I~iC’:\‘ELOPMP:NTAl.
IIIOI.O(;Y
188,
421-429
(19%))
Apparent
Absence of Na+/H+ Antiport Activity in the Two-Cell Mouse Embryo
We have used the pII-sensitive dye BCECF to investigate the regulation of intracellular pII (pII,) by twcwcell stage mouw embryos in bicarbonate-free medium. There is no indication of a Nat/H+ antiport active in regulating pH,, as rwovcry from acid&loading was inscnsitivc to amiloridc, cth~lisopropylamiloridc, or the ahscncc of cxtraccllular Na’. Instead, protons appear to be in equilibrium across the plasma membrane, as indicated by the response of pH, to changes in external K’. The embryos have an intracellular buffering power in the normal range (25.3 mhl/pH): their apparrnt permeability to protons is, howrwr, very high (0.22 cm/src). I IWO Aradem,r FWSi. Inc INTRODIJCTION
A number of mechanisms exist that regulate intracellular pH (pHi) (for reviews, see Roos and Boron, 1981; Boron, 1987). In mammalian cells, there is a nearly ubiquitous plasma membrane Nat/II+ antiport which uses the Nat gradient to export protons, thus alleviating acid loads in the cell. The Nat/H+ antiport has been found in nearly every mammalian cell examined so far, with the exception of the anucleate red blood cell (Murer it crl., 1976; Boron, 1983; Pouyssegur, 1985; Frelin et uI., 1985; Moolenaar, 1986), and, as recently reported, the rabbit pulmonary macrophage (Bidani et uh, 1989). Mutant cells have also been produced which lack the Na’/H’ antiport (Sardet et (xl., 1989). Another common set of systems for the regulation of pHi are the bicarbonate/Clexchangers. A sodium-dependent form uses the energy in the Na’ gradient to import HCO.; and export Cl-, thus alleviating intracellular acid loads. A sodium-independent form uses the Cl gradient to export HCO:] and import Cl-, reducing intracellular alkaline loads (see Boron, 1987). We have investigated the regulation of pHi by the blastomeres of the preimplantation mouse embryo, using the pH-sensitive fluorophore BCECF. In this paper we examine the two-cell mouse embryo’s ability to regulate pHi in a medium free from bicarbonate, in order to examine in isolation any activity due to a presumptive Na’ /Hi antiport in these cells. MATERIALS
2’,7’-Bis(2-carboxyethyl)-5(and cein acetoxymethyl ester
i4ND
METHODS
6)-carboxy fluores(RCECF-AM) (Molecular
Probes, Eugene, OR; 1 mg/ml stock in dimethyl sulfoxide, st,ored at -20°C) was added to solutions for a final concentration of 1.22 pM. Nigericin (Sigma; 10 mg/ml stock in ethanol, ~20°C) was added to solutions for a final concentration of 10 fig/ml. Valinomycin (Sigma; 10 mg/ml stock in DMSO, 4°C) was used at a final concentration of 5 pg/ml. Amiloride HCl (Sigma) was dissolved directly in solutions for a final concentration of 1 m&L Ethylisopropylamiloride (EIPA), the generous gift of Dr. Dale Benos (50 mM stock in DMSO, 4”C), was used at a final concentration of 50 WM. Pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were purchased from Sigma (10X stocks in water, -20°C) and diluted just before use. All other chemicals were purchased from Sigma, except ethanol (Aaper Alcohol and Chem. Co., Shelbyville, KY).
The media used were modified forms of M2 mouse embryo culture medium (Fulton and Whittingham, 1978; Hogan et (xl., 1986) which is buffered with Hepes. The modifications included the omission of bicarbonate, phenol red, and BSA from all solutions. The basic modified M2 was designated bfM2, for bicarbonate-free M2. The compositions of the various media are given in Table 1. BCECF-AM and all drugs were added to the solutions immediately prior to each experiment. The IJH; calibration medium contained 100 mM KCl, 25 mM NaCl, 21 mM Hepes, and 75 mM sucrose. Four different pH solutions-6.50, 6.85, 7.15, and 7.50-were used. Nigericin (10 pg/ml) and valinomycin (5 pg/ml) were added to the calibration solutions immediately before each experiment for use at its conclusion.
