The two-cell stage mouse embryo has been shown to exhibit HCO/Cl exchange activity which mediates recovery from intracellular alkalosis(1) . Biochemically, this activity is much like HCO/Cl exchange activities found in many cultured mammalian cells: it is inhibitable by the stilbene drug DIDS, ()is active above about pH 7.2 and has a K for external Cl in the millimolar range(1) . Surprisingly, there was no detectable corresponding activity of mechanisms to correct deviations of pH in the acid direction, such as the otherwise ubiquitous Na/H antiporter (2) or the Na,HCO/Cl exchanger(3) . Similarly, the unfertilized mouse egg has been reported to lack Na/H antiport activity(4) . Thus, HCO/Cl exchange appears to be the sole pH regulatory mechanism in the early embryo (at least at the two-cell stage).

Three genes encoding HCO/Cl exchangers have been identified in mammals(5) . All are related and are homologs of the erythroid anion exchanger, band 3, which functions as both a HCO/Cl exchanger and a membrane anchor of the cytoskeleton in erythrocytes. These homologs are designated AE1, AE2, and AE3 (``AE'' for ``anion exchanger''). The AE1 gene encodes at least two alternate polypeptides, erythroid band 3(6) , and an N terminally truncated kidney-specific form (7, 8) apparently active in renal acid secretion (9) . The AE2 gene encodes at least one polypeptide, which is widely distributed among various tissues and cultured cell lines(10, 11) where it may mediate pH regulation(12, 13) , and/or volume regulation(14) . The AE3 gene encodes at least two alternate transmembrane polypeptides with differing N termini. One was first cloned from brain and is termed the ``brain'' isoform(11, 15, 16) , while the other was cloned from heart and is termed the ``cardiac'' isoform(16, 17) . In addition, alternatively spliced mRNA encoding a polypeptide lacking the transmembrane domain has been described(18) . It is not yet known if all HCO/Cl exchangers are members of the AE family, or if unrelated proteins also serve this function. However, the physiological properties of HCO/Cl exchange in all those cell types where it has been examined resemble those of the proteins of the AE family.

pH regulation by HCO/Cl exchange has similar properties in various nucleated cell types and also when mediated by heterologously expressed AE polypeptides: HCO/Cl exchange is activated above a threshold pH which is usually around 7.1-7.3(1, 19, 20, 21, 22) . This threshold ``set point'' can be altered by metabolic alteration of the cell(12, 22) . The activity is inhibited by stilbene drugs such as DIDS, requires HCO in the cell, and requires an inwardly directed Cl gradient. HCO and Cl compete for the same transport sites, and the apparent K for both anions is similar (generally in the range of 1-10 mM; 1, 12, 26).

While HCO/Cl exchange activity has been demonstrated in the two-cell mouse embryo, neither the molecular basis of this HCO/Cl exchange, nor a requirement for HCO/Cl exchange activity in early development has been described. In addition, there has been no information available on the presence or absence of HCO/Cl exchange activity in other stages of preimplantation embryo. The studies presented here address these questions.

MATERIALS AND METHODS

Embryos

Media

Embryo Culture in Varying CO Concentrations with and without the Anion Exchange Inhibitor DIDS

The results of these experiments were expressed as the number of embryos reaching the expanded blastocyst stage after 68-70 h of culture. There were three or four replicates at each CO concentration (except for 0.4%, where there were two). These replicates were tested for homogeneity and found to be not significantly different (all p > 0.05 by Fisher's exact test, except for one anomalous replicate at 1.5% CO, control, which was nonetheless included). The replicates were therefore pooled for further analysis. The data were analyzed by logistic regression analysis, which allows estimates of the effects of each individual treatment variable as well as any interactions between them. The logistic regression parameters were calculated by exact calculation, rather than using asymptotic methods, since this allows for sparse data sets and data sets with numerous responses near 0 and 100%.

