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Originally published In Press as doi:10.1074/jbc.M700696200 on February 22, 2007

J. Biol. Chem., Vol. 282, Issue 16, 12127-12134, April 20, 2007
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Na/K-ATPase beta1 Subunit Expression Is Required for Blastocyst Formation and Normal Assembly of Trophectoderm Tight Junction-associated Proteins*

Pavneesh Madan, Keeley Rose, and Andrew J. Watson1

From the Departments of Obstetrics and Gynaecology, Physiology, and Pharmacology, University of Western Ontario, Children's Health Research Institute-Victoria Research Laboratories, London, Ontario N6A 4G5, Canada

Received for publication, January 24, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Na/K-ATPase plays an important role in mediating blastocyst formation. Despite the expression of multiple Na/K-ATPase {alpha} and beta isoforms during mouse preimplantation development, only the {alpha}1 and beta1 isoforms have been localized to the basolateral membrane regions of the trophectoderm. The aim of the present study was to selectively down-regulate the Na/K-ATPase beta1 subunit employing microinjection of mouse 1 cell zygotes with small interfering RNA (siRNA) oligos. Experiments comprised of non-injected controls and two groups microinjected with either StealthTM Na/K-ATPase beta1 subunit oligos or nonspecific StealthTM siRNA as control. Development to the 2-, 4-, 8-, and 16-cell and morula stages did not vary between the three groups. However, only 2.3% of the embryos microinjected with Na/K-ATPase beta1 subunit siRNA oligos developed to the blastocyst stage as compared with 73% for control-injected and 91% for non-injected controls. Na/K-ATPase beta1 subunit down-regulation was validated by employing reverse transcription-PCR and whole-mount immunofluorescence methods to demonstrate that Na/K-ATPase beta1 subunit mRNAs and protein were not detectable in beta1 subunit siRNA-microinjected embryos. Aggregation chimera experiments between beta1 subunit siRNA-microinjected embryos and controls demonstrated that blockade of blastocyst formation was reversible. The distribution of Na/K-ATPase {alpha}1 and tight junction-associated proteins occludin and ZO-1 were compared among the three treatment groups. No differences in protein distribution were observed between control groups; however, all three polypeptides displayed an aberrant distribution in Na/K-ATPase beta1 subunit siRNA-microinjected embryos. Our results demonstrate that the beta1 subunit of the Na/K-ATPase is required for blastocyst formation and that this subunit is also required to maintain a normal Na/K-ATPase distribution and localization of tight junction-associated polypeptides during preimplantation development.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Research investigating the mechanisms directing blastocyst formation has demonstrated that (a) the Na/K-ATPase assumes a polarized distribution confined to the trophectoderm basolateral membrane regions just before the onset of cavitation (1, 2), (b) expression of Na/K-ATPase subunit genes are up-regulated during the morula to blastocyst transition (3-7), (c) that Na/K-ATPase activity is significantly increased during the morula to blastocyst transition for a number of mammalian species (2, 8-10), (d) that treatment with ouabain (a potent and specific inhibitor of the Na/K-ATPase) affects cavitation and blastocyst formation in a number of mammalian species (5, 11-15), (e) that deletion of the Na/K-ATPase {alpha}1 subunit gene product is linked to aberrant blastocyst formation in vitro and likely peri-implantation lethality in vivo (16), and (f) that Na/K-ATPase also regulates the formation and function of trophectoderm tight junctions (17).

Taken together, these data support the hypothesis that the Na/K-ATPase contributes directly to the mechanism that regulates fluid movement across the trophectoderm resulting in the formation of the fluid-filled blastocoelic cavity. Our most recent efforts have initiated studies to investigate the individual roles of each Na/K-ATPase subunit during preimplantation development (16) and to explore whether the Na+ pump also regulates tight junction formation and function during blastocyst formation (17). Much to our initial surprise we discovered that it was possible to collect quite normal-looking day 3.5 post-human chorionic gonadotropin-injected Na/K-ATPase {alpha}1 null blastocysts from the reproductive tracts of heterozygous mice (16). However, these {alpha}1 null embryos struggle to attach and form trophoblast outgrowths in culture and do not form fully expanded blastocysts if collected and placed into culture at the eight-cell stage for determination of their developmental potential (16). Therefore, we have concluded that the {alpha}1 subunit of the Na/K-ATPase is required for normal development and initiation of pregnancy and that limited compensation perhaps by an alternative Na/K-ATPase {alpha} subunit isoform is able to allow for short term development of {alpha}1 null embryos to the blastocyst stage in vivo.

