures in KSOM in the presence of HCO₃^-/CO₂. For baseline pH_i measurements, the embryos were allowed to acclimatize for 10 min, and then a series of repeated pH_i measurements were made over a 10-min period at a frequency of one measurement per minute.

Probing Na⁺/H⁺ Antiporter Activity in Two-Cell Embryos

Recovery from acidosis After an initial 10-min period of baseline pH_i measurement, intracellular acidosis was induced using a 10-min pulse of pH-KSOM containing 25 mM NH₄Cl. This pulse produces an immediate alkalinization due to the rapid equilibration of NH₃ across the membrane, followed by a slower partial reacidification as a result of a slower influx of the less permeable NH₄⁺. Upon removal of external NH₄Cl, NH₃ rapidly exits the cell, leaving behind any H⁺ that has entered as NH₄⁺, thereby causing a net acidification of the cell [1, 14, 24]. The NH₄Cl pulse was followed by a 20- to 30-min period in pH-KSOM or other appropriate medium, during which the ability of the embryos to recover from acidosis was assessed. Measurements of pH_i were made at a frequency of two measurements per minute during recovery.

Four protocols were used in NH₄Cl pulse experiments. In the first, recovery from acidosis was assessed in normal pH-KSOM medium, with measurements obtained for 20 min after the NH₄Cl pulse. In the second, Na⁺-free pH-KSOM was used immediately after the pulse, and measurements were taken for 10 min in Na⁺-free medium. The medium was then replaced with normal pH-KSOM containing Na⁺, and pH_i was measured for an additional 20 min. In the third protocol, three media were used sequentially after the pulse: Na⁺-free pH-KSOM for 10 min, followed by pH-KSOM with amiloride (1 mM) for 10 min, followed by pH-KSOM for 10 min. In a variation of this protocol, the initial Na⁺-free medium was omitted, and medium containing amiloride was introduced immediately after the pulse, followed by amiloride-free medium for 20 min. In the fourth protocol, the medium containing NH₄Cl that was used during the pulse was Na⁺ free. Normal pH-KSOM was then used after the pulse, and pH_i measurements were made for 20 min.

Effect of external Na⁺ removal and readdition In one series of experiments, the effect on pH_i of removing and then reintroducing external Na⁺ was assessed. pH_i was measured for 10 min in pH-KSOM with normal Na⁺. After this set of measurements was complete, the medium was replaced with Na⁺-free pH-KSOM, and pH_i was measured for another 10 min. Finally, pH-KSOM containing Na⁺ was reintroduced, and pH_i measurements were made for an additional 20 min.

Data Analysis

The rates at which embryos recovered from induced acidosis were compared by determining the recovery rate immediately following the NH₄Cl pulse. Recoveries were approximately linear with time until pH_i began to plateau. Thus, the rate of recovery was calculated by linear regression to the data during the linear portion of the recovery. The first one or two points immediately following the solution change were not included in the regression to diminish the contribution of any perturbing effects of the solution change. In experiments where the medium was changed following the NH₄Cl pulse (e.g., from Na⁺-free medium to medium containing Na⁺), rates of change of pH_i following introduction of a new medium was determined in the same way, by linear regression to initial points of recovery data during the linear phase of recovery.

To determine the setpoint of the recovery from acidosis, recovery data were fit by nonlinear regression to a single exponential described by the following equation:

where t = 0 is the start of recovery and a, b, and c are constants determined by the fit: a corresponds to the difference between the initial and final pH_i, b is a rate constant for the recovery, and c gives the final pH_i upon complete recovery. The rate of recovery as a function of time is calculated by taking the first derivative of this equation:

Equations 1 and 2 are then combined to yield the dependence of rate of recovery on pH_i. The pH_i at which the rate falls to zero, which is the setpoint of the recovery, was determined graphically. Values for dpH_i/dt were calculated at pH_i 6.8, 6.9, 7.0, 7.1, and 7.2 and plotted, and a line was fit to the data by linear regression. The setpoint was determined visually from this graph as described previously [9, 11].

