(Received for publication, May 4, 1995; and in revised form, July 27, 1995)
From the
Bicarbonate/chloride
(HCO/Cl
) exchangers
regulate intracellular pH in the alkaline range. Previously, it has
been shown that mouse embryos at the two-cell stage exhibit this
activity, but that the otherwise ubiquitous mechanisms for regulating
intracellular pH in the acid-to-neutral range are undetectable. We have
examined mouse embryos during preimplantation development (one-cell
zygote through blastocyst) to determine whether
HCO
/Cl
exchange
activity exists at all stages, whether it is necessary for
preimplantation development, and whether messenger RNAs from the known
HCO
/Cl
exchanger genes
are expressed. We have found that all stages of preimplantation embryo
have detectable HCO
/Cl
exchange activity. In addition, inhibition of this activity with
the stilbene anion exchange inhibitor DIDS
(4,4`-diisothiocyanostilbene-2,2`-disulfonic acid) disrupts
intracellular pH homeostasis and markedly inhibits embryo development
from the two-cell stage to blastocysts in culture under conditions of
moderately high external pH. Finally, mRNA encoding two members of the
band 3-related AE anion exchanger gene family are expressed in
preimplantation embryos.
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.
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%.
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.
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.
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.
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.
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 pH
relative 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).
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).
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).
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.
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.