In the continuation of our pursuit of an understanding of the roles of each Na/K-ATPase subunit during preimplantation development, we have conducted the present study to characterize the consequences to early development and blastocyst and tight junction formation after the selective down-regulation of the Na/K-ATPase beta1 subunit by employing microinjection of 1 cell mouse zygotes with small interfering RNA (siRNA)2 oligos. Our results demonstrate that the beta1 subunit of the Na/K-ATPase is required for blastocyst formation and that this subunit is required to maintain a normal Na/K-ATPase distribution and localization of tight junction-associated polypeptides during preimplantation development.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Super-ovulation and Embryo Collection—Female CD-1 mice (Charles River, Saint-Constant, Quebec, Canada) 4-5 weeks of age were injected with 10 IU of pregnant mare's serum gonadotropin (Intervet, Whitby, Canada) followed by 10 IU of human chorionic gonadotropin (Intervet) 48 h later and just before mating with CD-1 males. Successful mating was determined the next morning by the presence of a vaginal plug and was considered day 0.5 of development. One-cell stage embryos were flushed from oviducts of female mice using flushing medium I (1.71 mM calcium lactate, 0.25 mM sodium pyruvate, 3 mg/ml human chorionic gonadotropin, and 10x Leibovitz-modified Hanks' balanced salt solution, all diluted with water to 1x) (45) containing hyaluronidase (1 mg/ml, Sigma). The embryos were washed 3x in potassium simplex optimized medium (KSOM) media (46) under paraffin oil in sterile culture dishes and subsequently cultured in KSOM medium plus amino acids (KSOMaa) (46) under a 5% CO2 in air atmosphere at 37 °C until transferred into experimental treatment groups as defined below. All medium components were purchased from Sigma unless stated otherwise. KSOMaa medium was made fresh before each collection and was sterile-filtered. The osmolarity of the medium was tested each time it was prepared and ranged between 288-298 milliosmolal. All experiments described in this study maintained a treatment drop volume-to-embryo ratio of 1 embryo/µl of KSOMaa culture medium. Animal care and handling was according to the guidelines of the University of Western Ontario Animal Care Committee.

Microinjection of siRNAs—Microinjection was performed under an inverted microscope using a mechanical micromanipulator (Leica) attached to Picoinjector PLI-100 (Harvard Apparatus, Saint-Laurent, Quebec, CA). Each injection delivered 10 pl of 20 µM siRNA duplexes into the cytoplasm of 1-cell-stage embryos. Microinjection of embryos was performed according to a standard procedure. One-cell embryos were placed in KSOMaa medium under light mineral oil. A holding pipette (Conception Technologies, San Diego, CA) was used to keep the one-cell embryos stationary during manipulation. An injection pipette (Conception Technologies) loaded with double-stranded (ds)R NA solution was inserted into the cytoplasm of each zygote followed by the microinjection of ~10 pl of dsRNA. After microinjection, embryos were cultured in KSOMaa medium as described above for up to 4 days to allow for an assessment of developmental capacity to the blastocyst stage. About 300 embryos were used for each experimental replicate, and in total a set of three replicates were conducted.

RNA Extraction, Reverse Transcription (RT), and RT-PCR—Total RNA was extracted from murine embryos (pools of 20 embryos/stage at 1-, 2-, 4-, 8-cell, morula, and blastocyst stages) using the phenol chloroform method of Chomczynski and Sacchi (18). The total RNA extracts were digested with deoxyribo-nuclease-1 to eliminate possible contamination from genomic DNA. The RT reactions were conducted using oligo-dT primers (Invitrogen, Burlington, Ontario, Canada) as previously described (19, 20). Briefly, samples were incubated for 90 min at 42 °C in a total volume of 20 µl consisting of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM dNTPs, and 200 units of Superscript II (Invitrogen) followed by heating the samples to 95 °C for 5 min for termination of reaction.

PCR was conducted using a previously described protocol (21). Briefly, two embryo equivalents for each stage of development under investigation were used per PCR reaction, which was repeated a minimum of three times from pools of three different developmental series of embryos. The conditions for each PCR reaction are given in Table 1. PCR products were resolved on 2.0% agarose gels containing 0.5 µg/ml ethidium bromide (Invitrogen). To confirm the specificity of each PCR product, representative amplicons were extracted from the gels and purified using a QIAquick gel extraction kit (Qiagen, Mississauga, ON) and submitted for nucleotide sequencing (DNA Sequencing Facility, Robarts Research Institute, London, ON, Canada). The nucleotide sequence was subsequently compared with sequences available in GenBankTM nucleotide sequence data base, and in all cases the specificity of each PCR product was confirmed. There was 98% sequence identity to mouse Na/K-ATPase beta1 subunit sequence.


