Na/K-ATPase β1 Subunit Expression Is Required for Blastocyst Formation and Normal Assembly of Trophectoderm Tight Junction-associated Proteins
2007; Elsevier BV; Volume: 282; Issue: 16 Linguagem: Inglês
10.1074/jbc.m700696200
ISSN1083-351X
AutoresPavneesh Madan, Keeley Rose, Andrew J. Watson,
Tópico(s)Reproductive Biology and Fertility
ResumoNa/K-ATPase plays an important role in mediating blastocyst formation. Despite the expression of multiple Na/K-ATPase α and β isoforms during mouse preimplantation development, only the α1 and β1 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 β1 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 Stealth™ Na/K-ATPase β1 subunit oligos or nonspecific Stealth™ 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 β1 subunit siRNA oligos developed to the blastocyst stage as compared with 73% for control-injected and 91% for non-injected controls. Na/K-ATPase β1 subunit down-regulation was validated by employing reverse transcription-PCR and whole-mount immunofluorescence methods to demonstrate that Na/K-ATPase β1 subunit mRNAs and protein were not detectable in β1 subunit siRNA-microinjected embryos. Aggregation chimera experiments between β1 subunit siRNA-microinjected embryos and controls demonstrated that blockade of blastocyst formation was reversible. The distribution of Na/K-ATPase α1 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 β1 subunit siRNA-microinjected embryos. Our results demonstrate that the β1 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. Na/K-ATPase plays an important role in mediating blastocyst formation. Despite the expression of multiple Na/K-ATPase α and β isoforms during mouse preimplantation development, only the α1 and β1 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 β1 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 Stealth™ Na/K-ATPase β1 subunit oligos or nonspecific Stealth™ 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 β1 subunit siRNA oligos developed to the blastocyst stage as compared with 73% for control-injected and 91% for non-injected controls. Na/K-ATPase β1 subunit down-regulation was validated by employing reverse transcription-PCR and whole-mount immunofluorescence methods to demonstrate that Na/K-ATPase β1 subunit mRNAs and protein were not detectable in β1 subunit siRNA-microinjected embryos. Aggregation chimera experiments between β1 subunit siRNA-microinjected embryos and controls demonstrated that blockade of blastocyst formation was reversible. The distribution of Na/K-ATPase α1 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 β1 subunit siRNA-microinjected embryos. Our results demonstrate that the β1 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. 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 (1Watson A.J. Kidder G.M. Dev. Biol. 1988; 126: 80-90Crossref PubMed Scopus (122) Google Scholar, 2Betts D.H. Barcroft L.C. Watson A.J. Dev. Biol. 1998; 197: 77-92Crossref PubMed Scopus (43) Google Scholar), (b) expression of Na/K-ATPase subunit genes are up-regulated during the morula to blastocyst transition (3Watson A.J. Pape C. Emanuel J.R. Levenson R. Kidder G.M. Dev. Genet. 1990; 11: 41-48Crossref PubMed Scopus (59) Google Scholar, 4Gardiner C.S. Williams J.S. Menino Jr., A.R. Biol. Reprod. 1990; 43: 788-794Crossref PubMed Scopus (30) Google Scholar, 5Betts D.H. MacPhee D.J. Kidder G.M. Watson A.J. Mol. Reprod. Dev. 1997; 46: 114-126Crossref PubMed Scopus (52) Google Scholar, 6Waelchli R.O. MacPhee D.J. Kidder G.M. Betteridge K.J. Biol. Reprod. 