422
DEVELOPMENTAL
BIOLOGY
VOL~JME
138, 1990
TABLE 1 COMPOSITION OF MEDIA Medium
Na+
K+
bfM2 Na’-free bfM2 NH&I-bfM2 High K’ bfM2 K+-free bfM2 WAF fbM2 WAF Na’-free bfM2 WAF NH&l bfM2 WAF high K’ bfM2
122 0 97.4 28.4 128 122 0 97.4 28.4
5.97 5.97 5.97 100 0 5.97 5.97 5.97 100
Mg”
Ca2+
NH;
cho’
cl-
PO:
so:
lac-
PYr-
HCO,
1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19
1.71 1.71 1.71 1.71 1.71 1.71 1.71 1.71 1.71
0 0 25.0 0 0 0 0 25.0 0
0 122 0 0 0 0 122 0 0
107 130 107 130 107 130 130 130 130
1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19
1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19
23.3 0 23.3 0 23.3 0 0 0 0
0.33 0 0.33 0.33 0.33 0 0 0 0
0 0 0 0 0 0 0 0 0
Note. Concentrations are given in mM (rounded off to three significant digits). All solutions mMKH,PO,, 1.19 mMMgS04, 23.3 mMNa-lactate, bfM2 was 98.8 mMNaCk4.78 mMKCl,1.19 CaCl,, 20.8 mMHepes. Solutions that were nominally Na+- or K’-free contained less than 1 and had 60 pg/ml penicillin G and 50 pg/ml streptomycin sulfate. Abbreviations: cho, choline; lac, acid free.
Animals
and Embryos
The embryos were obtained from matings of superovulated (5 IU PMSG followed 48 hr later by 5 IU hCG) CFl females with BDF males (Charles River, Canada). Two-cell embryos were collected 44-48 hr post hCG. pHi Measurements The embryos were loaded with a pH-sensitive fluorophore by exposing them to the nonfluorescent, membrane-permeant acetoxymethyl ester form of the dye (BCECF-AM; 1.22 PALM for 5-10 min). The ester groups are cleaved off intracellularly by endogenous esterases, trapping the fluorescent, membrane impermeant form, BCECF, in the cell (Thomas et al, 1979; Rink et al., 1982). The pHi of each blastomere is determined from the fluorescent emission of the dye as measured by a video image processor (Image 1, Interactive Video Systems, Concord, MA) operating on the image supplied by a double-intensified silicon target (ISIT) video camera (Dage-MTI, Inc., Michigan City, IN; gain, 3.0; 9.0 kV; and black level, 6.9-7.3) on a Zeiss IM35 inverted microscope. Custom software developed in the lab was used for the photometry which was done on 100 consecutive averaged frames. The intensity was measured within 10 x 10 pixel areas placed over each blastomere of each embryo. The ratio of the intensity of emission measured at 530 nm when excited at a pH-sensitive wavelength of the dye (500 nm) to that excited at a pH-insensitive wavelength (450 nm) varied linearly with pHi. We confirmed that the system had a linear response and that the ratio did not change with time or excitation intensity. Bleaching of the dye was negligible. This ratio was calibrated to pH by the nigericin/high K+ method (Thomas et al, 1979) using the four calibration solu-
glu
Hepes
5.56 5.56 5.56 5.56 5.56 5.56 5.56 5.56 5.56
20.8 20.8 20.8 20.8 20.8 20.8 20.8 20.8 20.8
were pH 7.27-7.34 at 37°C. The basic recipe for 0.33 mMNa-pyruvate, 5.56 mMglucose, 1.71 mM 0.2 mM, respectively, of these ions. All solutions lactate; pyr, pyruvate; glu, glucose; WAF, weak
tions, initially allowing 15 min for equilibration with the drug. We found it to be vital that a very low dye concentration and light exposure be used. Therefore, the extremely low light camera is essential. Higher dye levels, especially in conjunction with light, were toxic to the embryos; while two-cell embryos exposed to the dye/ light levels used in these experiments could be cultured to the blastocyst stage, those exposed to higher levels did not even undergo cleavage to the four-cell stage (data not shown). Each experiment consisted of a group of 10 randomly chosen embryos whose pHi was measured simultaneously (however, in a small number of experiments one or more embryos were lost-either washed away or burst-with the result that some groups had fewer than 10 embryos). The embryos were maintained at 3’7°C (kO.5”C) in a temperature-controlled chamber (Biophysica, Baltimore, MD) that had been modified to allow solution changes. Solutions were changed by flushing the 2.5 ml chamber with 20 ml of prewarmed solution. Acid Loading Embryos The embryos were acid-loaded by the NHICl pulse technique (Boron and DeWeer, 1976; Roos and Boron, 1981). Briefly, the NH: cation exists in equilibrium with NHs, the latter being much more permeant to cell membranes. Exposure to NH,Cl results in rapid equilibration of NH3 across the cell membrane, followed by a much slower entry of NH:. A marked intracellular acidification with respect to the original baseline is effected at the end of the NH,Cl pulse as protons carried in by the slow entry of NH,+ remain trapped when both NH, and NH: inside the cell exit as the more permeant
BAI,TZ,
BIGGERS,
AND LECHENE
Nu+/H+
Antipvrt
Absence
in
423
Embryos
Acid-Loading and Recovery
6.0 0
I
I
:
10
20
30
:
:
’ bfM2
40
I
:
50
: 60
70
TIME (min) FIG. 1. Acid-loading and recovery of two-cell embryos in bfM2. Ten two-cell embryos were acid-loaded by the NH,Cl pulse technique (pulse between arrows). The initial medium (before first arrow) was bfM2. Recovery occurred in bfM2 (after second arrow). Each point is the mean of pHi determinations of the 20 blastomeres of the 10 embryos, Error bars indicate one standard deviation. The curve, shown only for clarity, was fitted by cubic splines (one lit in each section, vertical lines added). The dotted line is the initial baseline (before manipulation of medium), fitted bg a least-squares linear regression.