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  		TABLE 1
Nucleotide sequences for polymerase chain reaction amplification reactions

 
Embryo Fixation and Whole-mount Indirect Immunofluorescence—To analyze the distribution of Na/K-ATPase {alpha} and beta isoforms, ZO-1, and occludin polypeptides during murine preimplantation development, a whole-mount immunofluorescence method previously described (21) was employed. Immunofluorescence was detected using laser scanning confocal microscopy as described (19). Briefly, embryos at timed stages of development (1-cell zygotes, 2-, 4-, and 8-cell, morula, and blastocyst stages) were washed in 1x phosphate-buffered saline (PBS) and then fixed in 2% paraformaldehyde in PBS for 20 min at room temperature. These fixed embryos were washed in 1x PBS and either processed immediately for immuno-staining or stored at 4 °C in PBS for a maximum of 4 weeks.

For immunostaining, fixed embryos were permeabilized and blocked in 1x PBS + 5% donkey serum + 0.01% Triton X-100 for 1 h at room temperature. Embryos were washed in 1x PBS and incubated with primary antibody diluted 1:100 in 1x PBS +1% donkey serum + 0.005% Triton X-100 for 1 h at room temperature followed by additional washes totaling 1 h at 37 °C. Primary antibodies were labeled by incubation for 1 h with fluorescein isothiocyanate-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:200. Embryos were then treated with rhodamine-conjugated phalloidin (5 µg/ml; 1:20) and 4,6-diamidino-2-phenylindole (1 mg/ml; 1:2000) for 30 min at 37 °C followed by 2 washes for 2 h each at 37 °C. Embryos were mounted in Fluoro-Guard Antifade Mounting Reagent (Bio-Rad, Mississauga, Ontario, Canada). Fluorescence patterns were examined using a Zeiss LSM 410 (laser scanning microscope) with an inverted Axiovert 100 microscope under 20-40x magnification. The images were then captured and stored as TIFF files by the Zeiss LSM software package.

Primary antibodies for Na/K-ATPase {alpha} and beta subunits and Z0-1 were obtained from Upstate Cell Signaling Solutions, Charlottesville, VA. Primary antibody for occludin was obtained from Zymed Laboratories Inc., San Francisco, CA.

siRNA Oligo Preparation—Double-stranded siRNA oligos were designed using BLOCK-iTTM RNAi Designer software (Invitrogen). Stealth siRNA duplex oligoribonucleotides against the beta1 subunit of Na/K-ATPase (GenBankTM accession number NM_009721 [GenBank] ) were synthesized by Invitrogen. The sequences were as follows: (i) sense 5'-CCC AAG AAU GAA UCC UUG GAG ACU U-3', antisense 5'-AAG UCU CCA AGG AUU CAU UCU UGG G-3'; (ii) control sense 5'-CCC AAG AGU CUA UUC AGG AGA ACU U-3', antisense 5'-AAG UUC UCC UGA AUA GAC UCU UGG G-3'. The duplex oligoribonucleotides were re-suspended in diethyl pyro-carbonate-treated water to make a 20 µM solution and stored at -20 °C until further use.

Reversal of Developmental Blockade by Aggregation Chimeras—Embryos from the three treatment groups (beta1 subunit siRNA-microinjected, random sequence siRNA-injected control, and non-injected control) were collected at the non-compacted eight-cell stage. These embryos were treated with acid Tyrode solution (pH 2.5) to remove zona pellucida. Denuded eight-cell embryos were washed three times in fresh KSOMaa drops and aggregated together for chimera production. The beta1 subunit siRNA-microinjected embryos were either paired with self-like (A:A) or with uninjected controls (A:C) or random sequence siRNA-injected controls (A:B). In addition, injected controls were also paired together (B:B) to serve as an additional control. In total, 76 chimeras were produced from 152 embryos over 3 replicates. Development of each aggregation chimera combination to the blastocyst stage was assessed. In addition, chimeras from each group were processed for immunofluorescence localization of Na/K-ATPase beta subunit polypeptides as described.

