Department of Physiology, The University of Western Ontario, London, Ontario N6A 5C1
The sodium/potassium pump, Na+,K+-ATPase, is generally understood to function as a heterodimer of two subunits, a catalytic subunit and a
noncatalytic, glycosylated
subunit. Recently, a putative third subunit, the
subunit, was cloned. This small protein (6.5 kD) coimmunoprecipitates with the
and
subunits and is closely associated with the ouabain
binding site on the holoenzyme, but its function is unknown. We have investigated the expression of the
subunit in preimplantation mouse development, where
Na+,K+-ATPase plays a critical role as the driving
force for blastocoel formation (cavitation). Using reverse transcriptase-polymerase chain reaction, we demonstrated that the
subunit mRNA accumulates continuously from the eight-cell stage onward and that it
cosediments with polyribosomes from its time of first
appearance. Confocal immunofluorescence microscopy
revealed that the
subunit itself accumulates and is localized at the blastomere surfaces up to the blastocyst
stage. In contrast with the
and
subunits, the
subunit is not concentrated in the basolateral surface of the polarized trophectoderm layer, but is strongly expressed at the apical surface as well. When embryos
were treated with antisense oligodeoxynucleotide complementary to the
subunit mRNA, ouabain-sensitive K+ transport (as indicated by 86Rb+ uptake) was reduced and cavitation delayed. However, Na+,K+-ATPase enzymatic activity was unaffected as determined by
a direct phosphorylation assay ("back door" phosphorylation) applied to plasma membrane preparations.
These results indicate that the
subunit, although not
an integral component of Na+,K+-ATPase, is an important determinant of active cation transport and that, as
such, its embryonic expression is essential for blastocoel formation in the mouse.
NA+,K+-ATPASE is a plasma membrane enzyme
that uses the energy from hydrolysis of ATP to
transport Na+ and K+ in opposite directions
across the membrane and against their electrochemical
gradients. This enzyme, also known as the sodium pump, is
required for normal functioning of all animal cells where the ion gradients maintained by the enzyme are used for
diverse functions such as regulation of cell volume and pH,
secondary active transport, and excitability (Jørgensen,
1986 A third putative subunit of Na+,K+-ATPase, the The preimplantation mouse embryo offers a unique opportunity to explore the function of the Embryo Collection and Subcellular Fractionation
Random-bred CF-1 females (Charles River Canada Ltd., St. Constant,
Québec or Harlan Sprague-Dawley, Indianapolis, IN) were superovulated
with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG)1 (both purchased from Sigma Chemical Co., St. Louis, MO;
5-10 IU each, separated by 46 to 48 h) and mated with CB6F1/J males (The Jackson Laboratory, Bar Harbor, ME). Embryos were flushed from
the reproductive tract on days 1-4 as described previously (MacPhee et
al., 1994 For fractionation into subribosomal and polyribosomal RNP fractions,
300-500 embryos were immediately lysed with detergent solution and subjected to differential centrifugation as described previously (De Sousa et
al., 1993 RNA Isolation and Reverse Trancriptase (RT)-PCR
RNA was isolated from embryo lysates and from embryo RNP fractions
by pelleting through CsCl, and the RNA reverse-transcribed, as described
previously (De Sousa et al., 1993 To confirm that Wholemount Immunofluorescence Microscopy
Embryos were fixed in a 2:1 dilution of methanol in PHEM buffer (60 mM
Pipes, 25 mM Hepes, 10 mM EGTA, 1 mM MgCl2, pH 6.9) for 2-3 min
followed by fixation in 100% methanol for an additional 3 min. They were
then washed three times in PHEM buffer followed by a final treatment for
45 min with blocking solution (0.01% Triton X-100, 0.1 M lysine, 1% goat
serum in PHEM). After washing in PHEM for 5 min, embryos were
treated overnight at 4°C with a 1:100 dilution of a rabbit antibody (Ab-G17; Mercer et al., 1993 Antisense Treatments
The antisense phosphorothioate oligodeoxynucleotide (ODN) used in this
study was designed using OligoTM software to be complementary to 15 bases including and downstream of the initiation codon of the mouse Ouabain-sensitive 86Rb Uptake Assay
Embryos treated with antisense or nonsense ODN were removed from
culture at 100 h post-hCG and transferred to a medium consisting of
KSOM with K+ salts replaced with equimolar NaCl, and BSA replaced
with 1.5 mg/ml polyvinylpyrrolidone. They were cultured in this medium with or without 1.0 mM ouabain (Sigma Chemical Co.), for 15 min before
the addition of 86RbCl (0.5-1.5 mCi/mg; Amersham Canada, Oakville,
Ontario). Embryos were assayed for 86Rb+ uptake as described previously
(Van Winkle and Campione, 1991 Membrane Isolation and "Back Door"
Phosphorylation Assay
Antisense- and nonsense-treated embryos were removed from culture at
100 h post-hCG, washed five times through PBS-PVP, and stored frozen
at Membranes were phosphorylated by the method of Resh (1982) Electrophoretic analysis of phosphorylated plasma membrane proteins
was carried out using acidic polyacrylamide gels as described by Blackshear (1984) The Our first step was to confirm that the
The identity of the 134-bp amplicon was confirmed by
direct sequencing. As shown in Fig. 2, the amplicon from
blastocyst cDNA showed 97% sequence identity with the
corresponding segment of the published sequence cloned
from mouse kidney cDNA (Mercer et al., 1993
The presence of
The developmental profile of the
Antisense Disruption of As a test of the hypothesis that embryonic synthesis of the
Results of one experiment, performed in triplicate, are
plotted in Fig. 5. Antisense treatment caused a delay in
cavitation, an effect that was apparent by 98 h post-hCG.