Data are presented as the mean ± SEM. In all cases, differences were considered significant at P < 0.05 . Throughout, n represents the total number of embryos and N represents the number of separate replicate experiments (each comprising a number of embryos). Descriptive statistics were obtained using SigmaPlot 1.0 (Jandel Scientific, San Rafael, CA) or SigmaPlot 5.0 (SPSS, Chicago, IL). Statistical comparisons were done with InStat (GraphPad, San Diego, CA). For statistical comparisons of three or more groups, a Bartlett test for homogeneity of variances was first used to determine whether a parametric ANOVA (pANOVA) or a nonparametric ANOVA (nANOVA) was appropriate. Comparisons were then made using pANOVA or nANOVA followed by the Tukey-Kramer multiple comparisons test or Dunn test, respectively. For comparing two groups of data, an F-test was performed to test for equality of variances prior to selecting a t-test for analysis. Statistical comparisons of two groups of data were made using the Student t-test for equal variances or the Mann-Whitney nonparametric t-test for unequal variances.

RESULTS

Effect of Manipulations on Embryo Viability

Loading mouse eggs or embryos with SNARF-1, placing them in the temperature-controlled chamber, and exposing them to the excitation wavelength for SNARF-1 does not affect the ability of eggs to be fertilized nor does it alter the electrophysiological properties of one-cell embryos or decrease the fraction that cleave normally in culture [20]. Preliminary experiments were carried out here to determine whether an NH₄Cl pulse and/or manipulation of external Na⁺ affected embryo viability. Embryos were placed into culture following acid loading and recovery using the same protocols as employed in pH_i measurements. Embryos (either one-cell or two-cell stage) derived from CF-1 females were subjected to a 10-min NH₄Cl pulse either 1) with Na⁺ present throughout or 2) followed by a 10-min period in Na⁺-free medium and then a 10-min period with Na⁺ reintroduced. The media used were those based on pH-KSOM. The embryos were then placed in microdrop culture in KSOM medium. When Na⁺ was present throughout, one-cell and two-cell embryos developed to the morula stage (93%, N = 4, n = 44; 98%, N = 4, n = 60, respectively) and further on to the blastocyst stage (32% and 95%, respectively). When Na⁺ was initially absent following the NH₄Cl pulse and then reintroduced, one-cell and two-cell embryos developed to the morula stage (95%, N = 4, n = 45; 93%, N = 4, n = 60, respectively) and blastocyst stage (48% and 68%, respectively). That is, development of two-cell embryos following NH₄Cl pulses and absence of external Na⁺ was essentially normal, and even the more sensitive one-cell embryos developed to morulae at a normal rate following these manipulations, although development to blastocysts was depressed to some extent. Thus, we concluded that the manipulations used here to determine the mechanisms of pH_i regulation did not have any major deleterious effects on two-cell mouse embryos.

Baseline pH_i

Baseline pH_i of two-cell embryos was measured while they were in microdrop culture in KSOM medium (5% CO₂/air, 37°C). The mean pH_i of CF-1 embryos was 7.17 ± 0.03 (N = 5, n = 68), that of BDF embryos was 7.10 ± 0.02 (N = 5, n = 71), and that of Balb/c embryos was 7.16 ± 0.04 (N = 5, n = 49). There was no significant difference in pH_i among strains (pANOVA, P = 0.13).

Baseline pH_i of two-cell embryos was also measured in pH-KSOM (37°C) in the temperature-controlled chamber at the beginning of each experiment. pH-KSOM is nominally HCO₃^- free, and thus there can be little HCO₃^-/Cl^- exchanger activity under these conditions. The baseline pH_i of CF-1 embryos was 7.23 ± 0.02 (N = 44, n = 771), that of BDF embryos was, 7.30 ± 0.03 (N = 24, n = 433), and that of Balb/c embryos was 7.33 ± 0.02 (N = 40, n = 566). Baseline pH_i in the chamber was not significantly different among strains (pANOVA, P = 0.81).

Recovery from Induced Intracellular Acidosis

A set of pilot experiments was performed to determine if there might be differences in pH_i regulation among mouse embryos derived from females of different strains. The NH₄Cl pulse technique was used to produce intracellular acidosis, and the ability of two-cell embryos derived from various strains to recover was assessed. These experiments were performed using instrumentation that had been used successfully to show robust Na⁺/H⁺ antiporter activity in hamster two-cell embryos (Madison, WI [17]). Several measurements with hamster embryos were interspersed with the mouse experiments, which were performed over a 2-wk period, and these experiments showed very active pH_i regulation by hamster two-cell embryos via Na⁺/H⁺ antiporter activity similar to that previously reported (data not shown). In contrast to the results with hamster embryos, we obtained variable results with mouse embryos in these initial experiments, with a substantial proportion of embryos showing little or no recovery. These pilot experiments indicated, however, that there was some Na⁺/H⁺ exchanger activity in mouse two-cell embryos (as indicated by inhibition of recovery in the absence of external Na⁺). In addition, the rate of recovery from acidosis appeared to differ among strains, especially in a protocol where the medium was Na⁺ free immediately following acidosis and then Na⁺ was replaced. Balb/c-derived embryos qualitatively appeared to be the most robust, and BDF-derived embryos had the least apparent activity. We used Balb/c-, BDF-, and CF-1-derived embryos for subsequent comparisons.