Statistical Analysis—The results are presented as the means ± S.E. from three independent experiments. Statistical differences between time points were assessed by analysis of variance. Differences were considered significant when p < 0.05. Significant differences between the means were determined using the least significant difference test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To investigate the consequences of Na/K-ATPase beta1 subunit down-regulation on preimplantation development, we employed microinjection of beta1 siRNAs into one-cell zygotes. Our control groups consisted of non-injected zygotes and those injected with a random sequence siRNA. In total, more than 300 zygotes were placed into each treatment group for evaluation of developmental outcomes. The experiment was repeated three times using zygotes collected from different mouse populations each time. The morphology of zygotes in each group 4 days after injection is displayed in Fig. 1A. Zygotes injected with the Na/K-ATPase beta1 siRNA developed through to the morula stage (Fig. 1, Aa and B). In contrast, zygotes injected with control siRNA or non-injected controls displayed a high frequency of development through to the blastocyst stage after injection (Fig. 1, A, b and c). In both injected groups we observed a nearly equal number of zygotes that did not develop, likely due to damage caused by the microinjection procedure (Fig. 1, Aa and b). When the data were plotted and analyzed, we clearly observed that injection with the Na/K-ATPase beta1 subunit siRNA resulted in a significant reduction in the proportion of zygotes that completed development to the blastocyst stage (Fig. 1B). Control-injected and control non-injected zygotes displayed a comparable blastocyst developmental frequency (Fig. 1B). The majority of beta1 siRNA-injected zygotes attained and then remained at the morula stage 4 days after injection (Fig. 1B). Attempts to provide morulae in the Na/K-ATPase beta1 siRNA treatment with additional time to progress beyond the morula stage by extending their culture interval for an additional 24 h did not increase the development of embryos in this group to the blastocyst stage (data not shown).

To ensure that the Na/K-ATPase beta1 siRNAs we employed were effective at down-regulating Na/K-ATPase beta1 gene expression during early development, we applied RT-PCR methods to detect Na/K-ATPase beta1 mRNAs and also whole mount immunofluorescence methods to map out Na/K-ATPase beta1 protein distribution. It was readily possible to detect transcripts encoding the Na/K-ATPase beta1 subunit in control-injected and non-injected control groups as indicated by the appearance of an expected size RT-PCR product in samples from both groups (Fig. 1C). In contrast it was not possible to detect the Na/K-ATPase beta1 RT-PCR product in samples prepared from Na/K-ATPase beta1 siRNA-injected zygotes (Fig. 1C). To ensure that Na/K-ATPase beta1 siRNA prepared samples still retained intact RNA, we employed RT-PCR to amplify transcripts encoding beta-actin. In all samples from all treatment groups it was readily possible to detect the expected size beta-actin cDNA product. The application of whole-mount immunofluorescence methods employing a Na/K-ATPase beta1 subunit-specific antiserum revealed a complete absence of detectable Na/K-ATPase beta1 subunit protein in beta1 siRNA-injected zygotes (Fig. 2, a-c). In contrast, the Na/K-ATPase beta1 subunit was detected in both control-injected and non-injected controls beginning at the 8-16-cell stage and also in both morulae and blastocyst stages as reported earlier (Fig. 2, e-h; Ref. 7). In control blastocysts the Na/K-ATPase beta1 subunit immunofluorescence maintained the expected polarized distribution in the trophectoderm and an apolar distribution in the inner cell mass (Fig. 2h). When we examined the few blastocysts that formed in the Na/K-ATPase beta1 siRNA-injected treatment group, no organized Na/K-ATPase beta1 subunit protein distribution could be detected (Fig. 2d); instead, a few small indistinct patches of green fluorescence were observed in these embryos (Fig. 2d). In total, these outcomes demonstrate that the Na/K-ATPase beta1 siRNA we employed in this study effectively down-regulated Na/K-ATPase beta1 subunit expression for at least 5 days during mouse preimplantation development.


