1 Loeb Research Institute, Ottawa Hospital and Departments of 2 Obstetrics and Gynecology, (Division of Reproductive Medicine), 3 Cellular and Molecular Medicine, University of Ottawa and 4 Human IVF Program, Ottawa Hospital, Ottawa, Ontario,Canada K1Y 4E9
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Abstract |
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Key words: embryo/HCO3/Cl exchanger/Na+/H+ antiporter/pH
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Introduction |
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HCO3/Cl exchangers mediate the electroneutral exchange of extracellular Cl for intracellular HCO3, thus decreasing pHi (Alper, 1991, 1994
). HCO3/Cl exchangers are members of the AE (anion exchanger) gene family, which includes the erythrocyte band 3 protein (AE1) and at least two other isoforms with wide tissue distributions (AE2 and AE3). We have shown that mRNA encoding two members of the AE family, AE2 and AE3, are expressed in preimplantation mouse embryos, with AE2 mRNA also present in the unfertilized egg (Zhao et al., 1995
; Phillips and Baltz, 1999
). HCO3/Cl exchanger activity is present in all stages of preimplantation embryo in the mouse and hamster and is required for maintenance of normal pHi and recovery from intracellular alkalosis in mouse embryos (Baltz et al., 1991
; Zhao et al., 1995
; Lane et al., 1999b
). Activity is very low in the unfertilized mouse and hamster egg and gradually becomes activated following fertilization, reaching maximal activity around the time of pronuclear formation (Lane et al., 1999b
; Phillips and Baltz, 1999
).
The Na+/H+ antiporter mediates the electroneutral exchange of extracellular Na+ for intracellular H+ to increase pHi. Na+/H+ antiporter is encoded by members of the NHE gene family (NHE16; Orlowski and Grinstein, 1997). NHE-1 mRNA expression has been demonstrated in mouse eggs and in all stages of mouse embryo development, and immunohistochemistry of mouse blastocysts has shown NHE-1 protein localization to the blastocoel membrane (Barr et al., 1998). NHE-3, whose mRNA has only been detected in unfertilized mouse eggs, immunolocalizes to the outer surface of the blastocyst (Barr et al., 1998
). pHi is regulated by Na+/H+ antiporter in hamster embryos (Lane et al., 1998
), and to varying extent in mouse embryos depending on the strain of mouse (Gibb et al., 1997; C.L.Steeves, M.Lane, K.P.Phillips, B.Bavister and J.M.Baltz, unpublished). Na+/H+ antiporter activity becomes activated following fertilization in the hamster (Lane et al., 1998
, 1999a
), similar to HCO3/Cl exchanger upregulation at fertilization in the mouse (Phillips and Baltz, 1999
) and hamster (Lane et al., 1999b
).
The Na+, HCO3/Cl exchanger imports HCO3 and Na+ in exchange for Cl and is amiloride-insensitive, inhibited instead by stilbene derivatives such as 4,4'-diisocyanatostilbene-2,2'-disulphonic acid, disodium salt (DIDS) (Grinstein et al., 1989). To date, Na+, HCO3/Cl exchanger activity has not been reported in mammalian embryos.
Recently, it has been shown that human preimplantation embryos, at all stages from zygote to blastocyst, have the ability to recover from intracellular alkalosis, which was induced by increased external pH (Dale et al., 1998). In contrast, Dale et al. could not detect any recovery from mild acidosis induced by decreased external pH in human embryos until the blastocyst stage. Thus, they concluded that preimplantation human embryos lack mechanisms needed for recovery from intracellular acidosis until the blastocyst stage, but possess a mechanism such as the HCO3/Cl exchanger for alleviating alkalosis throughout preimplantation development.
Here, we report the results of our investigations into the mechanisms used by human embryos to regulate pHi. We have used methods which were successfully employed in mouse and hamster embryos to reveal the identity and active ranges of pHi regulatory mechanisms present in human embryos.