The effect was still evident at 100 h, when the number of
cavitating embryos in the antisense group of this experiment was only ~50% of that in the groups treated with
nonsense ODN or lysolecithin alone ("control"). Those
embryos that had not started to cavitate at 100 h in the antisense treated group appeared otherwise normal when
viewed by phase contrast microscopy. In the course of nine
experiments, a delay in cavitation was consistently seen in
the antisense groups although the quantitative effect varied, with the frequency of cavitation in antisense groups
ranging between 45 and 73% of that in nonsense groups at
the 100 h time point (mean = 62%). The effect was not
sustained, however, because by 108 h post-hCG, virtually all of the embryos in all three treatment groups of each experiment were cavitating and there was no longer any difference between groups. In two experiments, some embryos were removed from culture at 80 h post-hCG and
RNA was isolated for semi-quantitative RT-PCR. The
amount of
Confocal immunofluorescence microscopy of embryos
taken from the treatment groups at 100 h post-hCG demonstrated that the antisense treatment had caused an obvious reduction in the amount of
The importance of trophectodermal Na+,K+-ATPase
for cavitation is assumed to reflect its ability to pump Na+
into the blastocoel in exchange for K+, which is pumped
into the trophectoderm cells. Previously, sodium pump activity in preimplantation embryos has been measured by monitoring ouabain-sensitive 86Rb+ uptake, Rb+ acting as
a surrogate for K+ (Van Winkle and Campione, 1991 Table I.
Effect of Antisense Treatment on 86Rb+ Uptake
; Fambrough et al., 1987
). Na+,K+-ATPase is generally considered to consist of two obligatory subunits: a catalytic
subunit and a noncatalytic, glycosylated
subunit (for review see Mercer, 1993
). In addition to ATP binding
and phosphorylation sites, the
subunit also bears a site
that binds the cardiac glycoside ouabain, a specific inhibitor of sodium pump activity. Although the
subunit lacks
catalytic activity, it is nonetheless required for production
of functional holoenzyme. Mammals have at least four isoforms of the
subunit and three of the
subunit, all encoded by separate genes (Mercer et al., 1986
; Kent et al.,
1987
; Martin-Vasallo et al., 1989
; Malo et al., 1990
; Shamraj and Lingrel, 1994
; Malik et al., 1996
).
subunit, was cloned more recently (Mercer et al., 1993
;
Béguin et al., 1997
). This small polypeptide (predicted
mass = 6.5 kD) copurifies and coimmunoprecipitates with
the
and
subunits and is closely associated with the ouabain binding site of the holoenzyme. It is a type I transmembrane protein (its NH2 terminus is extracellular) whose association with
/
heterodimers influences the
K+ activation of the enzyme (Béguin et al., 1997
). Its physiological function, however, is unknown. Experiments involving heterologous expression of combinations of
,
,
and
subunits in yeast failed to reveal any role of the
subunit in ouabain binding, enzymatic activity, or ion
transport (Scheiner-Bobis and Farley, 1994
). Yeasts do
not contain an endogenous Na+,K+-ATPase, however, so
it remains possible that the function of the
subunit will
only be revealed through experimentation with animal
cells. For example, the
subunit might be required for physiological regulation of sodium pump activity or for
polarized deployment of sodium pumps in epithelia.