Intracellular pH (pH) Measurements

pH Measurements of Embryos under Culture Conditions

Four to six replicates (36-74 embryos total/treatment) at each CO concentration with and without DIDS were used. The data from identical conditions were pooled and normal distribution confirmed by normal probability plots. To determine if there was a significant effect of CO on pH, the data were analyzed by two separate ANOVAs, one treating the data with [DIDS] = 0 at all three CO levels, and the other treating the data with [DIDS] = 100 µM at all three CO levels. These tests determine if pH depends on CO level in control medium or in DIDS-containing medium. To determine if DIDS had a significant effect on pH, the data were analyzed by an exact Wilcoxon Rank Sum test. The data were treated as comprising two groups (DIDS and control) stratified by CO (i.e. treating it as a confounding variable). This test determines if pH depends on the presence or absence of DIDS overall.

Detection of Functional HCO/Cl Exchangers in Embryos

SNARF-1-loaded embryos were placed into a temperature- and atmosphere-controlled chamber (37 °C and 5% CO; Biophysica Inc., Baltimore, MD) fitted with a perfusion apparatus (solution changed in <30 s). After monitoring pH for 10 min, the medium (KSOM with 9 mM sodium lactate replaced by NaCl, [Cl] = 110 mM) was replaced by medium that was identical except that Cl was replaced by gluconate and sulfate ([Cl] = 0, nominally); 100 µM DIDS was used to assess inhibition, and Na- and Cl-free medium was used to assess Na dependence (NaCl, sodium lactate, and sodium pyruvate replaced by isosmotic sucrose, remainder of Cl replaced by gluconate, and HCO supplied by choline HCO). Embryos at the one-cell, two-cell, morula, and blastocyst stages were used. Blastocysts were mechanically collapsed by passage through a narrow-bore pipette to allow DIDS access to the blastocoel cavity(27) . Three measurements, with and without DIDS, and two measurements without Na, were carried out at each stage (8-20 embryos/measurement group). The data, expressed as the mean pH of the group of embryos at each time point, were analyzed by determining the initial rate of pH increase after Cl removal, and by determining the maximum net change in pH. Initial rate of pH increase was determined by a linear regression performed on the linear portion of the increase, constituting the first seven (one-cell stage) or 10 (other stages) data points (taken at 30-s intervals). The net increase was determined as the difference between the base-line pH just prior to Cl removal, and the peak or plateau pH following Cl removal (both averaged over 5 min). Statistical analysis was performed to determine whether the initial rate of increase and/or net increase were significantly different in the presence or absence of DIDS; this was done by t test (assuming unequal variances) at each stage. The data were also analyzed to determine if the initial rate of increase or net increase in pH changed over development. Initial examination showed that there was no significant difference in either parameter (by t test) between the one- and two-cell stages, nor between the morula and blastocyst stages, so the data for one- and two-cell and for morula and blastocyst were pooled for further analysis. To determine if there was a change over development, t tests (two-tailed, assuming equal variances) were performed to compare initial rates and net changes between the pooled one- and two-cell, and the pooled morula and blastocyst, groups.

Isolation of mRNA and Preparation of cDNA from Embryos

Polymerase Chain Reaction (PCR) Determination of Anion Exchanger mRNA in Embryos

The Perkin Elmer Cetus PCR kit with Taq DNA polymerase was used with 1 µl of embryo cDNA (equivalent to 2.5 embryos) or diluted 30-cycle product, in 20 µl total volume. The temperature cycle was 93 °C (1 min), 55 °C (1 min), and 72 °C (3 min; or 13 min at end), controlled by a PTC-100 thermocycler (MJ Research, Watertown, MA). The products were analyzed on agarose gels (3:1 NuSieve:Seakem, FMC) and visualized with ethidium bromide. The predicted product sizes from cDNA were: for the 5`,3` pair (30-cycle PCR), AE1, 303; AE2, 280; AE3, 449; for the semi-nested PCR (5`, 3`-internal pair), AE1, 237; AE2, 210; AE3, 301.

