Na/K-ATPase β1 Subunit Expression Is Required for Blastocyst Formation and Normal Assembly of Trophectoderm Tight Junction-associated Proteins

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. 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 medium was before each and was The of the medium was each it was and between All experiments in this study a treatment of 1 of culture and was to the of the of of was under an using a to Quebec, 10 of siRNA into the of embryos. of embryos was to a One-cell embryos were placed in medium under was used to the embryos during with was into the of each followed by the microinjection of of embryos were cultured in medium as for to to allow for an of developmental to the blastocyst embryos were used for each experimental and in a of three were RNA and RNA was from embryos of at 2-, 4-, and blastocyst using the of and P. PubMed Scopus Google Scholar). The RNA were with to possible from The were conducted using Canada) as L.C. A. A. E.W. P. Watson A.J. J. Reprod. Fertil. 1998; PubMed Scopus Google Scholar, A.J. Watson A.J. Dev. Biol. 2004; PubMed Scopus Google Scholar). were for at °C in a of of mm mm 3 mm 10 mm 0.5 mm and of followed by the to °C for for of was conducted using a P. Watson A.J. PubMed Scopus Google Scholar). two for each stage of development under were used was a of three from of three developmental of embryos. The for each are in were containing 0.5 the of each were from the and using a and for Research The was subsequently compared with in data and in all the of each product was was to mouse Na/K-ATPase β1 subunit for 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 in a Embryo and the distribution of Na/K-ATPase α and β and occludin polypeptides during preimplantation development, a whole-mount immunofluorescence P. Watson A.J. PubMed Scopus Google Scholar) was was using as L.C. A. A. E.W. P. Watson A.J. J. Reprod. Fertil. 1998; PubMed Scopus Google Scholar). embryos at stages of development 2-, 4-, and and blastocyst were washed in phosphate-buffered and in in for at embryos were washed in and either for or at °C in for a of embryos were and in 5% serum for 1 h at were washed in and with diluted in serum for 1 h at followed by 1 h at 37 were by for 1 h with diluted were with and (1 for at 37 °C followed by for h each at 37 were in were using a with an under The were and as by the for Na/K-ATPase α and β and were from for occludin was from siRNA siRNA oligos were using siRNA the β1 subunit of Na/K-ATPase number were by The were as control The were in water to a and at °C until of by Aggregation from the three treatment groups subunit and non-injected were collected at the eight-cell embryos were with to eight-cell embryos were washed three in and for chimera The β1 subunit siRNA-microinjected embryos were either with or with controls or controls In injected controls were also to as an control. In were from embryos 3 Development of each chimera to the blastocyst stage was In from each were for immunofluorescence localization of Na/K-ATPase β subunit polypeptides as results are as the from three differences between were by of were considered differences between the were determined using the investigate the consequences of Na/K-ATPase β1 subunit down-regulation preimplantation development, we microinjection of β1 into Our control groups of non-injected zygotes and injected with a In zygotes were placed into each treatment for of developmental The was three using zygotes collected from mouse each The of zygotes in each after is displayed in injected with the Na/K-ATPase β1 siRNA developed to the morula stage and In zygotes injected with control siRNA or non-injected controls displayed a of development to the blastocyst stage after and In injected groups we observed a number of zygotes that did not likely to by the microinjection and the data were and we observed that with the Na/K-ATPase β1 subunit siRNA in a in the of zygotes that development to the blastocyst stage and control non-injected zygotes displayed a blastocyst developmental The of β1 zygotes and at the morula stage after to in the Na/K-ATPase β1 siRNA treatment with to the morula stage by their culture for an h did not the development of embryos in this to the blastocyst stage not that the Na/K-ATPase β1 we were at Na/K-ATPase β1 gene expression during early development, we methods to Na/K-ATPase β1 mRNAs and also immunofluorescence methods to Na/K-ATPase β1 protein was possible to the Na/K-ATPase β1 subunit in control-injected and non-injected control groups as by the of an product in from groups In it was not possible to the Na/K-ATPase β1 product in from Na/K-ATPase β1 zygotes that Na/K-ATPase β1 siRNA we to In all from all treatment groups it was possible to the The of whole-mount immunofluorescence methods employing a Na/K-ATPase β1 a of detectable Na/K-ATPase β1 subunit protein in β1 zygotes In the Na/K-ATPase β1 subunit was in control-injected and non-injected controls at the stage and also in and blastocyst stages as 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 the polarized distribution in the trophectoderm and an distribution in the cell we the blastocysts that in the Na/K-ATPase β1 treatment Na/K-ATPase β1 subunit protein distribution a small of were observed in these embryos In these demonstrate that the Na/K-ATPase β1 siRNA we in this study Na/K-ATPase β1 subunit expression for at during mouse preimplantation development. the of the developmental blockade displayed by β1 subunit siRNA-microinjected embryos by eight-cell embryos from all three treatment groups to chimera formation and to the blastocyst The β1 subunit siRNA-microinjected embryos did not to the blastocyst In β1 subunit siRNA-microinjected were with either siRNA-microinjected controls or injected controls all displayed a normal to the blastocyst stage In localization of Na/K-ATPase β subunit polypeptides in these by the of immunofluorescence methods the of β subunit immunofluorescence in subunit normal β subunit immunofluorescence in and β subunit immunofluorescence in that