subunit. After
approximately five cleavage divisions, the outer cells of
the embryo become specialized as a transporting epithelium, the trophectoderm (for review see Wiley et al.,
1990
). Na+,K+-ATPase becomes concentrated in the basolateral membranes of the trophectoderm where it takes on
a morphogenetic role: it drives fluid transport across the
cell layer to form the blastocoel (Watson and Kidder,
1988
; MacPhee et al., 1997
). This process, called cavitation, leads to the development of a blastocyst capable of initiating implantation. Cavitation is sensitive to a variety of perturbations affecting Na+,K+-ATPase, such as those
that interfere with the localization of the enzyme in the basolateral membranes (Watson et al., 1990a
) or those that
interfere with sodium pump activity, as in the case of ouabain treatment (Dizio and Tasca, 1977; Manejwala et al., 1989
; MacPhee et al., 1997
). Furthermore, cavitation requires expression of embryonic genes, and is accompanied
by de novo synthesis of the
and
subunits of the enzyme
(Kidder and McLachlin, 1985
; Watson et al., 1990b
; MacPhee
et al., 1994
, 1997
; Khidhir et al., 1995
). We reasoned that if
the putative
subunit is a determinant of Na+,K+-ATPase
function, then perturbing the embryonic synthesis of
subunits should have a noticeable effect on blastocyst development. Here we show that this is indeed the case. Our
results provide strong support that
subunit has an important influence on active cation transport in mammalian
cells.
Materials and Methods
). Embryos were washed five times through Ca2+- and Mg2+-free
PBS containing 3 mg/ml polyvinylpyrrolidone (PBS-PVP) before further
processing. The timing of embryo collection was as follows: oocytes, 18 h
post-hCG; two-cell, 48 h; four-cell, 60 h; eight-cell, 65 h; morula, 80 h;
early blastocyst, 90 h.
; MacPhee et al., 1994
). The resulting RNP fractions were used for
RNA isolation.
; MacPhee et al., 1994
; Davies et al.,
1996
). The absence of genomic DNA from each cDNA preparation was
confirmed by PCR using primers that amplify an intron of the
-actin gene
(De Sousa et al., 1993
). The
subunit upstream primer was 5
-CCCTTCGAGTACGACTATGA-3
and the downstream primer was 5
-TTGACCTGCCTATGTTTCTT-3
. These primers were designed using OligoTM
Primer Analysis Software (National Biosciences, Plymouth, MN) to amplify a 134-bp fragment of the mouse cDNA (Mercer et al., 1993
). A standard amplification cycle consisted of denaturation at 94°C for 45 s, annealing at 54°C for 40 s, and extension at 72°C for 45 s. For a profile of
subunit presence throughout preimplantation development, 10 embryo
equivalents were used for each stage at 40 cycles of amplification. The amplicons were analyzed on 3% agarose gels (3:1 low melting point agarose/
agarose) containing 0.75 µg/ml ethidium bromide. The identity of the amplicons was confirmed by restriction enzyme digestion (StuI) and by direct sequencing (GenAlyTic sequencing service, the University of Guelph, Guelph, Ontario) following purification using a QIAquick SpinTM PCR
purification kit (QIAGEN Inc., Chatsworth, CA).
subunit mRNA increases quantitatively during preimplantation development and is thus a product of embryonic transcription, a semi-quantitative RT-PCR method was used to compare mRNA
levels in different stages (Davies et al., 1996
). A fixed amount of rabbit
-globin mRNA (25 pg per 100 embryos; GIBCO BRL, Burlington, Ontario) was added to each embryo batch as an internal standard before
RNA extraction. After RNA isolation and reverse transcription, the
cDNA preparations were amplified using both
subunit and
-globin
primers in the same reaction tube. This necessitated using a "primer-dropping" method (Wong et al., 1994
) where
-globin primers were dropped
into reaction mixtures for 20 cycles of amplification after 20 cycles had already been completed with
subunit primers alone. The
subunit and
-globin amplicons were separated by electrophoresis, imaged using a fluorescent gel documentation system (Bio-Rad Gel Doc 1000), and the ratio between their peak areas determined using Molecular Analyst software (Bio-Rad Laboratories Canada Ltd., Mississauga, Ontario). Final ratios
were determined by averaging the data from five twofold serial dilutions
of each cDNA preparation.
) that was raised against a synthetic peptide corresponding to amino acids 6-22 of the sheep
subunit. This antibody was supplied by R. Mercer (Washington University, St. Louis, MO). Other
embryos were immunostained in the same way with a 1:50 dilution of a
mouse monoclonal antibody raised against the
1 subunit of Na+,K+-ATPase (supplied by M. Caplan, Yale University, New Haven, CT). After primary antibody treatment, embryos were washed three times for 10 min
each through a solution of 0.002% Triton X-100, 1% goat serum in
PHEM, and left in the final wash at 4°C for 2 h. This was followed by incubation for 1 h at 4°C in a 1:50 dilution of fluorescein isothiocyanate-conjugated goat anti-rabbit IgG or rat anti-mouse IgG, as appropriate (ICN
Pharmaceuticals Canada Ltd., Montréal, Québec). The embryos were
washed three times again through 0.002% Triton X-100, 1% goat serum in
PHEM, and kept in the final wash overnight at 4°C. Finally, they were
mounted in SlowFade (Molecular Probes, Inc., Eugene, OR) and viewed
with a Bio-Rad MRC 600 confocal microscope.