Figure 1
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  		FIGURE 1.
Morphology of Na/K-ATPase beta1 subunit siRNA-microinjected embryos. Panel A, morphological changes in mouse preimplantation development after Na/K-ATPase beta1 subunit siRNA microinjection into zygotes. Note a lack of complete blastocyst formation in Na/K-ATPase beta1 subunit siRNA-microinjected embryos; however, a few (2.3%) of the embryos did have multiple and small blastocoele cavities (a). The group of embryos microinjected with control siRNA or those representing non-injected controls developed normally to the blastocyst stage (b and c). Panel B, the percentage of embryos that developed into blastocysts after Na/K-ATPase beta1 subunit siRNA microinjection or control siRNA microinjection (CI) or non-injected controls (C) is shown. Only 2.3% of Na/K-ATPase beta1 subunit siRNA-injected zygotes progressed to the blastocyst stage compared with 73% (injected controls) or 91% (non-injected controls). n = 3 independent experiments. Values with different symbols represent significant differences. p value =<0.05. Panel C, validation of Na/K-ATPase beta1 siRNA efficacy at down-regulating beta1 gene expression. Transcripts encoding Na/K-ATPase beta1 subunit were not detected in embryos injected with beta1 siRNAs 4 days after microinjection. Embryos microinjected with control siRNA or non-injected embryos displayed the appropriate Na/K-ATPase beta1 mRNA product. Transcripts encoding beta-actin were readily detected in all three treatment groups. The negative control represents no cDNA sample, whereas the positive control is a mouse kidney cDNA sample.

 
We investigated the reversibility of the developmental blockade displayed by beta1 subunit siRNA-microinjected embryos by aggregating together eight-cell embryos from all three treatment groups to measure chimera formation and progression to the blastocyst stage. The beta1 subunit siRNA-microinjected embryos (Fig. 3A, A:A) did not progress to the blastocyst stage. In contrast, beta1 subunit siRNA-microinjected embryos, which were paired with either random sequence siRNA-microinjected (A:B), uninjected controls (A:C), or even injected controls paired together (B:B) all displayed a normal progression to the blastocyst stage (Fig. 3B). In addition localization of Na/K-ATPase beta subunit polypeptides in these chimeras by the application of immunofluorescence methods confirmed the complete absence of beta subunit immunofluorescence in A:A (beta subunit siRNA-injected) pairs (Fig. 4a), normal beta subunit immunofluorescence in B:B pairs (Fig. 4e), and reduced but obvious beta subunit immunofluorescence in A:B pairs (Fig. 4i). We would conclude that development of the A:B and A:C chimeras to the blastocyst stage (Fig. 3) was achieved by expression of Na/K-ATPase beta subunits by cells derived from the B or C embryo. This experiment, therefore, supports our initial conclusion that Na/K-ATPase beta subunit expression is required for progression to the blastocyst stage.


Figure 2
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  		FIGURE 2.
Na/K-ATPase beta1 subunit protein levels after microinjection of beta1 siRNAs. The distribution of Na/K-ATPase beta1 subunit polypeptides during murine preimplantation development was assessed (e, f, g, and h). Green, red, and blue colors in each representative photomicrograph indicate positive staining for the respective primary antibody, F-actin (rhodamine phalloidin), and nuclei (4,6-diamidino-2-phenylindole), respectively. Panels a, b, and c display a marked reduction in Na/K-ATPase beta1 subunit fluorescence in embryos microinjected with beta1 subunit siRNAs. Panel d shows the presence of multiple blastocoele cavities, which failed to coalesce to form a single blastocoele cavity in one of the rare (i.e. 2.3%) of Na/K-ATPase beta1 siRNA-injected zygotes that progressed to the blastocyst stage.

 


Figure 3
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  		FIGURE 3.
Morphology of chimeric embryos. Panel A, representative figures of chimeric embryos produced by pairing either beta1 subunit-deficient 8-cell embryos together (A:A) or to siRNA random sequence-injected controls (A:B) or to uninjected controls (A:C) or sequence siRNA-injected controls together (B:B). Although A:A embryos demonstrated a developmental blockade after compaction, A:B, A:C, and B:B embryos all progressed normally to the blastocyst stage. Panel B, the percentage of embryos that developed into blastocysts after eight-cell aggregation is shown. Values with different symbols represent significant differences. p value <0.05.