NH3. The embryos were exposed for approximately min to 25 mMNH4C1 (NH,Cl bfM2, Table 1).
Baseline pH, of Two-Cell Mouse Embryos in Bicarbonate-Free Medium We measured the baseline pHi of two-cell embryos in bfM2 in 18 experiments (171 embryos). The baseline pHi of each blastomere was determined by averaging three consecutive measurements taken before any experimental manipulations of the medium. The baseline pHi of individual blastomeres measured in this way ranged from 6.60 to 7.40. The mean pHi was 6.90 (SD = 0.15). The mean pHi’s of the 18 individual experiments ranged from 6.68 to 7.22 (there was substantial interexperimental variation in the mean pHJ.
bfM2 Amiloride or EIPA Na’ -free bfM2 K--free bfM2 WAF bfM2
+ bfM2”
constant, 0.18 0.18 0.25 0.17 0.14
(0.11) (0.10) (0.09) (-) (0.06)
min
- [H+],)e-“‘,
(1)
where [H’]i is the intracellular proton concentration at any time during recovery, [H+], is that after recovery, [H’la is that just after acid loading, i.e., at the lowest value of pHi, and t is time elapsed since the beginning of recovery. The corresponding half-time of the recovery is given by t1p2= In 2/k.
(2)
Ten experiments were done with a total of 83 embryos in which the recovery took place in bfM2. The mean rate constant of the recoveries obtained in these experiments (Table 2) was 0.18 mini’ (SD = 0.11; mean R2 value for the fit = 0.92 with log of both sides of Eq. (1) taken before fit). The mean half-time was 6.0 min (SD = 4.2). Intracellular
Buflering Power
Buffering power is the ratio of total amount of H+ added divided by the actual resulting pH change and is an important parameter in assessingthe ability of a cell to minimize perturbations in internal acid-base balance. The rise in pHi seen immediately upon introduction of NH4C1 gives a measure of the intracellular buff-
TABLE 2 RA’PES AFTEK A(:rn-Loal,
RECOVERY h- (rate
[H’], = [H’], + ([H’],
15
RESULTS
Medium
Two-cell embryos were acid-loaded by the NHdClpulse method as described above. Figure 1 shows one such experiment with 10 embryos in which the medium both before and after the pulse was bfM2. The expected transient alkalinization in the presence of NH*Cl (between arrows) can be seen. The cells become acid-loaded when the NH4C1 solution is replaced with bfM2 (second arrow), The embryos indeed recover from this acidload. The recovery was empirically found to follow an exponential timecourse and can therefore be described by a single first-order rate constant, k, given by
i)
t1,2 (half-time, 6.0 4.7 3.3 4.1 5.2
(4.2) (2.0) (2.0) (--, (2.3)
min)
n
N
83 48 39 10 20
10 5 4 1 2
R” 0.92 0.98 0.97 0.99 0.99
(0.13) (0.02) (0.03) (--, (0)
h;ote. Data are given as the means (standard deviation between the means of individual experiments). 7~ is the number of embryos. N is the number of experiments. R” is for the Iit of the recovery data to Eq. (1). The mean half-times were calculated using Eq. (2) with the rate constants for each individual experiment and then taking the mean. Thus, the mean k and tip:! are not themselves related by Eq. (2). None of the rate constants are significantly different from the control (t test, P < 0.05). ’ The data for amiloride and EIPA were pooled. The mean rate constant for the two amiloride experiments was 0.13 (0.06) and that for the three EIPA experiments was 0.21 (0.11). These are not significantly different.