subunit mRNA (Mercer et al., 1993
). The sequence of this ODN was
5
-CTICACAGCCACCAT-3
; inosine replaced guanine at the third residue in order to reduce the stability of self-complementarity within the
ODN. A randomized sequence ("nonsense") ODN (5
-CACCCTACIGACATC-3
) having the same base composition as the antisense
ODN was also used. To facilitate uptake of ODNs we used the strategy
developed by Khidhir et al. (1995)
that involves permeabilizing the embryos for a brief period with lysolecithin. Late 4-cell embryos, flushed from the oviducts at 60 h post-hCG, were exposed to 0.001% lysolecithin (Sigma Chemical Co.) for 2 min. They were then washed in potassium-augmented simplex optimization medium (KSOM; Erbach et al., 1994
)
and incubated continuously with 0.5 µM ODN in KSOM in a humidified
chamber with an atmosphere of 5% CO2 in air. Development of the embryos in microdrops under oil was monitored for 40 h; embryos were
scored at 2 h intervals for the presence or absence of a blastocoel (cavitation). At 100 h post-hCG the embryos were washed five times through
PBS-PVP and fixed immediately for wholemount immunofluorescence.
). Briefly, five embryos were incubated
for 30 min in drops of KSOM containing 0.35 mM 86RbCl and 0.35 mM
KCl, with or without ouabain, and with polyvinypyrrolidone replacing
BSA. The embryos were then washed four times in PBS-PVP, lysed in 2%
sodium dodecyl sulfate, and counted for 86Rb+ uptake. A sample of the final wash solution equal to the volume in which the embryos were transferred was also counted, with the counts per minute associated with the final wash being subtracted from the cpm for each group of embryos.
Because the epithelial trophectoderm of blastocysts acts as a barrier to
ouabain (DiZio and Tasca, 1977
), blastocysts were collapsed in 0.5 µg/ml
cytochalasin D (Sigma Chemical Co.) for 30 min before being assayed for
86Rb+ uptake as described above. The data from antisense- and nonsense-treated embryos were compared statistically using Student's t test. Some blastocysts were cultured for an additional 8 h after treatment with cytochalasin and 86Rb+ to look for deleterious effects on development; none
were noted.
80°C. Plasma membrane fractions were prepared using a modification
of the method of Resh (1982)
; all steps were carried out at 4°C. The embryos were homogenized by repeated pipetting in 500 µl buffer (255 mM
sucrose, 20 mM Tris-HCl, 1 mM EDTA, pH 7) and then the homogenate
was centrifuged at 16,000 g for 15 min using the JS 13.1 rotor of a Beckman J2-HS centrifuge. Pellets were resuspended in homogenization buffer
and centrifuged at 16,000 g for an additional 15 min. The resulting pellets
were again resuspended in homogenization buffer (100 µl), layered over
100 µl of a 33.5% sucrose cushion in Tris-EDTA buffer, and spun at
70,000 g for 1 h using the TLA-100 rotor of a Beckman tabletop ultracentrifuge. The top 150 µl of each supernatant was collected, diluted twofold with Tris-EDTA buffer, and centrifuged for 30 min at 50,000 g. Plasma
membrane pellets were suspended in a small amount of Hepes-Mg2+
buffer (100 mM Hepes-Tris, 5 mM MgCl2, pH 7.4) and stored at
80°C.
Kidney membranes were isolated by the same procedure. Protein concentration was determined with a protein assay kit (Bio-Rad Laboratories).
where
1 µg of membrane protein preparation was incubated in a volume of 80 µl
with 10 µM H3PO4, with or without 1 mM ouabain, for 30 min before
phosphorylation with 20 µCi of [32P]orthophosphate (NEN Life Science
Products, Guelph, Ontario), which had been prefiltered through a 0.22-µm filter. The reaction was quenched after 10 min by the addition of 50 µg
BSA (as carrier) and 500 µl ice-cold 5% trichloroacetic acid, 0.1 M H3PO4
and placed on ice for 5 min before centrifugation for 2 min at 10,000 g. Pellets were rinsed three times with 5% trichloroacetic acid, 0.1 M H3PO4
and then rinsed quickly with 0.3 ml of 0.15 M KH2PO4, pH 2. The final
pellets were suspended in sample buffer (250 mM sucrose, 35 mM 1-hexadecylpyridinium chloride, 100 mM KH2PO4, 128 mM
-mercaptoethanol,
pH 4) for liquid scintillation counting or for analysis by gel electrophoresis.
. The gels were run at 40 mA for 6 h, treated for 5 min in 1%
glycerol, dried for 45 min, and exposed to Kodak XAR-5 film with an intensifier screen at
80°C. Gel lanes run with marker proteins were separated from the gel, stained with 0.25% Coomassie blue in 10% acetic acid
and 10% methanol, and destained before being dried.