The ability of two-cell embryos derived from female mice of these three strains to recover from acidosis induced by NH₄Cl was assessed in a series of eight replicate experiments for each strain (Fig. 1). The initial rate of recovery of CF-1-derived two-cell embryos in pH-KSOM was 0.03 ± 0.006 pHU/min (N = 8, n = 138; Fig. 1, A and D). BDF-derived embryos recovered at 0.03 ± 0.003 pHU/min (N = 8, n = 115; Fig. 1, B and D). Balb/c-derived embryos recovered at 0.04 ± 0.009 pHU/min (N = 8, n = 72; Fig. 1, C and D). These rates were not significantly different among strains (Fig. 1D; nANOVA, P = 0.39).

View larger version (26K): [in this window] [in a new window]   FIG. 1. Recovery by two-cell mouse embryos from acidosis induced by NH4Cl pulse. A–C) Representative pHi traces obtained from two-cell embryos derived from CF-1, BDF, and Balb/c females. The initial part of each trace shows baseline pHi. An NH4Cl pulse was applied from 10 to 20 min, resulting in a net acidification after removal of NH4Cl. Recovery from the induced acidosis occurred in each case. The rate of recovery was determined from the slope of a line fit by linear regression, examples of which are shown. D) Mean rates of recovery from acidosis for embryos derived from each strain (no significant difference). E) Rate of recovery vs. pHi plots used to determine setpoints of the Na+/H+ antiporter for each mouse strain. The setpoint was taken to be where the line fit by linear regression reached the pHi axis (rate = 0). n, The number of embryos; N, the number of experiments

Effect of Absence and Reintroduction of Extracellular Na⁺ on Recovery from Acidosis

Two-cell embryos of all three strains failed to recover from NH₄Cl pulse-induced acidosis to any significant extent in the absence of external Na⁺ (Fig. 2, A–C). In eight replicate experiments performed with each strain, the initial rate of recovery in the absence of Na⁺ was 0.002 ± 0.0006 pHU/min (N = 8, n = 132) for CF-1-derived embryos, 0.003 ± 0.0007 pHU/min (N = 8, n = 163) for BDF-derived embryos, and 0.004 ± 0.001 pHU/min (N = 8, n = 86) for Balb/c-derived embryos (Fig. 2D).

View larger version (28K): [in this window] [in a new window]   FIG. 2. Effect of absence of external Na+ and its subsequent reintroduction on recovery from acidosis in mouse embryos. A–C) Representative traces of embryos derived from CF-1, BDF, and Balb/c females. The medium after induction of acidosis by NH4Cl pulse was Na+ free (0 Na+) for 10 min, after which Na+ was reintroduced (Na+). In all strains, absence of Na+ inhibited recovery from intracellular acidosis. Upon reintroduction of Na+, recovery occurred in each case. D) Mean recovery rates in the absence of Na+ (0 Na+) and upon its reintroduction (Na+). *P < 0.05, **P < 0.01, ***P < 0.001 (t-test) for difference between recovery in presence or absence of Na+ within each strain. Different letters indicate significantly different recovery rates among strains after Na+ reintroduction. E) Rate of recovery vs. pHi plots as in Figure 1. n, The number of embryos; N, the number of experiments. Although a shift in setpoint in Balb/c embryos is apparent (E), a much smaller shift was observed in a repeated set of experiments and may represent overshoot of the target pHi (C) rather than an actual change in setpoint

After a 10-min period in Na⁺-free medium, medium containing Na⁺ was reintroduced in each case. For all three strains, recovery began immediately when Na⁺ was introduced (Fig. 2, A–C). In BDF and CF-1 embryos, the initial rate of recovery was similar to that seen when Na⁺ was available immediately after the NH₄Cl pulse. CF-1 embryos recovered at a rate of 0.06 ± 0.02 pHU/min upon Na⁺ replacement, and BDF embryos recovered at a rate of 0.05 ± 0.01 pHU/min (Fig. 2, A, B, and D). However, the rate of recovery of Balb/c two-cell embryos upon reintroduction of Na⁺ was significantly higher (pANOVA; P < 0.001) than that seen in the other two strains, with a mean initial rate of recovery of 0.19 ± 0.02 pHU/min (Fig. 2, C and D).