 
These studies clearly indicated that down-regulation of Na/K-ATPase beta1 subunit expression resulted in a blockade of blastocyst formation. To begin to define the Na/K-ATPase beta1 subunit role in mediating blastocyst formation, we investigated the effects of beta1 subunit down-regulation on the distribution of Na/K-ATPase {alpha}1 polypeptides and tight junction-associated polypeptides, occludin and ZO-1, since these polypeptides are among the best characterized markers of trophectoderm differentiation and blastocyst formation. We confined our analysis to morula stage embryos since it was not possible to generate blastocysts after microinjection of Na/K-ATPase beta1 subunit siRNAs. In control-injected and also control non-injected morulae we observed normal patterns of Na/K-ATPase {alpha} and ZO-1 and occludin protein distribution (Fig. 5, b, d, and f). For Na/K-ATPase {alpha}1 protein, the fluorescence extended in an apolar fashion surrounding each morula blastomere (Fig. 5b). Like-wise both ZO-1 and occludin fluorescence maintained a tight cortical pattern surrounding the apical regions of each blastomere (Fig. 5, d and f). In complete contrast, Na/K-ATPase beta1 siRNA-injected morulae displayed a very aberrant Na/K-ATPase {alpha}1 fluorescence pattern in which the fluorescence became "discontinuous" around each cell periphery, consisting more of a general cytoplasmic distribution rather than a cortical distribution (Fig. 5a). The impact on ZO-1 and occludin protein distribution was even more dramatic, and the fluorescence pattern for both of these tight junction-associated proteins became very diffuse and cytoplasmic in nature (Fig. 5, c and e). Injection of Na/K-ATPase beta1 siRNAs resulted in a complete loss of the tight "continuous" cortical distribution that is so typical for both ZO-1 and occludin protein distributions in developing epithelia such as the trophectoderm.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our results demonstrate that the Na/K-ATPase beta1 subunit protein is required for blastocyst formation. In addition, our results indicate that the Na/K-ATPase beta1 subunit oversees the proper localization of Na/K-ATPase {alpha}1 subunit to the cortical membrane regions of each blastomere and also the proper distribution and assembly of tight junction-associated polypeptides (ZO-1 and occludin) to the apical membrane regions between differentiating trophectoderm cells. Interestingly, our results do not, however, indicate that the Na/K-ATPase beta1 subunit is required to support early development to the morula (16-32 cells) stage of mouse development. We conclude that the Na/K-ATPase beta1 subunit is instrumental in coordinating the proper insertion of Na/K-ATPase {alpha}/beta subunits to appropriate membrane domains and also the formation and establishment of trophectoderm tight junctions. Thus, the Na/K-ATPase beta1 subunit is an important mediator of cell polarity, trophectoderm differentiation, and blastocyst formation during mouse preimplantation development.

The Na/K-ATPase beta subunit has been generally prescribed to serve two primary roles including 1) a chaperone role that directs the insertion of the {alpha} subunit and functional enzyme unit to the appropriate membrane domain (22-25) and 2) the regulation of cation sensitivity of the Na/K-ATPase (26-28). Most recently, however, an additional role in regulating epithelial cell phenotype and cell motility has been defined for the Na/K-ATPase beta subunit (29-35). We would suggest that the results from our study support the likelihood that the Na/K-ATPase beta1 subunit contributes to all three roles during blastocyst formation in the mouse.


Figure 4
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  		FIGURE 4.
Na/K-ATPase beta1 subunit protein levels after chimera production. The distribution of Na/K-ATPase beta1 subunit polypeptides was assessed between chimeric embryos produced by pairing either beta1 subunit deficient eight-cell embryos together (A:A) or to siRNA random sequence-injected controls (A:B) or pairing random sequence siRNA-injected controls together (B:B). Green, red, and blue colors in each representative photomicrograph indicate positive staining for the respective primary antibody (panels a, e, and i), F-actin (rhodamine phalloidin) (b, f, and j), and nuclei (4,6-diamidino-2-phenylindole) (c, g, and k), respectively. Panels d, h, and l show a composite image of all three channels.

 


Figure 5
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  		FIGURE 5.
Distribution of Na/K-ATPase {alpha}1 subunit (a and b), ZO-1 (c and d), and occludin (e and f) proteins in Na/K-ATPase beta1 subunit siRNA-microinjected embryos or control-microinjected embryos. Note the discontinuous distribution of Na/K-ATPase {alpha}1 subunit in the Na/K-ATPase beta1 subunit siRNA-injected embryos. Loss of Na/K-ATPase beta1 subunit also resulted in the shift from a continuous to discontinuous distribution for both ZO-1 and occludin polypeptides. In addition, both ZO-1 and occludin immunofluorescence transitioned from a very tight focused signal adjacent to cell margins to become predominantly discontinuous and cytoplasmic in distribution.