Results
Subunit in Preimplantation Embryos Is a Product
of Embryonic Gene Expression
subunit, like the
and
subunits, is a product of embryonic gene expression.
PCR primers designed using the mouse cDNA sequence
(Mercer et al., 1993
) were used to amplify the expected
134-bp amplicon from reverse-transcribed RNA of selected stages of preimplantation development. Although
subunit mRNA was detected in unfertilized oocytes, it was
not detected in cleaving embryos until the eight-cell stage
(Fig. 1 A). It is then present continuously through the blastocyst stage. To confirm that
subunit mRNA present after the four-cell stage is a product of continuous embryonic transcription and to estimate its rate of accumulation,
we used a semi-quantitative RT-PCR approach (Davies et
al., 1996
) to compare mRNA levels between different stages. A fixed amount of
-globin mRNA was added to
each embryo batch before lysis to control for variation in
RNA recovery and the efficiency of reverse transcription
and PCR amplification. Because the amount of
-globin
mRNA added was the same for each stage on a per embryo basis, the ratio between the
subunit and
-globin
amplicons is a relative measure of the amount of
subunit mRNA. As shown in Fig. 1 B, the amount of
subunit
mRNA approximately doubles (on a per embryo basis) in
each succeeding stage from the eight-cell stage onward.
Thus the
subunit gene is actively transcribed throughout
this developmental period.
Fig. 1.
Subunit mRNA accumulates from the eight-cell stage
onward and is thus a product of embryonic transcription. (A) An
amplicon of the expected size (134 bp) was amplified from reverse-transcribed RNA of oocytes (OC), eight-cell embryos (8C),
morulae (MO), and early blastocysts (EB), but not of four-cell
embryos (4C). M, molecular size markers;
, negative control
(water blank); +, positive control (mouse kidney cDNA as template). 10 oocyte or embryo equivalents of cDNA were used for
each amplification (40 cycles). (B) Semi-quantitative RT-PCR
demonstrates that the amount of
subunit mRNA approximately
doubles on a per embryo basis in each successive stage. The mean
subunit/
-globin amplicon ratio was taken as an indication of
the relative mRNA level for each stage. The error bars indicate
standard deviation derived from five determinations.
[View Larger Versions of these Images (67 + 14K GIF file)]
). We presume that the few mismatches result from errors in PCR
amplification or sequencing, but we cannot rule out the possibility that the preimplantation embryo expresses an
isoform of the
subunit different from that found in kidney.
Fig. 2.
The sequence of the subunit amplicon from blastocysts is 97% identical to the sequence of the corresponding portion of the published mouse kidney cDNA (Mercer et al., 1993
).
The primer sequences are underlined. These sequence data are
available from Genbank/EMBL/DDBJ under accession number
X70060.
[View Larger Version of this Image (25K GIF file)]
subunit mRNA does not necessarily
indicate that de novo synthesis of the polypeptide is occurring. To eliminate this uncertainty, we used a previously
established protocol (De Sousa et al., 1993
) to prepare
subribosomal supernatant and polyribosomal pellet fractions from four- and eight-cell embryos and assayed them
for
subunit mRNA by RT-PCR (Fig. 3). Both fractions
from eight-cell embryos were found to contain the mRNA,
although we could not detect it in either fraction from
four-cell embryos. This result strongly suggests that
subunit mRNA is translated from the time of its first appearance in the eight-cell stage. The fact that a portion of the
mRNA does not cosediment with polyribosomes suggests
that the mRNA is not being translated with maximal efficiency, at least in the eight-cell stage. A similar finding was
reported for the mRNA encoding the
subunit (MacPhee
et al., 1994
).
Fig. 3.
Subunit mRNA cosediments with polyribosomes
from its time of first appearance in the eight-cell stage, indicating
de novo synthesis. Four- and eight-cell uncompacted embryos
were fractionated into subribosomal supernatant (S) and polyribosomal pellet (P) fractions which were assayed for
subunit
mRNA by RT-PCR. Each amplification reaction used 32.5 embryo equivalents of cDNA, with amplification for 40 cycles. M,
molecular size markers.
[View Larger Version of this Image (69K GIF file)]
subunit itself was explored by confocal immunofluorescence microscopy (Fig.
4). We first confirmed that the antibody produces a specific pattern of immunostaining of mouse kidney (Fig. 4 E;
Mercer et al., 1993
). As expected, strongest staining was
seen in individual nephron sections with much lighter
staining of glomeruli. The same antibody was then applied
to preimplantation embryos. As shown in Fig. 4 A, a low level of immunostaining, presumably resulting from a lingering oogenetic contribution, can be detected in four-cell
embryos. The intensity of immunostaining increases thereafter. Although there is some cytoplasmic immunoreactivity, the fluorescent signal is concentrated in the cell peripheries, including both apical (facing outward) and
basolateral (adjacent to the blastocoel) membranes of the
trophectoderm (Fig. 4 D). There does not appear to be a
significant difference in the amount of
subunit between inside and outside cells of morulae (Fig. 4 C) nor between
inner cell mass and trophectoderm of blastocysts. Embryos stained with preimmune serum (Fig. 4 F) showed no
specific immunoreactivity.