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Materials and methods |
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ICSI was performed on an inverted microscope at x400 magnification using Hoffman modulation contrast. 12 µl washed spermatozoa were placed in HEPES-buffered HTF-BSA containing 10% polyvinylpyrrolidone (PVP K-90; Irvine Scientific, Santa Ana, CA, USA) and injected using standard techniques. Following injection, each egg was cultured individually in a 20 µl drop of HTF-BSA covered with paraffin oil at 37°C, 5% O2/5% CO2/90% N2 for about 1719 h. Embryos were then assessed for the presence of pronuclei (PN). Pronuclei detected in embryos donated for research included (assessed 1719 h post-IVF/ICSI): 0PN, 26 embryos; 1PN, 43 embryos; 2PN, 120 embryos; 3 PN, 23 embryos; 4 PN, 1 embryo. Embryos were maintained in culture for 24 days post-IVF/ICSI during which time embryo cleavage was assessed. Embryos were graded from 1 to 5 (Rattanachaiyanont et al., 1999) according to the quality of blastomeres and the presence of fragmentation with 5 indicating the best-quality embryos. Embryos used for this study included 11 Grade 1; 53 Grade 2; 122 Grade 3; 21 Grade 4; and 6 Grade 5 embryos. Eggs were not rated.
Chemicals and solutions
All chemicals and drugs were obtained from Sigma (St Louis, MO, USA) unless otherwise noted. SNARF-1-AM (carboxyseminaphthorhodafluor-1-acetoxymethyl ester) and DIDS were obtained from Molecular Probes (Eugene, OR, USA).
Media used for pHi measurements were based on KSOM mouse embryo culture medium (Lawitts and Biggers, 1993). This medium supports the culture of human embryos, and its use allows direct comparisons with our previous results in the mouse. The modified KSOM contained (in mol/l) 104 NaCl, 2.5 KCl, 0.35 KH2PO4, 0.2 MgSO4, 1 Na+ lactate, 0.2 glucose, 0.2 Na+ pyruvate, 25 NaHCO3, 1.7 CaCl2, 1 glutamine, 0.01 tetrasodium EDTA, 0.03 streptomycin SO4 and 0.16 K penicillin G. Media were equilibrated with 5% CO2/ air except where noted. HEPES-KSOM (Lawitts and Biggers, 1993
), used for egg/embryo handling, was produced by replacing 21 mmol/l (of 25 mmol/l) NaHCO3 with equimolar HEPES (pH adjusted to 7.4 with NaOH or KOH, as appropriate). HCO3-free solutions were similarly produced by replacing 21 mmol/l NaHCO3 with equimolar HEPES and the remaining 4 mmol/l with equimolar NaCl. For Cl-free solutions, all Cl salts were replaced with corresponding gluconate salts. For Na+-free (< 1 mmol/l) solutions, NaCl was replaced with equimolar choline Cl, and Na+ pyruvate, Na+ lactate and NaHCO3 were replaced by K+ pyruvate, lactic acid and choline HCO3, respectively. To induce alkalosis, solutions containing 35 mmol/l NH4Cl were used (NH4+-KSOM), wherein 25 mmol/l NaHCO3 was reduced to 12 mmol/l; NaCl was 69 mmol/l. To inhibit recovery from alkalosis, 0 Cl NH4+-KSOM was used in which NH4Cl was replaced with NH4SO4 and all other Cl salts replaced with corresponding gluconate salts. Acidosis was induced by a 10 min pulse of 35 mmol/l NH4Cl (NH4+-KSOM or NH4+-HEPES-KSOM, as specified) containing 25 mmol/l NaHCO3.
pHi measurements
pHi was measured using the pH-sensitive fluorophore, SNARF-1, loaded into eggs/embryos by incubating them with 5.0 µmol/l SNARF-1-AM at 37°C for 30 min in HEPES-KSOM (House, 1994; Zhao et al., 1995
; Zhao and Baltz, 1996
; Phillips and Baltz, 1996
; Baltz and Phillips, 1998
; Phillips et al., 1998
). After SNARF-1 loading, eggs/embryos were washed several times with HEPES-KSOM and placed in a temperature-controlled chamber (Biophysica, Baltimore, MD, USA) that was modified to allow solution changes and control of the atmosphere. Complete exchange of solutions in the chamber was obtained after ~1 min (unpublished measurements). During measurements, eggs/embryos were maintained at 37°C (± 0.5°C). For most experiments, HEPES-KSOM was immediately replaced with KSOM.