424
DEVELOPMENTAL
BIOLOGY
ering power of the cell (for a review, see Roos and Boron, 1981). At the beginning of an NH4CI pulse the very permeant NH3 quickly equilibrates across the membrane. Essentially all this NH3 that initially enters the cell combined with intracellular H+ to form NH:. Therefore, the total intracellular H+ that has combined with NH3 (i.e., has been subtracted from the solution), divided by the actual change in pHi gives the buffering power (fi), in units of mM/pH. Using the dissociation constant for the equilibrium of NH; with NH3 and H+ (1.86 X 10. ’ M) (Weast, 1986) at an external pH of 7.30 and an external NH,Cl concentration of 25.0 mM allows NH3 concentration and hence the buffering power to be found as fl = 4.98 X lo”{ (lO~“H”)/ApHi},
(3)
where pH; is the pHi immediately after NH&l addition, and A pHi is the change in pHi upon NH4C1 addition. The intracellular buffering power was determined in 15 experiments (134 embryos). p was found to be 25.3 mM/pH (SD = 8.5). In other words, near the normal intracellular pH, the addition of 1 mM total protons is required for a decrease of 0.04 in pHi. This buffering power is, however, an average of the values of p over the range A pHi, since p may be a function of pH. The intrinsic buffering powers of a number of cells have been tabulated (Roos and Boron, 1981) and fall in the range 9-118 mM/pH; for comparison, Xenops embryo blastomere buffering is reported to be 18 mM/pH (Turin and Warner, 1980). Is pH, Recovery Mediated by Na+/H+ Antiport Activity? If the recovery of two-cell mouse embryos from acidloading is mediated by Nat/H+ antiport activity, it should be abolished by either (1) the lack of external
a
t No+-free 6.0-1 0
: 10
: 20
:
: 30
:
: 40
: . 50
VOLUME
138. 1990
Na+ or (2) the Na+/H+ antiport inhibitor amiloride, or its more potent and specific derivative ethylisopropylamiloride (EIPA) (see Benos, 1988, for a review of amiloride and its derivatives). Four experiments (39 embryos) were performed in which the NH4Cl solution was flushed with Na+-free bfM2 (choline replaced Nat, see Table 1). One of these is shown in Fig. 2a. pHi was still found to recover, even in the complete absence of Naf. The mean rate constant (Table 2) was 0.25 mini’ (SD = 0.09, mean R2 of fits = 0.97), corresponding to a half-time of 3.3 min (SD = 2.0), which is not significantly different from the control recoveries (t test, P < 0.05). Five experiments (48 embryos) were performed with either amiloride (1 mM) or EIPA (50 PM) present during recovery (Fig. 2b). These concentrations are more than sufficient to inhibit Na+/H+ antiport activity. Again, no effect on the recovery was observed: The mean rate constant (Table 2) was 0.18 (SD = 0.10, mean R2 of fits = 0.98), identical to that seen in the absence of these Na+/H’ antiport inhibitors. In addition, to eliminate interexperimental variations, three of the above experiments were performed (one with Na+-free recovery and two with amiloride recoveries) where first embryos were acid-loaded and the recovery rate was measured in bfM2, and then the same embryos were acid-loaded again and the recovery was measured either in Nat-free bfM2 or with amiloride (1 mM) present. In one experiment, k was 0.26 in bfM2 and subsequently 0.29 in Na+-free bfM2. This also indicates that the somewhat faster rates of recovery seen in Na+-free medium above were due to variation between experiments and not to any real effect of the medium. Similarly, in the two amiloride experiments, k was 0.10 and 0.13 in bfM2 and then 0.09 and 0.17, respectively, in the presence of amiloride. Therefore, it appears that the recovery of pHi after
6.0-
b
bfM2 :
: 60
t EIPA + bfM2 : 70
TIME (min) FIG. 2. Recovery from acid-loading under conditions which inhibit bfM2. Initial medium was bfM2; pulse was NH,CI bfM2. (b) Recovery In each case, recovery occurred even though any Na+/H+ antiport curve.
6.01 0
I 10
:
: 20
:
: 30
:
I 40
:
I 50
:
I 60
:
: 70
TIME (min) Na’/H’ antiport activity. (a) Recovery of 10 two-cell embryos in Na’-free in bfM2 with EIPA (50 PM). Initial and pulse media were the same as in a. activity would have been inhibited. See legend of Fig. 1 for explanation of
The Eflect
1
No+-free
0
10
20
40
50
60
70
TIME (min) FIG:. 3. Effect of replacing bfM2 with Na’-free bfM2. Ten two-cell embryos were initially in bfM2 (122 mM Na’). At the arrow, the medium was replaced by Nat-free bfM2 (~1 mM Na’). No marked change occurs. Note that in this experiment, the ratio was not calibrated to pH, and, thus, the data are reported as ratios. See legend of Fig. 1 for explanation of curve.
acid-loading is unaffected by the presence of amiloride or EIPA or the absence of Na+, indicating that a Na’/H+ antiport does not participate in recovery of two-cell mouse embryos from an acid load.