These primers flank at least one intron in each genomic sequence so that any product due to contaminating genomic DNA would be identifiably larger than that arising from cDNA, and embryo samples in which reverse transcriptase was omitted were all negative (not shown). Thus, any detected bands must arise from mRNA. To control for the possibility that our semi-nested PCR protocol was too sensitive and was detecting ``leaky transcription'' (see ``Discussion''), we used cDNA derived from tissues known not to express significant levels of a given AE message. Each negative control tissue was run in parallel with a positive tissue. For AE1, stomach was used as a negative control, and spleen as positive(11) ; for AE3, spleen was used as negative and heart as positive(11) . Unfortunately, there is no tissue which has been definitively shown to be negative for AE2, and thus no obvious negative control(10, 11) ; stomach, brain, heart, and kidney were all positive. Neither of the negative control tissues showed any detectable band of the expected size, while all positive controls were clearly positive (data not shown). Thus, any mRNAs detected by our PCR methods are unlikely to result from over-amplification and detection of non-physiological (``leaky'') transcription.

Restriction Digest Confirmation of PCR Products

Statistics

RESULTS

Effect of Alkaline pH and the Anion Exchange Inhibitor DIDS on Embryos in Culture

Figure 1: Development of two-cell mouse embryos to blastocysts as a function of CO concentration with and without the HCO/Cl exchange inhibitor DIDS. Two-cell embryos were cultured for 70 h, and the proportion reaching the expanded blastocyst stage was determined. The CO level was varied, and embryos were cultured in the presence or absence of the HCO/Cl exchange inhibitor DIDS (100 µM). Lowering CO (shown on bottom axis) and therefore raising the pH of the medium (shown on top axis) had little effect in the absence of DIDS (), but in the presence of DIDS (), higher pH was markedly inhibitory. Details of the data and analysis are given in the text.

The inclusion of the anion exchange inhibitor, DIDS (100 µM), in the culture medium with 5% CO (pH 7.35) had no effect on the proportion of embryos developing to blastocysts by 70 h in culture: 98% of the embryos developed to blastocysts in the presence of DIDS, which is not significantly different from the 93% obtained in the absence of DIDS (p = 0.62 by Fisher's exact test). Thus, DIDS alone is non-toxic.

However, at lower CO levels (2.0% down to 0.4%), DIDS greatly inhibited the development of embryos, with fewer than 20% reaching the blastocyst stage at 0.4 and 0.8% as compared to the 70-80% in the absence of DIDS (Fig. 1). To show that the effect of DIDS was significant, and that it depended on an interaction with CO, the full data set was analyzed by exact logistic regression analysis. The regression model assumed dependence on DIDS and on an interaction between CO and DIDS (model: Logit(proportion of blastocysts) = [DIDS] + [DIDS] [CO] + ; a very good fit was demonstrated by the Hosmer-Lemeshow test, p = 0.88, and Deviance test, p = 0.29). This model was chosen after models in which a [CO] term had been included failed to give an adequate fit by the Hosmer-Lemeshow test, and also failed to converge, indicating that dependence on [CO] alone does not contribute significantly to the overall fit. This analysis showed that both the effect of DIDS and the interaction between DIDS and CO were highly significant (both and were highly significantly different from zero, p < 10). Therefore, DIDS, but only in conjunction with lowered CO level, significantly decreases overall development.

Intracellular pH (pH) of Two-cell Embryos under Culture Conditions

Figure 2: Intracellular pH of two-cell embryos after 3-5 h in culture as a function of CO concentration with and without the HCO/Cl exchange inhibitor DIDS. Using the intracellular pH-sensitive fluorophore SNARF-1, the fluorescence emission intensity ratio (640-600 nm) and hence intracellular pH (pH) was measured under the same conditions in which embryos were cultured. The presence of the HCO/Cl exchange inhibitor DIDS () resulted in significantly elevated pHrelative to control (). The elevation was much more pronounced at lower CO levels. The vertical axes show the measured fluorescence emission intensity ratio (left) and the calculated pH (right). The small filled symbols represent the mean pH from four to six pooled replicates (36-74 embryos); the box plots superimposed at each point represent the population spread of the data: the center line is the median, while the upper and lower bounds of the box are the 75th and 25th percentiles, respectively, and the whiskers show the 10th and 90th percentiles. See text for experimental details and data analysis.