The methods used for pHi measurements have been previously described in detail (Baltz et al., 1990; Zhao et al., 1995
, 1997
; Baltz and Phillips, 1998
; Phillips et al., 1998
). Briefly, simultaneous measurements were made of groups of eggs/embryos with data recorded for each individual egg/embryo. SNARF-1 fluorescence was detected using an intensified CCD camera with output to an image storage and quantification system (Inovision, Durham, NC, USA). Two fluorescence emission wavelengths were detected, 640 nm (pHi-sensitive) and 600 nm (pHi-insensitive), using an excitation wavelength of 535 nm. The ratio of the two emission intensities (640/600), dependent only on pHi, was calculated by dividing the images after background subtraction. Ratio was calibrated to pHi using calibration solutions containing 10 µg/ml nigericin and 5 µg/ml valinomycin with 100 mmol/l K+ (Thomas et al., 1979
; Baltz et al., 1990
). pHi calibration curves were generated regularly and were indistinguishable between eggs and all embryo stages. SNARF-1 loading and exposure to excitation illumination does not adversely affect mouse eggs or embryos, as it was previously shown that mouse eggs could be fertilized by IVF and will cleave following pHi measurements (Phillips et al., 1998
).
Recovery from induced alkalosis
To determine whether eggs/embryos were able to recover from an increase in pHi, intracellular alkalosis was induced using the permeant weak base NH4Cl (Boron and DeWeer, 1976; Roos and Boron, 1981
; Zhao and Baltz, 1996
). Following steady-state pHi measurements, the solution was changed to NH4+-KSOM (12 mmol/l HCO3) for 20 min. HCO3 was reduced to 12 mmol/l to maximize recovery from intracellular alkalosis (external HCO3 acts as a competitive inhibitor; Baltz et al., 1991). Where appropriate, HCO3/Cl exchanger activity was inhibited by omitting external Cl. The rate of recovery from alkalosis was determined by fitting the recovery to a single exponential by non-linear regression. The first derivative was used to calculate the rate of recovery as a function of pHi. For statistical analysis, recovery rates were compared at every 0.1 pHi increment between 7.1 and 7.7.
Cl removal assay for HCO3/Cl exchanger activity
Upon exposure of cells to Cl-free solution, any HCO3/Cl exchanger present will run in reverse, resulting in intracellular alkalinization due to HCO3 influx coupled to Cl efflux (Nord et al., 1988). Intracellular alkalinization upon Cl removal is therefore indicative of HCO3/Cl exchanger activity and the initial rate of alkalinization serves as a measure of activity (Nord et al., 1988
; Zhao and Baltz, 1996
). In this assay, following 10 min of steady-state pHi measurement, the solution was changed to Cl-free KSOM for 20 min. The initial rate of intracellular alkalinization upon Cl removal was determined using linear regression. For some experiments, pHi was measured during Cl removal for 10 min, after which external Cl was replaced (KSOM) for 10 min and then a second Cl removal was performed for an additional 20 min.
Recovery from induced acidosis
To determine whether embryos were able to recover from a decrease in pHi, intracellular acidosis was induced by a 10 min pulse of NH4+-containing solution (NH4+-KSOM or NH4+-HEPES-KSOM) followed by a 15 min period in KSOM. This protocol results in the net acidification of the cytoplasm after the NH4+ pulse (Boron and DeWeer, 1976; Roos and Boron, 1981
; Zhao and Baltz, 1996
). Generally, any possible recovery from acidosis was assessed by measuring pHi for 15min following the NH4+ pulse. In some experiments, however, a 15min period in Na+-free solution was followed by another 15min in Na+-containing solution. The rate of recovery from acidosis was determined by fitting the recovery to a single exponential by non-linear regression. The first derivative was used to calculate the rate of recovery as a function of pHi. For statistical analysis, recovery rates were compared at every 0.1 pHi increment between 6.5 and 7.1.