1997; 57: 630-640Crossref PubMed Scopus (16) Google Scholar, 7MacPhee D.J. Jones D.H. Barr K.J. Betts D.H. Watson A.J. Kidder G.M. Dev. Biol. 2000; 222: 486-498Crossref PubMed Scopus (47) Google Scholar), (c) that Na/K-ATPase activity is significantly increased during the morula to blastocyst transition for a number of mammalian species (2Betts D.H. Barcroft L.C. Watson A.J. Dev. Biol. 1998; 197: 77-92Crossref PubMed Scopus (43) Google Scholar, 8Van Winkle L.J. Campione A.L. Dev. Biol. 1991; 146: 158-166Crossref PubMed Scopus (34) Google Scholar, 9Dumoulin J.C. Evers J.L. Michiels A.H. Pieters M.H. Bras M. Land J.A. Geraedts J.P. Mol. Reprod. Dev. 1993; 36: 320-327Crossref PubMed Scopus (14) Google Scholar, 10Houghton F.D. Humpherson P.G. Hawkhead J.A. Hall C.J. Leese H.J. Dev. Biol. 2003; 263: 360-366Crossref PubMed Scopus (51) Google Scholar), (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 (5Betts D.H. MacPhee D.J. Kidder G.M. Watson A.J. Mol. Reprod. Dev. 1997; 46: 114-126Crossref PubMed Scopus (52) Google Scholar, 11DiZio S.M. Tasca R.J. Dev. Biol. 1977; 59: 198-205Crossref PubMed Scopus (96) Google Scholar, 12Biggers J.D. Borland R.M. Lechene C.P. J. Physiol. (Lond.). 1978; 280: 319-330Crossref Scopus (24) Google Scholar, 13Benos D.J. Dev. Biol. 1981; 83: 69-78Crossref PubMed Scopus (16) Google Scholar, 14Wiley L.M. Dev. Biol. 1984; 105: 330-342Crossref PubMed Scopus (102) Google Scholar, 15Overstrom E.W. Benos D.J. Biggers J.D. J. Reprod. Fertil. 1989; 85: 283-295Crossref PubMed Scopus (18) Google Scholar), (e) that deletion of the Na/K-ATPase α1 subunit gene product is linked to aberrant blastocyst formation in vitro and likely peri-implantation lethality in vivo (16Barcroft L.C. Moseley A.E. Lingrel J.B. Watson A.J. Mech. Dev. 2004; 121: 417-426PubMed Google Scholar), and (f) that Na/K-ATPase also regulates the formation and function of trophectoderm tight junctions (17Violette M.I. Madan P. Watson A.J. Dev. Biol. 2006; 289: 406-419Crossref PubMed Scopus (57) Google Scholar). 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 (16Barcroft L.C. Moseley A.E. Lingrel J.B. Watson A.J. Mech. Dev. 2004; 121: 417-426PubMed Google Scholar) and to explore whether the Na+ pump also regulates tight junction formation and function during blastocyst formation (17Violette M.I. Madan P. Watson A.J. Dev. Biol. 2006; 289: 406-419Crossref PubMed Scopus (57) Google Scholar). 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 α1 null blastocysts from the reproductive tracts of heterozygous mice (16Barcroft L.C. Moseley A.E. Lingrel J.B. Watson A.J. Mech. Dev. 2004; 121: 417-426PubMed Google Scholar). However, these α1 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 (16Barcroft L.C. Moseley A.E. Lingrel J.B. Watson A.J. Mech. Dev. 2004; 121: 417-426PubMed Google Scholar). Therefore, we have concluded that the α1 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 α subunit isoform is able to allow for short term development of α1 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 β1 subunit by employing microinjection of 1 cell mouse zygotes with small interfering RNA (siRNA) 2The 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. oligos. Our results demonstrate that the β1 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. 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 10× Leibovitz-modified Hanks' balanced salt solution, all diluted with water to 1×) (45Spindle A. In Vitro. 1980; 16: 669-674Crossref PubMed Scopus (122) Google Scholar) containing hyaluronidase (1 mg/ml, Sigma). The embryos were washed 3× in potassium simplex optimized medium (KSOM) media (46Summers M.C. Bhatnagar P.R. Lawitts J.A. Biggers J.D. Biol. Reprod. 1995; 53: 431-437Crossref PubMed Scopus (118) Google Scholar) under paraffin oil in sterile culture dishes and subsequently cultured in KSOM medium plus amino acids (KSOMaa) (46Summers M.C. Bhatnagar P.R. Lawitts J.A. Biggers J.D. Biol. Reprod. 1995; 53: 431-437Crossref PubMed Scopus (118) Google Scholar) 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 (18Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63171) Google Scholar). 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 (19Barcroft L.C. Hay-Schmidt A. Caveney A. Gilfoyle E. Overstrom E.W. Hyttel P. Watson A.J. J. Reprod. Fertil. 