Analysis of these data was performed in two steps. First, the effect of varying CO alone at constant [DIDS] was tested by ANOVA, treating the [DIDS] = 0 and [DIDS] = 100 µM groups separately. For each group, it was found that varying CO had a highly significant effect on pH (both p < 10). However, the effect was small in the absence of DIDS, and occurred only at 0.8% CO, but not at 1.7 or 5% CO (Fig. 2). The second analysis tested the significance of the effect of DIDS and was performed on the entire data set. For this purpose, the data set can be considered to be stratified by CO level, and the effect of CO eliminated by considering it a confounding variable, and so the Wilcoxon Rank-Sum test for stratified data was used. The effect of DIDS was found to be highly significant, with p < 10. Thus, the presence of DIDS in culture raises pH significantly. Pairwise t tests (assuming unequal variances, since all F tests showed significant difference in variances) performed at each of the three CO levels showed highly significant differences in mean pH at each CO (all p < 10). Thus, pH is raised by DIDS even in 5% CO; however, the effect is much greater at lower CO, with pH being very high at 1.7 and 0.8% CO in the presence of DIDS (Fig. 2).

Presence of Functional HCO/Cl Exchange at Each Stage

Figure 3: HCO/Cl exchanger activity during preimplantation development. A, plots of pHi versus time in 1-cell, 2-cell, morula, and blastocyst stage embryos monitored before and after switching to Cl-free medium. pH was measured (as described in the text) in embryos in normal Cl medium () and then the medium was replaced by Cl-free medium (), which caused an increase in pH at each stage. The increase was not affected by lack of external Na (middle set of plots) but was completely inhibited by DIDS (100 µM; lowest set of plots). Each trace represents the mean pH in a group of embryos in one representative experiment. Points are separated by 1 min, except immediately following the removal of Cl, where they are separated by 30 s. B, initial rate of pHi increase at each stage. The initial rate of increase of pH upon replacement of the medium with Cl-free medium was determined by linear regression (as described in the text). The bars represent the mean rate (± S.E.) of this increase at each stage. Cross-hatched bars represent the mean initial rates in control medium, bars with horizontal lines represent the rates in the absence of external Na, while the open bars represent the rates in the presence of 100 µM DIDS. See text for data analysis. C, net increase in pH. The net increase in pH from normal medium to its new steady-state value in Cl-free medium is shown for each stage (mean ± S.E.). Cross-hatched bars represent the mean increase in control medium, bars with horizontal lines represent the increase in the absence of external Na, while the open bars represent the increase in the presence of 100 µM DIDS. See text for data analysis.

An increase in pH upon Cl removal could potentially be mediated by either HCO/Cl exchange or Na,HCO/Cl exchange. To ensure that we were detecting HCO/Cl exchange, Cl removal was performed in nominally Na-free medium (Fig. 3). In the absence of external Na, pH still increased upon removal of Cl at each stage. The mean initial rates of increase (Fig. 3B) were not significantly different from the control recoveries at each stage (p = 0.89, 0.15, 0.64, and 0.42, respectively, by two-tailed t test). The mean extents of the increase (Fig. 3C) were also not significantly different from control at any stage (p = 0.75, 0.57, 0.53, and 0.10). Therefore, there was no evidence of significant Na,HCO/Cl exchange activity at any stage tested, indicating that the increase in pH upon Cl removal at each stage demonstrates that there is HCO/Cl exchange activity present at each stage.

To determine whether there was a change in the initial rate of increase of pH, or in net increase in pH, over the course of preimplantation development, a t test (two-tailed) was performed to test for a significant difference between the pooled results for one- and two-cell stages, and the pooled results for morula and blastocysts (one- and two-cell, and morula and blastocyst stage results were pooled after determining that there was no significant difference in either parameter within pooled stages; see ``Materials and Methods''). Both the initial rate of increase (Fig. 3B) and the net increase (Fig. 3C) decreased significantly from the one- and two-cell stages to the morula and blastocyst stages (p = 0.039 for initial rate and 0.0015 for net increase).