Statistics
Data are presented as the mean ± SEM. In all cases, P < 0.05 was considered significant. Descriptive statistics were obtained using SigmaPlot 1.0 (Jandel Scientific, San Rafael, CA, USA) or SigmaPlot 5.0 (SPSS, Chicago, IL, USA). Throughout, `n' indicates the total number of eggs or embryos. Statistical comparisons were done using InStat (GraphPad, San Diego, CA, USA). For statistical comparisons of three or more groups Bartlett's test for homogeneity of variances was first used to determine whether parametric ANOVA (pANOVA) or non-parametric ANOVA (nANOVA) was appropriate. Comparisons were then made using pANOVA or nANOVA followed by the TukeyKramer multiple comparisons test or Dunn's test, respectively. For 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 Student's t-test or Welch's alternate t-test for equal or unequal variances, respectively. Linear regression was used to determine the initial rate of alkalinization upon Cl removal and to determine the rate of pHi change when recovery from acidosis was inhibited. Non-linear regression, using the appropriate exponential curves as specified, was used to determine the rates of recovery from alkalosis and acidosis.
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Results |
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Recovery from intracellular alkalosis in embryos
Cleavage-stage embryos were alkalinized by exposure to NH4Cl to assess whether embryos could recover from intracellular alkalosis (Figure 1A). Recovery rates were determined by fitting a single exponential to the recovery by non-linear regression for every 0.1 pHi increment between pHi 7.1 and 7.7 (Figure 1C
). There was no significant difference in recovery rates between stages of embryo development (compared at pHi 7.6; P > 0.05; nANOVA), and thus, the recovery rates for cleavage-stage embryos were pooled (n = 15; Figure 1A
). When embryos (n = 8) were alkalinized in the absence of external Cl (Figure 1B,C
) recovery from alkalosis was significantly inhibited (P = 0.001, Welch's t-test). Thus, embryos demonstrate Cl-dependent recovery from alkalosis, which was active above about pHi 7.27.3 (Figure 1C
).
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Embryos also recovered from acidosis in the presence of external HCO3 (Figure 3A,C). Recovery from acidosis in the pHi range 6.56.9 was significantly inhibited by the absence of external Na+ (Figure 3A, C
; P < 0.0001; nANOVA). However, neither inhibition of Na+/H+ antiporter by amiloride (1 mmol/l) nor the presence of anion transport inhibitor DIDS (500 µmol/l) significantly reduced the recovery rate (Figure 3A,C
; P > 0.05; nANOVA).
pHi regulation in egs
HCO3/Cl exchanger activity and recovery from acidosis were only assessed in the few eggs available to us, and thus no statistical tests were performed. HCO3/Cl exchanger activity in eggs was assessed by the Cl removal assay (Figure 4A). The mean rate of intracellular alkalinization during Cl removal (± SEM) was 0.075 ± 0.025 pHU/min in GV eggs (n = 2), 0.028 ± 0.010 pHU/min for MI eggs (n = 3), and 0.017 ± 0.002 pHU/min in MII eggs (n = 5) which appear similar to the rates observed in cleavage-stage embryos. For MI eggs, a second Cl removal was performed in which Cl was replaced following the first Cl removal (Figure 4C
). Results suggest that the initial rate of intracellular alkalinization (0.028 pHU/min) was reduced when the anion transport inhibitor DIDS was present during the second Cl removal (0.003 ± 0.001 pHU/min).
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Discussion |
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We also found that human cleavage-stage embryos rapidly recovered from acidosis. Recovery from acidosis, induced by an NH4Cl pulse, in the absence of external HCO3 and CO2, was Na+-dependent and completely inhibited by the presence of the Na+/H+ antiporter inhibitor amiloride. This suggests that Na+/H+ antiporter activity is present in the human cleavage-stage embryo which is consistent with the presence of Na+/H+ antiporter activity in hamster (Lane et al., 1998) and mouse embryos of various strains (Gibb et al., 1997; C.L.Steeves, M.Lane, K.P.Phillips, B.D.Bavister and J.M.Baltz, unpublished data).