The &?iect cf External
Na’ on Steady-State
Ki
pH
IIIOI.O(;Y
188,
421-429
(19%))
Apparent
Absence of Na+/H+ Antiport Activity in the Two-Cell Mouse Embryo
We have used the pII-sensitive dye BCECF to investigate the regulation of intracellular pII (pII,) by twcwcell stage mouw embryos in bicarbonate-free medium. There is no indication of a Nat/H+ antiport active in regulating pH,, as rwovcry from acid&loading was inscnsitivc to amiloridc, cth~lisopropylamiloridc, or the ahscncc of cxtraccllular Na’. Instead, protons appear to be in equilibrium across the plasma membrane, as indicated by the response of pH, to changes in external K’. The embryos have an intracellular buffering power in the normal range (25.3 mhl/pH): their apparrnt permeability to protons is, howrwr, very high (0.22 cm/src). I IWO Aradem,r FWSi. Inc INTRODIJCTION
A number of mechanisms exist that regulate intracellular pH (pHi) (for reviews, see Roos and Boron, 1981; Boron, 1987). In mammalian cells, there is a nearly ubiquitous plasma membrane Nat/II+ antiport which uses the Nat gradient to export protons, thus alleviating acid loads in the cell. The Nat/H+ antiport has been found in nearly every mammalian cell examined so far, with the exception of the anucleate red blood cell (Murer it crl., 1976; Boron, 1983; Pouyssegur, 1985; Frelin et uI., 1985; Moolenaar, 1986), and, as recently reported, the rabbit pulmonary macrophage (Bidani et uh, 1989). Mutant cells have also been produced which lack the Na’/H’ antiport (Sardet et (xl., 1989). Another common set of systems for the regulation of pHi are the bicarbonate/Clexchangers. A sodium-dependent form uses the energy in the Na’ gradient to import HCO.; and export Cl-, thus alleviating intracellular acid loads. A sodium-independent form uses the Cl gradient to export HCO:] and import Cl-, reducing intracellular alkaline loads (see Boron, 1987). We have investigated the regulation of pHi by the blastomeres of the preimplantation mouse embryo, using the pH-sensitive fluorophore BCECF. In this paper we examine the two-cell mouse embryo’s ability to regulate pHi in a medium free from bicarbonate, in order to examine in isolation any activity due to a presumptive Na’ /Hi antiport in these cells. MATERIALS
2’,7’-Bis(2-carboxyethyl)-5(and cein acetoxymethyl ester
i4ND
METHODS
6)-carboxy fluores(RCECF-AM) (Molecular
Probes, Eugene, OR; 1 mg/ml stock in dimethyl sulfoxide, st,ored at -20°C) was added to solutions for a final concentration of 1.22 pM. Nigericin (Sigma; 10 mg/ml stock in ethanol, ~20°C) was added to solutions for a final concentration of 10 fig/ml. Valinomycin (Sigma; 10 mg/ml stock in DMSO, 4°C) was used at a final concentration of 5 pg/ml. Amiloride HCl (Sigma) was dissolved directly in solutions for a final concentration of 1 m&L Ethylisopropylamiloride (EIPA), the generous gift of Dr. Dale Benos (50 mM stock in DMSO, 4”C), was used at a final concentration of 50 WM. Pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were purchased from Sigma (10X stocks in water, -20°C) and diluted just before use. All other chemicals were purchased from Sigma, except ethanol (Aaper Alcohol and Chem. Co., Shelbyville, KY).
The media used were modified forms of M2 mouse embryo culture medium (Fulton and Whittingham, 1978; Hogan et (xl., 1986) which is buffered with Hepes. The modifications included the omission of bicarbonate, phenol red, and BSA from all solutions. The basic modified M2 was designated bfM2, for bicarbonate-free M2. The compositions of the various media are given in Table 1. BCECF-AM and all drugs were added to the solutions immediately prior to each experiment. The IJH; calibration medium contained 100 mM KCl, 25 mM NaCl, 21 mM Hepes, and 75 mM sucrose. Four different pH solutions-6.50, 6.85, 7.15, and 7.50-were used. Nigericin (10 pg/ml) and valinomycin (5 pg/ml) were added to the calibration solutions immediately before each experiment for use at its conclusion.