1998; 114: 327-339Crossref PubMed Scopus (69) Google Scholar, 20Natale D.R. Paliga A.J. Beier F. D'Souza S.J. Watson A.J. Dev. Biol. 2004; 268: 76-88Crossref PubMed Scopus (83) Google Scholar). 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 (21Madan P. Calder M.D. Watson A.J. Reproduction. 2005; 130: 41-51Crossref PubMed Scopus (31) Google Scholar). 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 GenBank™ 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 β1 subunit sequence.TABLE 1Nucleotide sequences for polymerase chain reaction amplification reactionsGene productPrimerPrimer sequenceSizebpNa/K-ATPase β1 subunit (5Betts D.H. MacPhee D.J. Kidder G.M. Watson A.J. Mol. Reprod. Dev. 1997; 46: 114-126Crossref PubMed Scopus (52) Google Scholar)5′TTCAGCCCAGAAGGACGACATG3783′AGGGAAGCCGTAGTATCCGCCCAβ-Actin5′CGTGGGCCGCCCTAGGCACCA2483′GGGGGGACTTGGGATTCCGGTT Open table in a new tab Embryo Fixation and Whole-mount Indirect Immunofluorescence—To analyze the distribution of Na/K-ATPase α and β isoforms, ZO-1, and occludin polypeptides during murine preimplantation development, a whole-mount immunofluorescence method previously described (21Madan P. Calder M.D. Watson A.J. Reproduction. 2005; 130: 41-51Crossref PubMed Scopus (31) Google Scholar) was employed. Immunofluorescence was detected using laser scanning confocal microscopy as described (19Barcroft L.C. Hay-Schmidt A. Caveney A. Gilfoyle E. Overstrom E.W. Hyttel P. Watson A.J. J. Reprod. Fertil. 1998; 114: 327-339Crossref PubMed Scopus (69) Google Scholar). Briefly, embryos at timed stages of development (1-cell zygotes, 2-, 4-, and 8-cell, morula, and blastocyst stages) were washed in 1× phosphate-buffered saline (PBS) and then fixed in 2% paraformaldehyde in PBS for 20 min at room temperature. These fixed embryos were washed in 1× 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 1× PBS + 5% donkey serum + 0.01% Triton X-100 for 1 h at room temperature. Embryos were washed in 1× PBS and incubated with primary antibody diluted 1:100 in 1× 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 20-40× magnification. The images were then captured and stored as TIFF files by the Zeiss LSM software package. Primary antibodies for Na/K-ATPase α and β 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-iT™ RNAi Designer software (Invitrogen). Stealth siRNA duplex oligoribonucleotides against the β1 subunit of Na/K-ATPase (GenBank™ accession number NM_009721) 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 (β1 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 β1 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 β 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. To investigate the consequences of Na/K-ATPase β1 subunit down-regulation on preimplantation development, we employed microinjection of β1 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 β1 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 β1 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 β1 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 β1 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 β1 siRNAs we employed were effective at down-regulating Na/K-ATPase β1 gene expression during early development, we applied RT-PCR methods to detect Na/K-ATPase β1 mRNAs and also whole mount immunofluorescence methods to map out Na/K-ATPase β1 protein distribution. It was readily possible to detect transcripts encoding the Na/K-ATPase β1 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 β1 RT-PCR product in samples prepared from Na/K-ATPase β1 siRNA-injected zygotes (Fig. 1C). To ensure that Na/K-ATPase β1 siRNA prepared samples still retained intact RNA, we employed RT-PCR to amplify transcripts encoding β-actin. In all samples from all treatment groups it was readily possible to detect the expected size β-actin cDNA product. The application of whole-mount immunofluorescence methods employing a Na/K-ATPase β1 subunit-specific antiserum revealed a complete absence of detectable Na/K-ATPase β1 subunit protein in β1 siRNA-injected zygotes (Fig. 2, a-c). In contrast, the Na/K-ATPase β1 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. 