To determine whether any HCO3-dependent mechanisms mediating recovery from acidosis were also present in cleavage-stage embryos, recoveries from acidosis were measured in the presence of external HCO3 with CO2 present. In the presence of HCO3/CO2, human embryos exhibited a rapid, Na+-dependent recovery from acidosis to a higher pHi (~7.07.2) than in the absence of external HCO3/CO2 where pHi recovered to only ~6.8. This suggests that a second, Na+-dependent mechanism, which requires HCO3, contributes to the alleviation of acidosis in human cleavage-stage embryos. The presence of a second system is also suggested by the inability of amiloride to inhibit recovery from acidosis in the presence of external HCO3/CO2, in contrast to the complete inhibition seen in HCO3/CO2-free medium. Such Na+- and HCO3-dependent, but amiloride-insensitive, recovery would seem to implicate Na+, HCO3/Cl exchanger activity. We could not, however, detect a component of the recovery in the presence of HCO3/CO2 which was DIDS-sensitive, although DIDS would be expected to partially inhibit recovery by eliminating the contribution of Na+, HCO3/Cl exchange, leaving only Na+/H+ antiporter activity. It is possible, however, that the variability in recovery rates seen with human embryos precluded such detection of partial inhibition.
We have shown that pHi regulation in the human embryo results from the concerted efforts of at least three exchangers: HCO3/Cl exchanger, Na+/H+ antiporter and an Na+, HCO3-dependent, amiloride-insensitive transporter which may be the Na+, HCO3/Cl exchanger, similar to reports in other cell types (Vaughan-Jones, 1988). The presence of two acid-alleviating systems (Na+/H+ antiporter and an Na+, HCO3-dependent system) in the human cleavage stage embryo is similar to the presence of redundant pathways to alleviate pHi in other cell types (Vaughan-Jones, 1988
; Alper, 1991
, 1994
). Na+/H+ antiporter activity is important for the formation of the blastocoel cavity in the blastocyst (Barr et al., 1998
) and may also mediate cell volume regulation or ion homeostasis in addition to pHi regulation in the developing embryo.
Our data indicate that human cleavage-stage embryos have the ability to effectively maintain pHi within a range of ~7.07.3. We have found that the embryo's HCO3/Cl exchanger is activated when pHi rises above ~7.27.3, which therefore prevents pHi from increasing beyond this level. We also found that, in the presence of HCO3/CO2, there appear to be two mechanisms which will prevent pHi from becoming too low. Na+/H+ antiporter activity becomes activated below a threshold of ~6.8, while a second HCO3-dependent mechanism which may be an Na+, HCO3/Cl exchanger, is activated below 7.0 (Figure 3C). Together, these would effectively maintain pHi above 7.0. The hypothesis that pHi is maintained within a range of 7.07.3 by the concerted activities of these mechanisms is consistent with the baseline pHi of ~7.1 we measured for cleavage-stage human embryos under our conditions.
In the absence of HCO3/CO2, our data indicate that human embryos would be less able to regulate pHi. Since defence against alkalosis by HCO3/Cl exchanger requires intracellular HCO3, alkalosis could not be opposed in media without HCO3/CO2 where there would be little intracellular HCO3. Acidosis would still be opposed, but only below ~6.8 where the Na+/H+ antiporter is active; the second HCO3-dependent mechanism which maintains pHi above 7.0 would be inactive since it would require external HCO3. This has implications for the handling of human embryos in vitro, which are routinely manipulated in HEPES-buffered media with low HCO3 concentrations at atmospheric CO2. This would be predicted to impair the ability of embryos to maintain pHi since the low CO2 would result in very low intracellular HCO3 concentrations which would inhibit HCO3/Cl exchange, and low external HCO3 would slow any HCO3-dependent mechanism for alleviating acidosis. Thus, media containing sufficient HCO3 with appropriate CO2 tension (i.e. 25 mmol/l HCO3/5% CO2) would be preferable.