We decided to repeat this comparison in a separate set of experiments to confirm that Balb/c embryos indeed recovered at a faster rate than did embryos of either of the other strains. Therefore, approximately 6 mo after the first set of data were obtained, we have again measured recovery rates of Balb/c and CF-1 embryos using this protocol. Again, we found that Balb/c embryos recovered at a faster rate of 0.18 ± 0.03 pHU/min (N = 8, n = 89) compared with CF-1 embryos, which recovered at a rate of 0.05 ± 0.02 pHU/min (N = 8, n = 104). These rates were similar to those obtained in the first set of experiments (0.19 and 0.06 pHU/min, respectively) and were again significantly different from one another (Student t-test, P = 0.0004).

Setpoint of the Recovery Mechanism

To obtain values for the setpoint, the dependence of the rate of recovery on pH_i was determined for all three strains (as described in Materials and Methods) immediately following the NH₄Cl pulse for the protocol where Na⁺ was present throughout (Fig. 1) and upon reintroduction of Na⁺ for the protocol where there was an initial Na⁺-free period followed by Na⁺ replacement (Fig. 2). When Na⁺ was present throughout, the setpoints for all three strains were similar (Fig. 1E): 7.15 for CF-1, 7.17 for BDF, and 7.18 for Balb/c. Similarly, after reintroduction of Na⁺, CF-1 and BDF embryos had setpoints of about 7.10 (Fig. 2E). In contrast, however, the setpoint of Balb/c embryos in these experiments had increased somewhat to about 7.35 after reintroduction of Na⁺ (Fig. 2E). In addition, the rate of recovery was higher at any given pH_i for Balb/c than for CF-1 or BDF embryos.

When measurements comparing recoveries in Balb/c and CF-1 embryos were repeated, an almost identical finding of higher rates of recoveries at any given pH_i for Balb/c than for CF-1 embryos was obtained (data not shown), but the apparent difference in setpoint was not nearly as pronounced, being near 7.15 for CF-1 embryos and nearer 7.25 for Balb/c embryos.

pH_i Following Recovery from Acidosis

The maximum pH_i attained following recovery was determined by taking an average of 10 consecutive pH_i measurements at the plateau of the pH_i curve. In experiments where Na⁺ was present throughout (Fig. 1), the pH_i after recovery was 7.12 ± 0.04 for CF-1 embryos, 7.13 ± 0.03 for BDF embryos, and 7.10 ± 0.06 for Balb/c embryos. These values were not significantly different among strains (pANOVA, P = 0.10). The recovery pH_i was slightly lower for each strain than the baseline pH_i had been before acidosis was induced (7.23–7.33), and this difference was significant (Student t-test; P = 0.0061 and 0.0021 for CF-1 and BDF embryos, respectively, P < 0.0001 for Balb/c embryos).

In the experiments where Na⁺-free medium was present after the NH₄Cl pulse and then Na⁺ was reintroduced (Fig. 2), pH_i of CF-1 and BDF embryos recovered to 7.08 ± 0.03 and 7.10 ± 0.04, respectively. These levels were slightly lower than baseline pH_i had been before the pulse (Student t-test; P = 0.0004 and 0.0007 for CF-1 and BDF embryos, respectively). After reintroduction of Na⁺, embryos of these two strains recovered to essentially the same pH_i as when Na⁺ was present throughout. Balb/c embryos recovered to a significantly higher pH_i when Na⁺ was reintroduced, reaching 7.34 ± 0.04. This value was significantly higher than that shown after recovery in embryos derived from the other two strains (pANOVA, P < 0.001) and was significantly higher than that for Balb/c embryos when Na⁺ had been continually present (Student t-test, P = 0.0067).

Effect of Amiloride on Recovery from Acidosis

The presence of the Na⁺/H⁺ antiporter inhibitor amiloride (1 mM) completely blocked recovery from acidosis in embryos derived from all three strains regardless of whether Na⁺ was present throughout or whether amiloride was added upon Na⁺ reintroduction. When Na⁺ was present throughout, the mean rate of recovery with amiloride present (Fig. 3, A, B, F, and G) for CF-1 two-cell embryos was 0.007 ± 0.001 pHU/min (N = 7, n = 107). After amiloride was washed out, the rate was 0.09 ± 0.005 pHU/min. Balb/c embryos recovered at 0.01 ± 0.002 pHU/min (N = 8, n = 137) in the presence of amiloride and at 0.10 ± 0.02 pHU/min after washout. The increase in recovery rate after amiloride was washed out was significant in both cases (paired t-test; P < 0.0001 and 0.001 for CF-1 and Balb/c embryos, respectively), indicating that amiloride inhibition was reversible. Recovery rates after washout did not differ significantly among strains (Student t-test, P = 0.60).