HCO/Cl (Anion) Exchanger mRNAs in Preimplantation Embryos

Figure 4: RT-PCR assays for known HCO/Cl exchanger isoform mRNA expression in preimplantation embryos. A, 30- cycle PCR. The lanes are marked as follows: 1 = one-cell embryos; 2 = two-cell embryos, m = morulae; b = blastocysts; all four stages are shown for each of AE1, AE2, and AE3, as marked. Expected sizes of PCR products are marked at right of gel. The unmarked lanes at right and left are phiX-174 HindIII digest markers (sizes shown at left). AE2 products are seen at the one-cell and blastocyst stages, faintly at the morula stage, and are barely visible at the two-cell stage. B, semi-nested PCR. The first two lanes of each gel are positive controls consisting of RT-PCR products of tissues known to express the relevant mRNA (AE1: kidney, spleen; AE2: stomach, kidney; AE3: brain, heart). Embryo lanes are marked as in A. The lanes marked e are PCR products derived from PCRs starting with cDNA from the equivalent of 2.5 embryos; lanes marked w are negative controls consisting of a sample of the drop in which the embryos were washed, treated identically to the embryo samples (i.e. RNA isolation, RT-PCR). Expected sizes of PCR products are marked at right of gels. The unmarked lanes at right and left are markers as described in A.

To ensure that the PCR products visualized were indeed derived from specific AE mRNAs, we used restriction enzymes to cleave the products and show that the fragments generated were of the expected sizes. Fig. 5shows these results: the PCR products from embryos all gave the expected restriction fragments, and therefore arise from the designated AE mRNAs.

Figure 5: Restriction digest confirmation of identity of HCO/Cl exchanger isoform mRNAs. For restriction digests of PCR products, seminested PCR reactions similar to those used to generate the products shown in Fig. 4B were run, except that five times larger reactions were run, 10 additional cycles of PCR were used, and the entire product was used for restriction digest (see ``Materials and Methods''). The PCR products were digested with appropriate restriction enzymes and the sizes of the resulting fragments determined from the gel. The AE isoform is specified at the top. Embryo stages are marked at the top as 1c, 2c, M, and Bl for one-cell, two-cell, morula, and blastocyst, respectively. The restriction enzyme, expected fragment sizes, and size of original PCR product (in parentheses) is indicated under the gel. Marker lanes at right and left are phi X 174 HinfI digest (sizes shown at left).

DISCUSSION

Is HCO/Cl Exchange Present throughout Preimplantation Embryo Development?

()

Is HCO/Cl Exchange Activity Necessary for Preimplantation Development?

A rise in pH is the most likely cause of the decreased development observed in low CO when HCO/Cl exchange activity is inhibited. Without DIDS, the mean pH is maintained below 7.2 even when the external pH is well above 8.0. This in itself is indicative of activity of the embryo HCO/Cl exchanger, whose activation threshold is about 7.2(1) . However, in the presence of DIDS, pH is higher at all CO levels tested (Fig. 2). With the exchanger inhibited, mean pH rises to above 7.5, with the pH in individual embryos rising to above 7.8 (the box plots at each point show the population distribution, see figure legend). The greatest pH increases correlate with the most significantly decreased embryo viability, as can be seen from Fig. 1and Fig. 2. Taken together, the data shown in these figures suggest the possibility that the lethal level of pH is about 7.45; the percentages of embryos in each treatment group which fail to develop is approximately equal to the proportion whose pH values are above 7.45 (although a direct correspondence between those embryos with highest pH and those which fail to develop has not been shown).