Dale et al. (1998) previously found that the baseline pHi of human eggs and zygotes was ~7.4 in HCO3/CO2-buffered medium. This value for eggs is different from the 7.07.1 which we found; the reason for this discrepancy could be due to differences in the media used by Dale et al. (1998) and those used here, but we can only speculate as to why the value for oocyte pHi obtained by Dale et al. was higher than that which we have measured here. Dale et al. probed the ability of cleavage-stage embryos to recover from an alkalosis produced by exposing them to pH 8.0 medium (Medium 199) buffered with HEPES at atmospheric CO2. Although the low CO2, and hence very low intracellular HCO3, might be expected to reduce HCO3/Cl exchanger activity, the amount of intracellular HCO3 remaining within the embryos under their conditions (apparently used soon after removal from CO2-buffered medium) was sufficient to permit HCO3/Cl exchanger activity. Using this method, recovery from alkalosis was detected at every stage of embryo development, which is consistent with our findings here. In contrast, Dale et al. (1998) did not detect an ability in human cleavage-stage embryos to recover from acidosis until the blastocyst stage, using a protocol in which pHi was reduced to ~7.0 by decreasing external pH to the same level, again in HEPES-buffered medium at atmospheric CO2. This is also consistent with our findings, which indicated that Na+-dependent recovery from acidosis in human embryos is fairly low until pHi falls below 6.9 and thus would not have been detected with their protocol. It is unclear if the media used by Dale et al. (1998) for pHi measurements contained HCO3. Low or absent external HCO3 may also have contributed to difficulty detecting recovery from acidosis. As we have shown here, recovery from acidosis was dependent on the presence of external HCO3/CO2 between pHi 6.8 and 7.1. We did not perform measurements on later-stage human embryos (morulae and blastocysts), and thus can only speculate that a change in transport kinetics occurs after blastocyst formation, allowing HCO3 independent recovery from mild acidosis in blastocysts.
We also examined pHi regulation in a few available human eggs. pHi of GV, MI and MII eggs was not significantly different from cleavage-stage embryos, with pHi ranging from 7.0 to 7.1. All egg stages examined exhibited intracellular alkalinization upon Cl removal, similar to cleavage-stage embryos. The rate of alkalinization in MI eggs appeared to be reduced by DIDS, which suggests that HCO3/Cl exchanger activity is also present in eggs. MII eggs exhibited a robust recovery from alkaline load, which contrasts with the recent finding that HCO3/Cl exchanger activity in the mouse and hamster is activated upon egg activation or fertilization, with eggs (MII) having very low or barely detectable HCO3/Cl exchanger activity (Lane et al., 1999b; Phillips and Baltz, 1999
). However, the data are consistent with the finding by Dale et al. (1998) that fresh MII eggs recovered from alkalosis and that recovery was inhibited by DIDS. In contrast, recovery from induced acidosis, measured in the presence of external HCO3, was barely detectable in MI eggs and did not appear to be Na+-dependent. MII eggs demonstrated low rates of recovery from acidosis that appeared Na+-dependent, although the recovery appeared to be much slower compared to the rates measured in cleavage-stage embryos. This is consistent with the recent finding that Na+/H+ antiporter activity is low in unfertilized hamster eggs (MII) and is subsequently activated upon fertilization (Lane et al., 1999a
). Thus, it appears that although human eggs may regulate pHi against alkalosis, they do not demonstrate robust pHi regulation in the acid range.
Robust pHi regulation may be particularly important for the developing cleavage-stage embryo, as the Fallopian tube has been reported to be quite alkaline in several mammalian species (rhesus: pH 7.7, Maas et al., 1977; rabbit: pH 7.9, Maas et al., 1987; rat: pH 8.08.2, Ben-Yosef et al.; 1996; mouse: pH 7.7, Y.Zhao, P.J-P.Chauvet and J.M.Baltz, unpublished data). Acid-alleviating systems may also be required by the growing embryo during this period of development to correct perturbations in pHi due to increased metabolism and the production of intracellular protons resulting from processes such as ATP hydrolysis. We have demonstrated that human cleavage-stage embryos correct deviations in steady-state pHi by HCO3/Cl exchanger activity, Na+/H+ antiporter activity and an Na+, HCO3-dependent system which together maintain embryo pHi between 7.0 and 7.3. Thus, cleavage-stage human embryos possess the ability to maintain pHi within a narrow physiological range, but this ability requires the presence of HCO3 and CO2.
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Acknowledgments |
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Notes |
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References |
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Submitted on August 9, 1999; accepted on December 21, 1999.