7MacPhee D.J. Jones D.H. Barr K.J. Betts D.H. Watson A.J. Kidder G.M. Dev. Biol. 2000; 222: 486-498Crossref PubMed Scopus (47) Google Scholar). In control blastocysts the Na/K-ATPase β1 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 β1 siRNA-injected treatment group, no organized Na/K-ATPase β1 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 β1 siRNA we employed in this study effectively down-regulated Na/K-ATPase β1 subunit expression for at least 5 days during mouse preimplantation development. We investigated the reversibility of the developmental blockade displayed by β1 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 β1 subunit siRNA-microinjected embryos (Fig. 3A, A:A) did not progress to the blastocyst stage. In contrast, β1 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 β subunit polypeptides in these chimeras by the application of immunofluorescence methods confirmed the complete absence of β subunit immunofluorescence in A:A (β subunit siRNA-injected) pairs (Fig. 4a), normal β subunit immunofluorescence in B:B pairs (Fig. 4e), and reduced but obvious β 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 β subunits by cells derived from the B or C embryo. This experiment, therefore, supports our initial conclusion that Na/K-ATPase β subunit expression is required for progression to the blastocyst stage.FIGURE 4Na/K-ATPase β1 subunit protein levels after chimera production. The distribution of Na/K-ATPase β1 subunit polypeptides was assessed between chimeric embryos produced by pairing either β1 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) These studies clearly indicated that down-regulation of Na/K-ATPase β1 subunit expression resulted in a blockade of blastocyst formation. To begin to define the Na/K-ATPase β1 subunit role in mediating blastocyst formation, we investigated the effects of β1 subunit down-regulation on the distribution of Na/K-ATPase α1 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 β1 subunit siRNAs. In control-injected and also control non-injected morulae we observed normal patterns of Na/K-ATPase α and ZO-1 and occludin protein distribution (Fig. 5, b, d, and f). For Na/K-ATPase α1 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 β1 siRNA-injected morulae displayed a very aberrant Na/K-ATPase α1 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 β1 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. Our results demonstrate that the Na/K-ATPase β1 subunit protein is required for blastocyst formation. In addition, our results indicate that the Na/K-ATPase β1 subunit oversees the proper localization of Na/K-ATPase α1 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 β1 subunit is required to support early development to the morula (16-32 cells) stage of mouse development. We conclude that the Na/K-ATPase β1 subunit is instrumental in coordinating the proper insertion of Na/K-ATPase α/β subunits to appropriate membrane domains and also the formation and establishment of trophectoderm tight junctions. Thus, the Na/K-ATPase β1 subunit is an important mediator of cell polarity, trophectoderm differentiation, and blastocyst formation during mouse preimplantation development. The Na/K-ATPase β subunit has been generally prescribed to serve two primary roles including 1) a chaperone role that directs the insertion of the α subunit and functional enzyme unit to the appropriate membrane domain (22Geering K. J. Bioenerg. Biomembr. 2001; 33: 425-438Crossref PubMed Scopus (270) Google Scholar, 23Geering K. Theulaz I. Verrey F. Hauptle M.T. Rossier B.C. Am. J. Physiol. 1989; 257: C851-C858Crossref PubMed Google Scholar, 24Beguin P. Hasler U. Beggah A. Horisberger J.D. Geering K. J. Biol. 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