The results of culture at 5% CO with DIDS indicate that, under normal culture conditions, HCO/Cl exchange activity is not needed for development in vitro. However, it is likely to be necessary for development in vivo. The pH values of oviductal and uterine fluids have not been determined in the mouse, but in other species where this has been measured, the oviductal fluid surrounding embryos has been found to have a high bicarbonate content and high pH, with up to pH 7.7 measured in the rhesus monkey (28) , and 7.8-8.2 in the rabbit (29, and references therein). Thus, while artificial culture conditions may allow embryos to grow in the absence of HCO/Cl exchange activity, it may be continually needed in vivo at least in the portion of preimplantation development that occurs in the oviduct. It is also clear from Fig. 2that pH is affected even at 5% CO; this may place a stress on the embryos which would compromise their viability in the face of any additional adverse conditions, even if not lethal by itself.

Are AE Family HCO/Cl Exchangers Present in Preimplantation Embryos?

RT-PCR is an extremely sensitive technique. In some cases leaky transcription can occur, in which a very few copies of an mRNA are transcribed nonspecifically. These non-physiological transcripts can potentially be detected by RT-PCR if amplification continues for enough cycles. However, the AE2 and AE3 transcripts which we detected are probably expressed at physiologically significant levels. First, we have shown that the seminested PCR protocol which we used does not detect leaky transcription of AE mRNA in negative control tissues known not to express significant levels of an AE transcript (see ``Materials and Methods''). Second, the transcripts are detected from cDNA derived from very few cells. AE2 message is detected after only 30 cycles of PCR from the equivalent of as few as 2.5 cells (one-cell stage), and AE3 is detected from as few as 5 cells (two-cell stage) using semi-nested PCR. Third, every AE mRNA was not detected at every embryo stage indiscriminately, as would be expected for leaky transcription.

AE2 message must necessarily be produced from both the maternal and embryonic genomes, since it is present before and after the two-cell stage, where the overall switch from maternal to embryonic gene expression occurs in the mouse. AE3 would thus seem to be a product of the embryonic genome only.

It appears that preimplantation stage mouse embryos make mRNA for at least two members of the AE HCO/Cl exchanger family AE2 and AE3. This makes the polypeptides encoded by the AE2 and AE3 genes good candidates for mediating the pH regulatory HCO/Cl exchange activity which we have demonstrated in the preimplantation embryo. It is evident that, of these two anion exchangers, AE2 is the most likely to be responsible for HCO/Cl exchange activity at the one-cell stage: there is robust HCO/Cl exchange activity at the one-cell stage (Fig. 3), but little or no AE3 message detectable at this stage, while in contrast there is a strong AE2 RT-PCR signal. However, it still must be shown directly which AE mRNAs are translated into proteins in the plasma membranes of embryos and that these proteins are responsible for the observed HCO/Cl exchange activity and pH regulation.

Conclusions

FOOTNOTES

*

This work was supported by Medical Research Council of Canada Operating Grant MT12040 (to J. M. B. and National Institutes of Health Grants RO1 HD29533 (to J. M. B.) and RO1 DK43495 (to S. L. A.). A preliminary account of a portion of this work has been published as an abstract(30) . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. A perliminary account of a portion of this work has been published ad an abstract(30) .

§

These authors contributed equally.

¶

Funded by the Loeb Medical Research Institute.

**

Recipient of a Loeb Medical Research Institute Summer Studentship.

§§

Established Investigator of the American Heart Association.

¶¶

MRC Scholar. To whom correspondence should be addressed: Loeb Medical Research Institute, Ottawa Civic Hospital, 1053 Carling Ave., Ottawa, Ontario K1Y 4E9, Canada. Tel.: 613-798-5555 (ext. 3714); Fax: 613-761-5327; jay{at}civich.ottawa.on.ca.

()

The abbreviations used are: DIDS, 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid; SNARF-1, carboxyseminapthorhodafluor-1; SNARF-1-AM, acetoxymethylester derivative of SNARF-1; PMSG, pregnant mare serum gonadotropin; hCG, human chorionic gonadotropin; ANOVA, analysis of variance; PCR, polymerase chain reaction; RT, reverse transcribed.

()

Y. Zhao and J. M. Baltz, unpublished results.

ACKNOWLEDGEMENTS

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