View larger version (38K): [in this window] [in a new window]   FIG. 3. Effect of amiloride. A and B) Representative traces of CF-1 and Balb/c mouse embryos. Na+ was present throughout. Amiloride was initially present following induction of acidosis (Amil) and then was washed out with identical amiloride-free medium (washout). C–E) Representative traces of CF-1, BDF, and Balb/c embryos. Medium after induction of acidosis was initially Na+ free (0 Na+), after which medium containing Na+ was introduced. This latter medium initially contained 1 mM amiloride (Amil), which was then washed out (washout) with identical medium devoid of amiloride. F) Mean rates of recovery for the protocol described in A and B. G) Mean rates of recovery for protocol described in C–E. Significant differences are indicated as in Figure 2. n, The number of embryos; N, the number of experiments.

When there was a period of Na⁺-free medium following the NH₄Cl pulse, recovery in the absence of Na⁺ was again completely inhibited (Fig. 3, C–E and H). When Na⁺ was reintroduced in the presence of amiloride, there was no significant increase in the rate of recovery over what was seen in the absence of Na⁺, with mean recovery rates of 0.01 ± 0.002 pHU/min (N = 8, n = 168) in CF-1 embryos, 0.02 ± 0.004 pHU/min (N = 8, n = 155) in BDF embryos, and 0.008 ± 0.004 pHU/min (N = 5, n = 91) in Balb/c embryos (Fig. 3H). When amiloride was then washed out in the continued presence of Na⁺, the embryos recovered from acidosis, with mean rates of 0.06 ± 0.02 pHU/min for CF-1, 0.05 ± 0.01 pHU/min for BDF, and 0.13 ± 0.01 pHU/min for Balb/c (Fig. 3H). However, in contrast to the results when Na⁺ was present throughout, the rate of recovery for Balb/c embryos was in this case significantly higher than those for CF-1 or BDF embryos (pANOVA, P < 0.05). Thus, despite the presence of Na⁺ during the 10-min period after the end of the NH₄Cl pulse while recovery was inhibited by amiloride, Balb/c embryos exhibited a high rate of recovery upon amiloride washout similar to that seen when there was no intervening period between the Na⁺-free medium and recovery.

Effect of Na⁺-Free Medium During the NH₄Cl Pulse

The increase in recovery rate exhibited by Balb/c but not by CF-1 or BDF embryos after Na⁺ reintroduction may have been due to exposure to Na⁺-free medium alone or to exposure to Na⁺-free medium combined with acidosis. To test the effect of exposure to Na⁺-free medium uncoupled from acidosis, acidosis was induced by an NH₄Cl pulse using Na⁺-free medium containing NH₄Cl, followed by recovery in normal medium containing Na⁺. In this way, exposure to Na⁺-free medium occurred while pH_i was alkaline instead of acidic (i.e., Na⁺ was absent while NH₄Cl was present and hence the cytoplasm was alkaline). After acidosis was induced using Na⁺-free medium containing NH₄Cl (Fig. 4), CF-1 embryos recovered from acidosis at a mean rate of 0.03 ± 0.002 pHU/min (N = 8, n = 133), and Balb/c embryos recovered at a mean rate of 0.06 ± 0.008 pHU/min (N = 6, n = 88). These rates were not significantly different (Mann-Whitney, P = 0.11).

View larger version (24K): [in this window] [in a new window]   FIG. 4. Effect of absence of Na+ during NH4Cl pulse. A and B) Representative traces of CF-1 and Balb/c mouse embryos. Na+ was absent during NH4Cl pulse (0 Na+ NH4Cl) and then present during recovery from acidosis (Na+). C) Mean rates of recovery from acidosis. There were no significant differences. n, The number of embryos; N, the number of experiments

Effect of Na⁺ Removal and Reintroduction on Baseline pH_i

Two-cell embryos derived from both CF-1 and Balb/c females (BDF embryos not assessed) acidified very slowly upon removal of external Na⁺ (Fig. 5), with identical rates of acidification of -0.011 ± 0.002 pHU/min for CF-1 (N = 8, n = 133) and Balb/c (N = 6, n = 88). In contrast, upon reintroduction of Na⁺, a much faster change in pH_i occurred, with pH_i generally overshooting the initial baseline pH_i. The mean rates of alkalinization following Na⁺ reintroduction were 0.08 ± 0.04 pHU/min for CF-1 and 0.06 ± 0.01 pHU/min for Balb/c. These rates did not differ significantly (Mann-Whitney, P = 0.84).