Fig. 4.
The subunit accumulates from the eight-cell stage onward and is localized in both apical and basolateral membrane
domains of trophectoderm. Confocal images were made at the
same magnification and using comparable microscope settings for
all stages to allow comparison of signal intensities between
stages. Shown are representative four- (A) and eight-cell (B) embryos, a compacted morula (C), and a blastocyst (D). ICM, inner
cell mass; TR, trophectoderm. A section of mouse kidney showing a glomerulus (G), stained with the same antibody is shown in
E (Bar, 25 µm). The image in F, in which the signal intensity was
enhanced for better visibility, shows a blastocyst treated with preimmune serum. Bar, 10 µm.
[View Larger Version of this Image (107K GIF file)]
Subunit Accumulation
Delays Cavitation
subunit is required for cavitation, we treated embryos
with antisense ODN designed to disrupt translation of
subunit mRNA. Treatment began in the four-cell stage,
before the mRNA begins to accumulate, to maximize the
effect. A nonsense (scrambled sequence) ODN was used
as a control for nonspecific toxicity. Embryos were permeabilized with lysolecithin to facilitate uptake of the ODN. In preliminary experiments, we determined that a lysolecithin concentration of 0.001% is optimal; greater concentrations proved toxic. This concentration differs from that
used by Khidhir et al. (1995)
, probably reflecting differences in the strain of mice used and between particular
lots of lysolecithin.
subunit mRNA in the antisense-treated
groups was found to have been reduced by 34% in one experiment and 46% in the other, as compared with the respective nonsense-treated groups.
Fig. 5.
Antisense disruption of subunit accumulation interferes with blastocyst development. Embryos treated with antisense ODN, but not nonsense ODN or lysolecithin alone (control), suffer a delay in the onset of cavitation. The experiment was
done in triplicate with each embryo drop containing 20 embryos.
[View Larger Version of this Image (34K GIF file)]
subunit (Fig. 6, A and B)
as compared with embryos treated with nonsense ODN
(Fig. 6, D and E). In contrast, we could not detect any effect on the
subunit after disrupting accumulation of the
subunit (Fig. 6, C and F). This finding makes it unlikely that, in this system, the
subunit is required for deployment of the enzyme in plasma membranes.
Fig. 6.
Treatment with antisense oligodeoxynucleotide caused
an obvious reduction in the amount of subunit (but not
subunit) in the blastomere membranes. Confocal images were made
at the same magnification and using comparable microscope settings for all stages in order to allow comparison of signal intensities between treatments. Embryos in A-C were treated with antisense ODN whereas those in D-F were treated with nonsense
ODN; all were taken out of culture at 100 h post-hCG. The embryos were immunostained with
subunit antibody except those
in C and F, which were immunostained with
subunit antibody.
Embryos in A and D were from one experiment whereas embryos
in B, C, E, and F were from a separate experiment. Bar, 10 µm.
[View Larger Version of this Image (135K GIF file)]
). We
used this same method to determine the extent to which
the antisense treatment had affected the function of the
sodium pump. As summarized in Table I, the rate of ouabain-sensitive 86Rb+ uptake in the first two experiments
was severalfold lower in late morulae in which the onset of
cavitation had been delayed by the antisense treatment; in
the third experiment, the reduction was 43%. In the second and third experiments we also analyzed embryos that
had succeeded in initiating cavitation by 100 h despite the
antisense treatment, and found similar reductions. These
data indicate that the
subunit is an important determinant
of the function of the sodium pump and that embryonic
expression of the
subunit is a requirement for cavitation.
The Rb+ uptake assay is a measure of Na+,K+-ATPase
activity in intact cells and, as such, can reflect changes
brought about by effectors of pump activity independent
of the number of active enzyme molecules. Hence, a reduction in ouabain-sensitive Rb+ uptake could indicate a
direct effect on the enzyme itself or some change in a
physiological effector. In order to ascertain whether the
enzymatic activity of the enzyme itself had been affected by the reduction of subunit accumulation, we used the
"back door" phosphorylation assay (Resh, 1982
) to measure Na+,K+-ATPase activity in isolated plasma membrane preparations. This assay is based on the fact that, in
the presence of ouabain and Mg2+, the
subunit of
Na+,K+-ATPase is uniquely phosphorylated to form an alkali-labile intermediate that is identical to that which is
generated during the enzyme's forward reaction cycle.