 
Our results certainly indicate that we were able to specifically down-regulate Na/K-ATPase beta1 subunit expression by the microinjection of beta1 subunit-specific siRNAs as we were unable to detect the presence of either Na/K-ATPase beta1 subunit mRNAs or polypeptides in beta1 subunit siRNA-microinjected zygotes. This result coupled with the aberrant Na/K-ATPase {alpha}1 subunit distribution that we observed in the Na/K-ATPase beta1 subunit siRNA-microinjected embryos has lead us to conclude that the Na/K-ATPase beta1 subunit is required to direct the proper targeting and insertion of the Na/K-ATPase {alpha}1 subunit into the trophectoderm cell membranes, and without this occurring, blastocyst formation is blocked.

But how might the Na/K-ATPase beta1 subunit-microinjected zygotes survive to the morula stage? We have known for some time that the Na/K-ATPase beta1 subunit gene products display a very dramatic up-regulation just before blastocyst formation, suggesting that the up-regulation of this gene is required for cavitation to occur (3-5). Na/K-ATPase {alpha} subunit mRNAs are present throughout preimplantation development and display a much more gradual increase as the embryo progresses to the blastocyst stage (3-5). It is very likely that Na/K-ATPase {alpha} subunits outnumber beta subunits by a wide margin in early embryo stages and, thus, the full up-regulation of the Na/K-ATPase required to support cavitation cannot occur until Na/K-ATPase beta1 subunits increase to more closely match {alpha} subunit levels (3-5). As well, mRNAs and polypeptides encoding the other Na/K-ATPase beta subunits (beta2, beta3) are also present in preimplantation embryos (5, 7). We would suggest that the "housekeeping" and early cleavage stage (1-cell to morula) requirements for the Na/K-ATPase may be handled by primarily {alpha}1/beta2or {alpha}1/beta3 isoenzymes but that at the morula stage the up-regulation of Na/K-ATPase beta1 subunit gene products provides for the overall increase in enzyme ({alpha}1/beta1 isozymes) that is required to drive blastocyst formation. Alternatively, despite not detecting mRNAs and polypeptides encoding the Na/K-ATPase beta1 subunit within beta1 subunit siRNA-microinjected embryos, there may still be sufficient protein present that is below the level of immunofluorescence detection but is sufficient to pair up with Na/K-ATPase {alpha}1 subunits to satisfy the housekeeping requirement of the enzyme up to the morula stage. Our results clearly indicate that the Na/K-ATPase beta1 subunit is required to support blastocyst formation and proper insertion of Na/K-ATPase {alpha}1 subunits into trophectoderm membranes.

We have employed the eight-cell aggregation chimera method (36) to investigate the reversibility of the developmental blockade produced by the loss of Na/K-ATPase beta1 subunit embryos and confirm the presence of beta subunit protein with development to the blastocyst stage. Our results have demonstrated that when beta1 subunit siRNA-microinjected eight-cell embryos are paired with either random sequence siRNA or uninjected controls, they go on to form blastocysts in normal proportions, whereas those beta1 subunit embryos that were paired together failed to develop to the blastocyst stage. This finding is consistent with our previous observation that down-regulation of beta1 subunit expression is associated with failure to support development to the blastocyst stage.

More recently, a series of important studies primarily from Rajasekaran et al. (29-31, 37) have demonstrated that the Na/K-ATPase also has roles in regulating cell motility, oncogenic transformation, epithelial to mesenchymal cell transition, and tight junction formation and function. Madin-Darby canine kidney cells (MDCK) transformed by Moloney sarcoma virus (MSV-MDCK) express reduced levels of the Na/K-ATPase beta1 subunit compared with non-transformed MDCK cells (31). When the Na/K-ATPase beta1 subunit levels are increased in MSV-MDCK cells, one observes a corresponding increase in Na/K-ATPase {alpha}1 subunit protein due to enhanced {alpha}1 subunit translation (31). In addition, MSV-MDCK cells display a reduced cell motility (correlated with reduced Na/K-ATPase beta1 levels) that is overcome by also increasing Na/K-ATPase beta1 subunit expression in these cells (32, 34). Further studies have indicated that Na/K-ATPase beta1 subunit levels may be regulated by the transcription factor "snail," as increased snail levels result in reduced Na/K-ATPase beta1 subunit levels, and blockade of snail levels increased Na/K-ATPase beta1 levels (37). Collectively these studies demonstrate that the Na/K-ATPase beta1 subunit is a critical regulator of Na/K-ATPase {alpha}1 subunits and, thus, enzyme function and that Na/K-ATPase subunit levels are important mediators of the epithelial cell phenotype and cell motility.