View larger version (27K): [in this window] [in a new window]   FIG. 5. Effect of removal and reintroduction of Na+ on baseline pHi in mouse embryos. A and B) Representative traces of CF-1 and Balb/c embryos. Rates of change of pHi upon removal and reintroduction of Na+ were determined from slopes of lines fit by linear regression. C) Mean rates of change of pHi after Na+ removal (0 Na+) and after its subsequent reintroduction (Na+). There were no significant differences. n, The number of embryos; N, the number of experiments

DISCUSSION

Two-cell-stage mouse embryos derived from CF-1, BDF, or Balb/c strain females recover from acidosis by means of Na⁺/H⁺ antiporter activity. This conclusion is supported by the following evidence: recovery occurred in the HCO₃^--free medium (Fig. 1), recovery was completely blocked in the absence of external Na⁺ (Fig. 2), and recovery was reversibly blocked by amiloride (Fig. 3). The presence of active Na⁺/H⁺ antiporters in two-cell mouse embryos was also indicated by the effect on baseline pH_i of removing and then reintroducing Na⁺ (Fig. 5). There was a slow decrease upon removal of Na⁺ and a fast increase upon reintroduction of Na⁺, consistent with activation of the antiporter by lower pH_i.

Although two-cell embryos derived from all three strains could recover from acidosis via Na⁺/H⁺ antiporter activity, the rate of recovery was slow (only 0.02–0.04 pHU/min), in sharp contrast with the 7- to 10-fold higher rates in hamster and cow and 5-fold higher rate in QS strain mouse embryos (Table 1). The difference in initial recovery rates was not due to a less severe acidosis in these mice than in hamster or QS mouse embryos; the minimum pH_i reached in both species was comparable to that achieved in these three mouse strains [15, 17]. The pH_i setpoints of Na⁺/H⁺ antiporter activity in two-cell embryos were around 7.15 in embryos derived from females of all three strains (Fig. 1E), essentially the same as reported for the hamster (7.14) [17]. The HCO₃^-/Cl^- exchanger, the major pH_i regulatory mechanism that mediates recovery from alkalosis, is present in two-cell mouse embryos and has a setpoint that was measured as 7.15 in BDF-derived embryos [7] and 7.20 in CF-1-derived embryos [9]. Thus, the combination of both mechanisms would maintain pH_i near this range, accounting for the baseline pH_i of about 7.10–7.17, which we measured in microdrop culture in the presence of HCO₃^-/CO₂, and indicating that both mechanisms probably contribute to maintaining normal pH_i in embryos. The somewhat higher baseline pH_i that we measured in the chamber (7.23–7.33) likely reflects the lack of HCO₃^-/Cl^- exchanger activity under these nominally HCO₃^-/CO₂-free conditions.

A surprising difference in pH_i regulation among two-cell embryos from different strains of females was revealed by experiments in which Na⁺ was initially absent and then was reintroduced during recovery from acidosis (Fig. 2). After Na⁺ was reintroduced, CF-1 and BDF embryos recovered from acidosis at about the same slow rate as when Na⁺ had been present throughout (Figs. 1 and 2 and Table 1). In contrast, however, embryos derived from Balb/c females recovered at a much faster rate, with a fivefold higher mean initial recovery rate than when Na⁺ was present throughout (Figs. 1 and 2 and Table 1). A plot of the rate of recovery vs. pH_i revealed that the pH_i setpoint may shift somewhat in Balb/c embryos but not in those from BDF or CF-1 females (Fig. 2E), although the extent of the apparent shift differed between two sets of similar experiments performed 6 mo apart. The pH_i achieved after recovery from acidosis was also somewhat different. Although pH_i recovered to about 7.10 for CF-1 and BDF embryos and in Balb/c embryos where Na⁺ was present throughout, it instead recovered to about 7.3 in Balb/c embryos after reintroduction of Na⁺. This difference could reflect a change in setpoint in Balb/c embryos. Alternatively, it could result from an overshoot of recovery unopposed by HCO₃^-/Cl^- exchanger activity in these nominally HCO₃^--free conditions. Because pH_i is increasing more quickly in Balb/c embryos, it would overshoot to a greater extent during any lag time between sensing pH_i and the consequent modulation of Na⁺/H⁺ antiporter activity.