422
DEVELOPMENTAL
BIOLOGY
VOL~JME
138, 1990
TABLE 1 COMPOSITION OF MEDIA Medium
Na+
K+
bfM2 Na’-free bfM2 NH&I-bfM2 High K’ bfM2 K+-free bfM2 WAF fbM2 WAF Na’-free bfM2 WAF NH&l bfM2 WAF high K’ bfM2
122 0 97.4 28.4 128 122 0 97.4 28.4
5.97 5.97 5.97 100 0 5.97 5.97 5.97 100
Mg”
Ca2+
NH;
cho’
cl-
PO:
so:
lac-
PYr-
HCO,
1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19
1.71 1.71 1.71 1.71 1.71 1.71 1.71 1.71 1.71
0 0 25.0 0 0 0 0 25.0 0
0 122 0 0 0 0 122 0 0
107 130 107 130 107 130 130 130 130
1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19
1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19 1.19
23.3 0 23.3 0 23.3 0 0 0 0
0.33 0 0.33 0.33 0.33 0 0 0 0
0 0 0 0 0 0 0 0 0
Note. Concentrations are given in mM (rounded off to three significant digits). All solutions mMKH,PO,, 1.19 mMMgS04, 23.3 mMNa-lactate, bfM2 was 98.8 mMNaCk4.78 mMKCl,1.19 CaCl,, 20.8 mMHepes. Solutions that were nominally Na+- or K’-free contained less than 1 and had 60 pg/ml penicillin G and 50 pg/ml streptomycin sulfate. Abbreviations: cho, choline; lac, acid free.
Animals
and Embryos
The embryos were obtained from matings of superovulated (5 IU PMSG followed 48 hr later by 5 IU hCG) CFl females with BDF males (Charles River, Canada). Two-cell embryos were collected 44-48 hr post hCG. pHi Measurements The embryos were loaded with a pH-sensitive fluorophore by exposing them to the nonfluorescent, membrane-permeant acetoxymethyl ester form of the dye (BCECF-AM; 1.22 PALM for 5-10 min). The ester groups are cleaved off intracellularly by endogenous esterases, trapping the fluorescent, membrane impermeant form, BCECF, in the cell (Thomas et al, 1979; Rink et al., 1982). The pHi of each blastomere is determined from the fluorescent emission of the dye as measured by a video image processor (Image 1, Interactive Video Systems, Concord, MA) operating on the image supplied by a double-intensified silicon target (ISIT) video camera (Dage-MTI, Inc., Michigan City, IN; gain, 3.0; 9.0 kV; and black level, 6.9-7.3) on a Zeiss IM35 inverted microscope. Custom software developed in the lab was used for the photometry which was done on 100 consecutive averaged frames. The intensity was measured within 10 x 10 pixel areas placed over each blastomere of each embryo. The ratio of the intensity of emission measured at 530 nm when excited at a pH-sensitive wavelength of the dye (500 nm) to that excited at a pH-insensitive wavelength (450 nm) varied linearly with pHi. We confirmed that the system had a linear response and that the ratio did not change with time or excitation intensity. Bleaching of the dye was negligible. This ratio was calibrated to pH by the nigericin/high K+ method (Thomas et al, 1979) using the four calibration solu-
glu
Hepes
5.56 5.56 5.56 5.56 5.56 5.56 5.56 5.56 5.56
20.8 20.8 20.8 20.8 20.8 20.8 20.8 20.8 20.8
were pH 7.27-7.34 at 37°C. The basic recipe for 0.33 mMNa-pyruvate, 5.56 mMglucose, 1.71 mM 0.2 mM, respectively, of these ions. All solutions lactate; pyr, pyruvate; glu, glucose; WAF, weak
tions, initially allowing 15 min for equilibration with the drug. We found it to be vital that a very low dye concentration and light exposure be used. Therefore, the extremely low light camera is essential. Higher dye levels, especially in conjunction with light, were toxic to the embryos; while two-cell embryos exposed to the dye/ light levels used in these experiments could be cultured to the blastocyst stage, those exposed to higher levels did not even undergo cleavage to the four-cell stage (data not shown). Each experiment consisted of a group of 10 randomly chosen embryos whose pHi was measured simultaneously (however, in a small number of experiments one or more embryos were lost-either washed away or burst-with the result that some groups had fewer than 10 embryos). The embryos were maintained at 3’7°C (kO.5”C) in a temperature-controlled chamber (Biophysica, Baltimore, MD) that had been modified to allow solution changes. Solutions were changed by flushing the 2.5 ml chamber with 20 ml of prewarmed solution. Acid Loading Embryos The embryos were acid-loaded by the NHICl pulse technique (Boron and DeWeer, 1976; Roos and Boron, 1981). Briefly, the NH: cation exists in equilibrium with NHs, the latter being much more permeant to cell membranes. Exposure to NH,Cl results in rapid equilibration of NH3 across the cell membrane, followed by a much slower entry of NH:. A marked intracellular acidification with respect to the original baseline is effected at the end of the NH,Cl pulse as protons carried in by the slow entry of NH,+ remain trapped when both NH, and NH: inside the cell exit as the more permeant
BAI,TZ,
BIGGERS,
AND LECHENE
Nu+/H+
Antipvrt
Absence
in
423
Embryos
Acid-Loading and Recovery
6.0 0
I
I
:
10
20
30
:
:
’ bfM2
40
I
:
50
: 60
70
TIME (min) FIG. 1. Acid-loading and recovery of two-cell embryos in bfM2. Ten two-cell embryos were acid-loaded by the NH,Cl pulse technique (pulse between arrows). The initial medium (before first arrow) was bfM2. Recovery occurred in bfM2 (after second arrow). Each point is the mean of pHi determinations of the 20 blastomeres of the 10 embryos, Error bars indicate one standard deviation. The curve, shown only for clarity, was fitted by cubic splines (one lit in each section, vertical lines added). The dotted line is the initial baseline (before manipulation of medium), fitted bg a least-squares linear regression.