In addition, recent studies have also demonstrated that the Na/K-ATPase has an important role in regulating tight junction formation and function (32-35). Tight junctions are composed of several transmembrane proteins including claudins and occludin and several associated proteins including ZO-1, ZO-2, cingulin, and 7H6 that link the junctional unit to the cell's actin cytoskeleton (38-41). Inhibition of Na/K-ATPase by ouabain treatment is associated with aberrant distribution of tight junction-associated proteins and also tight junction function, as indicated by increased permeability to fluorescein isothiocyanate-labeled dextran molecules in both cell lines (32, 34) and mouse blastocysts (17). We discovered that the disruption of ZO-1 tight junction protein distribution after ouabain treatment of early mouse embryos was correlated with re-arrangements in occludin distribution, and these effects provided a basis for understanding the increased permeability to fluorescein isothiocyanate-dextran that ouabain-treated embryo displayed (17). We have, therefore, concluded that Na+/K+-ATPase is a regulator of tight junction paracellular permeability in mouse blastocysts as well as epithelial cell lines. The outcomes from the present study also support this conclusion as we observed a dramatic shift in normal protein distribution for both ZO-1 and occludin in all Na/K-ATPase beta1 subunit siRNA-microinjected embryos. It is our expectation that the aberrant protein distribution patterns we observed in these embryos are indicative of a disrupted tight junction formation in Na/K-ATPase beta1 subunit siRNA-microinjected embryos that would result in a failure in establishing a proper trophectoderm tight junction seal that is required to allow for expansion of the blastocyst cavity during cavitation. Thus, the failure of Na/K-ATPase beta1 subunit siRNA-microinjected embryos to form blastocysts could have resulted from both a mis-targeting and proper insertion of Na/K-ATPase {alpha} subunits into the appropriate trophectoderm domains and also due to a disruption of tight junction assembly and establishment of the trophectoderm tight junctional seal.

How might the Na/K-ATPase regulate tight junction formation? It seems likely that influences are mediated via regulation of the actin cytoskeleton. A model proposed by Barwe et al. (29) suggests that the Na/K-ATPase {alpha} subunit is able to phosphorylate p85, which recruits this subunit to the plasma membrane and results in activation of phosphatidylinositol 3-kinase and the generation of inositol 1,4,5-trisphosphate, which induces the binding of annexin II to the Na/K-ATPase beta subunit cytoplasmic tail, activation of the Rac1 RhoGTPase, and modifications to the actin cytoskeleton. Studies have shown that loss of tight junction structure and increased paracellular permeability is often linked to the disruption of the circumferential actin ring that is localized at the apical pole of polarized epithelial cells (42-44). In MDCK cells, loss of tight junction permeability upon Na+/K+-ATPase inhibition also is associated with reduced stress fiber content and reduced RhoA activity (34). Overexpression of RhoA can bypass the inhibitory effect of Na+/K+-ATPase inhibition on tight junction formation, indicating that RhoA is essential and may be downstream of Na+/K+-ATPase in regulating tight junction function (34). These possibilities certainly offer exciting directions to follow in our efforts to understand Na/K-ATPase function during early development and, in particular, its role in regulating tight junction formation and function in the early embryo.

In conclusion, blockade of Na/K-ATPase beta1 subunit expression by microinjection of Na/K-ATPase beta1 siRNAs resulted in failure of microinjected embryos to develop to the blastocyst stage, which was associated with aberrant expression of Na/K-ATPase {alpha}1 and tight junction ZO-1 and occludin polypeptides, indicating that the Na/K-ATPase beta1 subunit is a potent regulator of both Na/K-ATPase {alpha}1 subunit membrane insertion and also tight junction protein assembly during preimplantation development.


   FOOTNOTES
 
* This work was supported by a Canadian Institutes of Health Research operating grant (to A. J. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: CHRI-VRL (5th floor), 800 Commissioners Rd. East, London, Ontario N6A 4G5, Canada. Tel.: 519-685-8500 (ext. 55068); Fax: 519-685-8186; E-mail awatson{at}uwo.ca.

2 The abbreviations used are: siRNA, small interfering RNA; KSOM, potassium simplex optimized medium; KSOMaa, KSOM amino acids; RT, reverse transcription; PBS, phosphate-buffered saline; MDCK, Madin-Darby canine kidney cells. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Gerald M. Kidder and Michele Calder for constructive comments and critically reviewing the manuscript. We appreciate the assistance of Barry Fong and Jenny Hickson for assistance with embryo collections.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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