Because the increased recovery rate in Balb/c embryos occurred only after a period in Na⁺-free medium during acidosis but not when Na⁺ was present throughout, exposure to Na⁺-free medium alone may have been sufficient to elicit increased Na⁺/H⁺ antiporter activity in Balb/c embryos. To test the effect of exposure to Na⁺-free medium separately from acidosis, we used medium containing NH₄Cl, which was also Na⁺ free, for the NH₄Cl pulse. In this way, the length of exposure to Na⁺-free medium was identical, but it occurred while pH_i was alkaline rather than acidic because pH_i is markedly alkaline while NH₄Cl is present. Exposure to Na⁺-free medium alone did not activate Na⁺/H⁺ antiporter in Balb/c embryos (Fig. 4). The activity of the Na⁺/H⁺ antiporter was also probed by exposing embryos to Na⁺-free medium and then reintroducing Na⁺ (Fig. 5). Again, no difference was seen between CF-1 and Balb/c embryos. Thus, Na⁺-free medium alone does not induce an activation of the antiporter. Alternatively, activation might also have been due solely to the prolonged acidosis experienced by the embryos during the period in Na⁺-free medium and may not have been influenced by the absence of Na⁺. However, a prolonged acidosis produced in the presence of Na⁺ by amiloride did not result in stimulation of antiporter activity in Balb/c embryos (Fig. 3). Thus, it appears that a combination of Na⁺-free medium and acidosis is required for the Na⁺/H⁺ antiporter in Balb/c embryos to exhibit increased recovery rates.

A tentative explanation for this effect in Balb/c embryos is that it is mediated by depletion of intracellular Na⁺ during exposure to Na⁺-free medium. Depletion of intracellular Na⁺ can stimulate Na⁺/H⁺ antiporter activity in some cells. In some instances, a drop in intracellular Na⁺ causes a shift in setpoint, possibly mediated by signaling via an intracellular Na⁺ receptor [25, 26]. In other cases, depletion of intracellular Na⁺ stimulates Na⁺/H⁺ antiporter activity directly (with no shift in setpoint), because the Na⁺ gradient, which powers antiporter activity, is steeper after depletion [26–28]. The Na⁺/H⁺ antiporter itself is a major route for intracellular Na⁺ efflux into Na⁺-free medium because the reversed Na⁺ gradient causes the antiporter to run in "reverse" (Fig. 5). Because the antiporter is stimulated by intracellular acidosis, Na⁺ efflux and hence intracellular Na⁺ depletion would occur more rapidly when the cytoplasm is acidic. Thus, if the cell is subjected to both Na⁺-free medium and intracellular acidosis simultaneously, intracellular Na⁺ could be depleted much more quickly, leading to higher Na⁺/H⁺ antiporter activity in the normal direction when external Na⁺ is restored. Such a scenario would provide a possible explanation for the apparent requirement for both Na⁺-free medium and acidosis.

It is not clear why only Balb/c of the three strains tested should produce embryos that exhibit this effect. There may be a difference in signaling pathways such that Balb/c embryos respond more robustly to depletion of intracellular Na⁺. Another possibility would be that embryos derived from different strains express a different proportion of NHE isoforms, with possible isoform-specific differences in the response to the absence of Na⁺ during recovery from acidosis (although such a difference between isoforms has not been reported) underlying the observed difference in overall recoveries. Although CF-1 embryos express both the NHE1 and NHE3 isoforms [16], the expression pattern in other strains is not known. Further investigation is needed to elucidate the mechanism behind the shift of pH_i setpoint in Balb/c embryos and to determine why this shift did not occur in embryos derived from the other strains.