Since the enzyme must pass through at least a partial reaction cycle to be phosphorylated, only active enzyme is detected. As shown in Fig. 7, a single polypeptide of ~95,000
Mr was phosphorylated when plasma membrane preparations from embryos (100 h post-hCG) or from adult kidney were incubated with [32P]orthophosphate and Mg2+ in
the presence of ouabain. Phosphorylation of the 95-kD
polypeptide was minimal in the absence of ouabain. As expected, the bound phosphate was released when the membranes were incubated in 2 mM ATP (Resh, 1982
).
Measurements of Na+,K+-ATPase activity in antisense-treated embryos using this phosphorylation assay are summarized in Table II. Four experiments were carried out; in
the first, duplicate assays were performed for each treatment whereas in the other three experiments the assays
were done in triplicate. In each experiment, the assay
failed to demonstrate any difference in enzymatic activity
between antisense- and nonsense-treated groups. These
data make it clear that the reduced availability of subunits in antisense-treated embryos had no effect on
Na+,K+-ATPase activity per se, making it doubtful that
the
subunit is an integral component of the enzyme.
Table II. Effect of Antisense Treatment on Na+,K+-ATPase Activity |
In preimplantation embryos, Na+,K+-ATPase is both a
"housekeeping" enzyme, responsible for maintaining the
electrochemical gradients of Na+ and K+ across the plasma
membrane, and an agent of morphogenesis. Sodium pumps deployed in the basolateral membranes of the epithelial trophectoderm work in concert with a variety of
Na+ entry routes arrayed in the apical domain to generate
a transepithelial flow of Na+ and water to form the blastocoel (Watson and Kidder, 1988; Manejwala et al., 1989
).
Hence, treatments that interfere with the establishment of
a polarized distribution of trophectodermal sodium pumps
or with the activity of those pumps can prevent cavitation
(Manejwala et al., 1989
; Watson et al., 1990a
; MacPhee et
al., 1997
). We have taken advantage of the dependence of
blastocyst development on embryonic expression of sodium pump subunits to test the hypothesis that the putative
subunit of Na+,K+-ATPase is an important determinant of the enzyme's function and, as such, is required for
cavitation. Embryonic transcription of the
subunit gene,
cosedimentation of the mRNA with polyribosomes, and
increasing
subunit immunoreactivity with development
after the eight-cell stage all served as evidence that the
subunit is embryonically expressed in the mouse, a prerequisite finding before an antisense approach could be applied. The temporal pattern of expression of the
subunit
gene, with mRNA present in oocytes being degraded after
fertilization to reappear during cleavage of the zygote, is
typical of genes expressed in preimplantation embryos and
reflects the transition from oogenetic to embryonic control
of development (for review see Kidder, 1993
).
Antisense disruption of subunit accumulation delayed
cavitation, indicating that synthesis of the
subunit is essential for blastocyst development. Antisense-treated morulae collected at 100 h post-hCG, when the cavitation delay
was most apparent, were found to have reduced immunoreactivity for the
subunit as well as reduced sodium
pump function as indicated by ouabain-sensitive 86Rb+ uptake. Failure of the antisense ODN to completely block
cavitation probably reflects the fact that synthesis of the
subunit was not completely abolished; this could be due to
incomplete destruction of the mRNA, although we do not
know to what extent translation of the remaining mRNA
might have been impaired. In any case, antisense-treated morulae retained some capacity for active cation transport. Those embryos that were able to initiate extracellular fluid accumulation on schedule with nonsense-treated
or control embryos are likely to have been the most advanced in their cohort and as such would have had more
time to accumulate fluid using their reduced Na+ transport
capacity. It is also possible that the more advanced embryos, which would have been the first to cleave to the
eight-cell stage, might have initiated
subunit expression
before the antisense inhibition had become fully established. This interpretation is supported by the observation
that when the antisense treatment was delayed until the
eight-cell stage, there was no discernable effect on the timing of cavitation (results not shown).
Using the Xenopus oocyte expression system, Béguin et
al. (1997) demonstrated that the
subunit is not required
for the assembly of
/
heterodimers or their insertion
into the plasma membrane as functional enzyme molecules. These results are in agreement with our observation
that antisense attenuation of
subunit accumulation did
not affect the level of
subunit immunoreactivity at the
cell surface. However, Béguin et al. (1997)
also showed that stable expression and plasma membrane insertion of
the
subunit in oocytes depends on its association with
nascent
/
heterodimers. If the same is true in preimplantation mouse embryos, then it remains to be discovered
how the
subunit could accumulate in both the apical and
basolateral surfaces of the trophectoderm when
1 and
1
subunits are concentrated in the basolateral surface (Watson and Kidder, 1988
; MacPhee et al., 1997
). One explanation might be that nascent
subunits are delivered to both
surfaces in association with
/
heterodimers, but that this
association is lost in the apical domain when the latter are
not retained there. In polarized MDCK cells, for example,
localization of Na+,K+-ATPase is achieved by preferential
retention of nascent
/
heterodimers in the basolateral
domain whereas they are selectively removed from the
apical domain (Hammerton et al., 1991
).