NH3. The embryos were exposed for approximately min to 25 mMNH4C1 (NH,Cl bfM2, Table 1).
Baseline pH, of Two-Cell Mouse Embryos in Bicarbonate-Free Medium We measured the baseline pHi of two-cell embryos in bfM2 in 18 experiments (171 embryos). The baseline pHi of each blastomere was determined by averaging three consecutive measurements taken before any experimental manipulations of the medium. The baseline pHi of individual blastomeres measured in this way ranged from 6.60 to 7.40. The mean pHi was 6.90 (SD = 0.15). The mean pHi’s of the 18 individual experiments ranged from 6.68 to 7.22 (there was substantial interexperimental variation in the mean pHJ.
bfM2 Amiloride or EIPA Na’ -free bfM2 K--free bfM2 WAF bfM2
+ bfM2”
constant, 0.18 0.18 0.25 0.17 0.14
(0.11) (0.10) (0.09) (-) (0.06)
min
- [H+],)e-“‘,
(1)
where [H’]i is the intracellular proton concentration at any time during recovery, [H+], is that after recovery, [H’la is that just after acid loading, i.e., at the lowest value of pHi, and t is time elapsed since the beginning of recovery. The corresponding half-time of the recovery is given by t1p2= In 2/k.
(2)
Ten experiments were done with a total of 83 embryos in which the recovery took place in bfM2. The mean rate constant of the recoveries obtained in these experiments (Table 2) was 0.18 mini’ (SD = 0.11; mean R2 value for the fit = 0.92 with log of both sides of Eq. (1) taken before fit). The mean half-time was 6.0 min (SD = 4.2). Intracellular
Buflering Power
Buffering power is the ratio of total amount of H+ added divided by the actual resulting pH change and is an important parameter in assessingthe ability of a cell to minimize perturbations in internal acid-base balance. The rise in pHi seen immediately upon introduction of NH4C1 gives a measure of the intracellular buff-
TABLE 2 RA’PES AFTEK A(:rn-Loal,
RECOVERY h- (rate
[H’], = [H’], + ([H’],
15
RESULTS
Medium
Two-cell embryos were acid-loaded by the NHdClpulse method as described above. Figure 1 shows one such experiment with 10 embryos in which the medium both before and after the pulse was bfM2. The expected transient alkalinization in the presence of NH*Cl (between arrows) can be seen. The cells become acid-loaded when the NH4C1 solution is replaced with bfM2 (second arrow), The embryos indeed recover from this acidload. The recovery was empirically found to follow an exponential timecourse and can therefore be described by a single first-order rate constant, k, given by
i)
t1,2 (half-time, 6.0 4.7 3.3 4.1 5.2
(4.2) (2.0) (2.0) (--, (2.3)
min)
n
N
83 48 39 10 20
10 5 4 1 2
R” 0.92 0.98 0.97 0.99 0.99
(0.13) (0.02) (0.03) (--, (0)
h;ote. Data are given as the means (standard deviation between the means of individual experiments). 7~ is the number of embryos. N is the number of experiments. R” is for the Iit of the recovery data to Eq. (1). The mean half-times were calculated using Eq. (2) with the rate constants for each individual experiment and then taking the mean. Thus, the mean k and tip:! are not themselves related by Eq. (2). None of the rate constants are significantly different from the control (t test, P < 0.05). ’ The data for amiloride and EIPA were pooled. The mean rate constant for the two amiloride experiments was 0.13 (0.06) and that for the three EIPA experiments was 0.21 (0.11). These are not significantly different.