The results reported here clearly differ from those of previous reports, in which Baltz et al. [14, 29] claimed an absence of Na⁺/H⁺ antiporter-mediated recovery from acidosis in two-cell mouse embryos derived from BDF and CF-1 strain females. In those studies, recovery occurred and was not inhibited by absence of external Na⁺ or by amiloride [14, 29]. This type of recovery was typical of those in QS strain embryos [15] under conditions in which Na⁺/H⁺ antiporter activity had been completely inhibited (by amiloride derivatives or lack of Na⁺). The simplest explanation for the divergent results is that the embryos assessed by Baltz et al. [14, 29] actually did not have detectable Na⁺/H⁺ antiporter activity, consistent with their low baseline pH_i, low pH_i after recovery, and lack of sensitivity to amiloride or absence of external Na⁺. Alternatively, Gibb et al. [15] proposed that Na⁺/H⁺ antiporter activity existed but was not detected for two major reasons. First, amiloride or lack of external Na⁺ actually had inhibited recovery from acidosis, resulting in a smaller extent of recovery (i.e., only partial recovery) that was not reflected in the parameters reported (rate constant and half-time of the exponential recovery). Second, the effects of H⁺-lactate cotransport on pH_i masked the contribution of Na⁺/H⁺ antiporter activity to recovery from acidosis. However, calculation of the extents of recoveries from original data [14] indicated that there was no significant difference, with pH_i recovering to a mean of 0.11 ± 0.04 pHU below the initial pH_i (measured just before the NH₄Cl pulse) in control embryos compared with 0.09 ± 0.06 pHU with amiloride and 0.10 ± 0.02 pHU in the absence of Na⁺. Thus, the inability to demonstrate Na⁺/H⁺ antiporter activity in two-cell embryos cannot have been due to a failure to detect differences in the extent of recoveries, as proposed by Gibb et al. [15]. Two-cell-stage mouse embryos possess an H⁺-monocarboxylate cotransporter [30] that transports lactate and pyruvate, and transport via this mechanism can affect pH_i because of its cotransport of H⁺ across the plasma membrane, even though this mechanism is not a pH_i regulatory mechanism per se [15, 31]. However, H⁺-monocarboxylate transport does not mask Na⁺/H⁺ antiporter activity. Na⁺/H⁺ antiporter activity was easily detectable in QS strain mouse embryos in the presence of lactate, and H⁺-monocarboxylate transport supports only a partial recovery, which plateaus well below the Na⁺/H⁺ antiporter setpoint [15]. Thus, it is unlikely that the apparent absence of Na⁺/H⁺ antiporter activity in two-cell embryos that was initially reported was due to masking of such activity by the H⁺-monocarboxylate transporter. In the present study, low (1 mM) lactate media were used to prevent any possibility of contribution by lactate transport.

The question thus still remains of why two-cell embryos did not appear to possess Na⁺/H⁺ antiporter activity in the earliest studies [14, 29]. One possibility is that the purity of pH-sensitive fluorophores has greatly increased since the late 1980s, and the BCECF batches used for pH_i measurements in the earliest studies may have caused a loss of Na⁺/H⁺ antiporter activity, whereas more recent BCECF does not. It is possible that this BCECF (or excitation of its fluorescence) induced the reported permeability to H⁺ in two-cell embryos [14, 32] that might have masked the low level of Na⁺/H⁺ antiporter activity in embryos derived from CF-1 and BDF females. Contrary to the suggestions of Gibb et al. [15], this apparent H⁺ permeability did not require the presence of monocarboxylic acids such as lactate or pyruvate or alterations in their concentrations [14, 32]. Thus, this permeability was not related to H⁺-monocarboxylic acid transport nor could it have been a manifestation of Na⁺/H⁺ antiporter activity, as H⁺ permeability was apparent in the alkaline pH_i range where Na⁺/H⁺ antiporter would be quiescent [32].

We demonstrated that mouse two-cell embryos have Na⁺/H⁺ antiporter activity, as shown earlier by Gibb et al. [15] in embryos derived fro the QS strain. However, we also demonstrated that embryos derived from different strains of female mice exhibit differences in pH_i regulation by this mechanism. These differences illustrate the pitfalls in generalizing physiological findings from embryos derived from only one mouse strain and highlight the even greater difficulties of generalizing across species.

ACKNOWLEDGMENTS

The authors thank Mrs. Mary-Anne Hammer for excellent technical support.

FOOTNOTES

First decision: 26 December 2000.

¹ This work was supported by an operating grant (MOP 12040) from the Canadian Institutes of Health Research (CIHR, formerly the Medical Research Council of Canada) and as part of the U.S. National Institutes of Health NICHD National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Embryo Development (HD 22023). C.L.S. was supported by a CIHR studentship award.

² Correspondence: Jay M. Baltz, Loeb Research Institute, Ottawa Hospital Civic Campus, 725 Parkdale Ave., Ottawa, ON, Canada K1Y 4E9. FAX: 613 761 5327 or 5365; jbaltz{at}lri.ca

³ Current address: Colorado Center for Reproductive Medicine, Englewood, CO 80110.

⁴ Current address: Department of Biological Sciences, University of New Orleans, New Orleans, LA 70148.

Accepted: March 6, 2001.

Received: November 20, 2000.

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