The fact that the subunit is present in both apical and
basolateral trophectoderm surfaces suggests that it may
also function separately from Na+,K+-ATPase. Although
there are few studies examining the cellular distribution of
the
subunit, it has consistently been found in association
with the
and
subunits in those cells and tissues where it
has been detected (Mercer et al., 1993
). Hence we expected to find the
subunit concentrated in the basolateral (juxtacoelic) surface of the trophectodermal epithelium. The fact that it is not suggests that the
subunit may
associate with other membrane proteins or have functional properties not requiring the
and
subunits. Expression studies using Xenopus oocytes (Noguchi et al.,
1987
; Béguin et al., 1997
), yeast (Scheiner-Bobis and Farley, 1994
; Pedersen and Jørgensen, 1992
), and insect cells (DeTomaso et al., 1993) have all indicated that functional
Na+,K+-ATPase activity can be obtained by expressing
only the
and
subunits. Interestingly, the amino acid sequence of the
subunit places it in a recently recognized
family of membrane proteins, characterized by single
transmembrane domains that mediate transmembrane ion
movements. Other members of the family include phospholemman (Palmer et al., 1991
), channel-inducing factor
(Attali et al., 1995
), and Mat-8 (Morrison et al., 1995
).
When expressed in Xenopus oocytes, phospholemman and
Mat-8 induce Cl
selective currents while channel-inducing factor induces a K+ current. Early evidence suggested
that these proteins may activate endogenous channels;
however, it is now known that phospholemman itself
forms a taurine-selective ion channel when incorporated into a synthetic lipid bilayer (Moorman et al., 1995
). More
recently, it has been demonstrated that in the absence of
taurine, phospholemman can function as a cation or anion
selective channel (Kowdley et al., 1997
). The channel undergoes voltage-dependent transitions among conformations with distinct ion selectivities, possibly explaining the
very different ion selectivity properties of the members of
this family of proteins. Consistent with these observations, it has been determined that the
subunit induces monovalent cation (Na+, or K+) selective channels when expressed
in Xenopus oocytes (Mercer, R., personal communication). The possible functioning of the
subunit as a cation
channel is compatible with our observations and could explain the discrepancy between our assay data obtained from intact cells and those from isolated plasma membranes. In the intact embryo, the
subunit may increase
Na+,K+-ATPase activity by supplying intracellular Na+ to
the enzyme such that, when its expression is inhibited,
transepithelial Na+ transport is adversely affected; such an
effect could not be detected by assaying plasma membrane
preparations in vitro. In this respect, trans-trophectodermal Na+ movement in blastocysts may be similar to transepithelial transport in the kidney in which Na+ entry at the
apical membrane is often rate limiting (for review see
Stanton and Kaissling, 1989
). Thus the presence of the
subunit in the apical membranes of the blastocyst may be
relevant to its function during cavitation. Alternatively,
the
subunit associated with the sodium pumps in the basolateral membranes may serve to recycle intracellular K+
into the blastocoel to sustain pump function.
Whatever the role of the subunit turns out to be, it has
an important influence on Na+,K+-ATPase activity and
transepithelial Na+ transport, though probably not as an
integral component of the enzyme. Furthermore, our results demonstrate that the
subunit is an essential component of the cellular machinery that drives fluid transport
during blastocoel formation. Future experiments will be
aimed at trying to clarify what role the
subunit plays in
influencing sodium pump activity during preimplantation
development.
Received for publication 21 March 1997 and in revised form 27 August 1997.
Address all correspondence to Gerald M. Kidder, Department of Physiology, The University of Western Ontario, London, Ontario N6A 5C1. Tel.: (519) 661-3132. Fax: (519) 661-3827. E-mail: gkidder{at}physiology.uwo.caWe gratefully acknowledge the assistance of K. Barr, I. Craig, and L. Barcroft as well as the many useful comments of D. MacPhee, F. Houghton,
P. DeSousa, and A. Watson. We thank M. Caplan for the 1 antibody. We
are especially grateful to R. Mercer for supplying the
subunit antibody,
for sharing his insights into the function of the
subunit, and for allowing
us to quote his unpublished results.
This work was supported by a grant from the Medical Research Council of Canada (MT-11302) to G.M. Kidder.
hCG, human chorionic gonadotropin; KSOM, potassium-augmented simplex optimization medium; ODN, oligodeoxynucleotide; PVP, polyvinylpyrrolidone; RT, reverse transcription.
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