424
DEVELOPMENTAL
BIOLOGY
ering power of the cell (for a review, see Roos and Boron, 1981). At the beginning of an NH4CI pulse the very permeant NH3 quickly equilibrates across the membrane. Essentially all this NH3 that initially enters the cell combined with intracellular H+ to form NH:. Therefore, the total intracellular H+ that has combined with NH3 (i.e., has been subtracted from the solution), divided by the actual change in pHi gives the buffering power (fi), in units of mM/pH. Using the dissociation constant for the equilibrium of NH; with NH3 and H+ (1.86 X 10. ’ M) (Weast, 1986) at an external pH of 7.30 and an external NH,Cl concentration of 25.0 mM allows NH3 concentration and hence the buffering power to be found as fl = 4.98 X lo”{ (lO~“H”)/ApHi},
(3)
where pH; is the pHi immediately after NH&l addition, and A pHi is the change in pHi upon NH4C1 addition. The intracellular buffering power was determined in 15 experiments (134 embryos). p was found to be 25.3 mM/pH (SD = 8.5). In other words, near the normal intracellular pH, the addition of 1 mM total protons is required for a decrease of 0.04 in pHi. This buffering power is, however, an average of the values of p over the range A pHi, since p may be a function of pH. The intrinsic buffering powers of a number of cells have been tabulated (Roos and Boron, 1981) and fall in the range 9-118 mM/pH; for comparison, Xenops embryo blastomere buffering is reported to be 18 mM/pH (Turin and Warner, 1980). Is pH, Recovery Mediated by Na+/H+ Antiport Activity? If the recovery of two-cell mouse embryos from acidloading is mediated by Nat/H+ antiport activity, it should be abolished by either (1) the lack of external
a
t No+-free 6.0-1 0
: 10
: 20
:
: 30
:
: 40
: . 50
VOLUME
138. 1990
Na+ or (2) the Na+/H+ antiport inhibitor amiloride, or its more potent and specific derivative ethylisopropylamiloride (EIPA) (see Benos, 1988, for a review of amiloride and its derivatives). Four experiments (39 embryos) were performed in which the NH4Cl solution was flushed with Na+-free bfM2 (choline replaced Nat, see Table 1). One of these is shown in Fig. 2a. pHi was still found to recover, even in the complete absence of Naf. The mean rate constant (Table 2) was 0.25 mini’ (SD = 0.09, mean R2 of fits = 0.97), corresponding to a half-time of 3.3 min (SD = 2.0), which is not significantly different from the control recoveries (t test, P < 0.05). Five experiments (48 embryos) were performed with either amiloride (1 mM) or EIPA (50 PM) present during recovery (Fig. 2b). These concentrations are more than sufficient to inhibit Na+/H+ antiport activity. Again, no effect on the recovery was observed: The mean rate constant (Table 2) was 0.18 (SD = 0.10, mean R2 of fits = 0.98), identical to that seen in the absence of these Na+/H’ antiport inhibitors. In addition, to eliminate interexperimental variations, three of the above experiments were performed (one with Na+-free recovery and two with amiloride recoveries) where first embryos were acid-loaded and the recovery rate was measured in bfM2, and then the same embryos were acid-loaded again and the recovery was measured either in Nat-free bfM2 or with amiloride (1 mM) present. In one experiment, k was 0.26 in bfM2 and subsequently 0.29 in Na+-free bfM2. This also indicates that the somewhat faster rates of recovery seen in Na+-free medium above were due to variation between experiments and not to any real effect of the medium. Similarly, in the two amiloride experiments, k was 0.10 and 0.13 in bfM2 and then 0.09 and 0.17, respectively, in the presence of amiloride. Therefore, it appears that the recovery of pHi after
6.0-
b
bfM2 :
: 60
t EIPA + bfM2 : 70
TIME (min) FIG. 2. Recovery from acid-loading under conditions which inhibit bfM2. Initial medium was bfM2; pulse was NH,CI bfM2. (b) Recovery In each case, recovery occurred even though any Na+/H+ antiport curve.
6.01 0
I 10
:
: 20
:
: 30
:
I 40
:
I 50
:
I 60
:
: 70
TIME (min) Na’/H’ antiport activity. (a) Recovery of 10 two-cell embryos in Na’-free in bfM2 with EIPA (50 PM). Initial and pulse media were the same as in a. activity would have been inhibited. See legend of Fig. 1 for explanation of
The Eflect
1
No+-free
0
10
20
40
50
60
70
TIME (min) FIG:. 3. Effect of replacing bfM2 with Na’-free bfM2. Ten two-cell embryos were initially in bfM2 (122 mM Na’). At the arrow, the medium was replaced by Nat-free bfM2 (~1 mM Na’). No marked change occurs. Note that in this experiment, the ratio was not calibrated to pH, and, thus, the data are reported as ratios. See legend of Fig. 1 for explanation of curve.
acid-loading is unaffected by the presence of amiloride or EIPA or the absence of Na+, indicating that a Na’/H+ antiport does not participate in recovery of two-cell mouse embryos from an acid load.
The &?iect cf External
Na’ on Steady-State
Ki
pH