development of the and to the blastocyst stage was by expression of Na/K-ATPase β by from the or our initial that Na/K-ATPase β subunit expression is required for to the blastocyst β1 subunit protein after chimera The distribution of Na/K-ATPase β1 subunit polypeptides was between embryos by either β1 subunit eight-cell embryos or to siRNA controls or controls and in each for the and and and and and a of all three studies that down-regulation of Na/K-ATPase β1 subunit expression in a blockade of blastocyst formation. to the Na/K-ATPase β1 subunit role in mediating blastocyst we the of β1 subunit down-regulation the distribution of Na/K-ATPase α1 polypeptides and tight junction-associated occludin and these polypeptides are among the of trophectoderm and blastocyst formation. confined our to morula stage embryos it was not possible to blastocysts after microinjection of Na/K-ATPase β1 subunit In control-injected and also control non-injected we observed normal of Na/K-ATPase α and ZO-1 and occludin protein distribution and Na/K-ATPase α1 the in an each morula ZO-1 and occludin a tight the regions of each and In Na/K-ATPase β1 displayed a aberrant Na/K-ATPase α1 in the each cell of a distribution a distribution The ZO-1 and occludin protein distribution was and the for of these tight junction-associated proteins and in and of Na/K-ATPase β1 in a of the tight distribution that is for ZO-1 and occludin protein in as the trophectoderm. Our results demonstrate that the Na/K-ATPase β1 subunit protein is required for blastocyst formation. In our results that the Na/K-ATPase β1 subunit the localization of Na/K-ATPase α1 subunit to the membrane regions of each and also the distribution and of tight junction-associated polypeptides and to the membrane regions between trophectoderm cells. our results do however, that the Na/K-ATPase β1 subunit is required to support early development to the morula stage of mouse development. that the Na/K-ATPase β1 subunit is in the of Na/K-ATPase to membrane and also the formation and of trophectoderm tight the Na/K-ATPase β1 subunit is an important of cell trophectoderm and blastocyst formation during mouse preimplantation development. The Na/K-ATPase β subunit has been to two roles a role that the of the α subunit and to the membrane J. PubMed Scopus Google Scholar, J. Physiol. 1989; PubMed Google Scholar, P. A. J.D. J. Biol. 1998; PubMed Scopus Google Scholar, P. Mol. Biol. 2000; 11: PubMed Scopus (52) Google Scholar) and the of of the Na/K-ATPase A. P. P. J. Biol. PubMed Scopus Google Scholar, P. J.D. J. Physiol. PubMed Scopus Google Scholar, P. J.D. J. Biol. 1998; PubMed Scopus Google Scholar). however, an role in cell and cell has been defined for the Na/K-ATPase β subunit J.P. Mol. Biol. 16: PubMed Scopus Google Scholar, J. 2003; PubMed Scopus Google Scholar, J. C. J.L. Mol. Biol. 2004; PubMed Scopus Google Scholar, Jr., A. Mol. Biol. PubMed Scopus Google Scholar, J. Physiol. Physiol. 2003; PubMed Scopus Google Scholar, A. Mol. Biol. PubMed Scopus Google Scholar, Scholar). that the results from our study support the that the Na/K-ATPase β1 subunit contributes to all three roles during blastocyst formation in the Our results that we were able to down-regulate Na/K-ATPase β1 subunit expression by the microinjection of β1 as we were to the presence of either Na/K-ATPase β1 subunit mRNAs or polypeptides in β1 subunit siRNA-microinjected with the aberrant Na/K-ATPase α1 subunit distribution that we observed in the Na/K-ATPase β1 subunit siRNA-microinjected embryos has to that the Na/K-ATPase β1 subunit is required to the and of the Na/K-ATPase α1 subunit into the trophectoderm cell and this blastocyst formation is the Na/K-ATPase β1 zygotes to the morula have for that the Na/K-ATPase β1 subunit gene a just before blastocyst that the of this gene is required for cavitation to (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). Na/K-ATPase α subunit mRNAs are present preimplantation development and a as the to the blastocyst stage (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). is likely that Na/K-ATPase α β by a in early stages the of the Na/K-ATPase required to support cavitation until Na/K-ATPase β1 to α subunit (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). mRNAs and polypeptides the Na/K-ATPase β are also present in preimplantation embryos (5Betts D.H. MacPhee D.J. Kidder G.M. Watson A.J. Mol. Reprod. Dev. 1997; 46: 114-126Crossref PubMed Scopus (52) 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). that the and early stage to for the Na/K-ATPase by that at the morula stage the of Na/K-ATPase β1 subunit gene for the in that is required to blastocyst formation. not mRNAs and polypeptides the Na/K-ATPase β1 subunit β1 subunit siRNA-microinjected protein present that is the of immunofluorescence is to with Na/K-ATPase α1 to the of the to the morula Our results that the Na/K-ATPase β1 subunit is required to support blastocyst formation and of Na/K-ATPase α1 into trophectoderm have the eight-cell chimera Mol. Biol. 2000; Google Scholar) to investigate the of the developmental blockade by the of Na/K-ATPase β1 subunit embryos and the presence of β subunit protein with development to the blastocyst Our results have demonstrated that β1 subunit siRNA-microinjected eight-cell embryos are with either siRNA or to form blastocysts in normal β1 subunit embryos that were to to the blastocyst is with our that down-regulation of β1 subunit expression is with to support development to the blastocyst a of important studies from J.P. Mol. Biol. 16: PubMed Scopus Google Scholar, J. 2003; PubMed Scopus Google Scholar, J. C. J.L. Mol. Biol. 2004; PubMed Scopus Google Scholar, J. Mol. Biol. 2004; PubMed Scopus Google Scholar) have demonstrated that the Na/K-ATPase also has roles in cell to cell and tight junction formation and Madin-Darby canine kidney by of the Na/K-ATPase β1 subunit compared with J. C. J.L. Mol. Biol. 2004; PubMed Scopus Google Scholar). the Na/K-ATPase β1 subunit are increased in a in Na/K-ATPase α1 subunit protein to α1 subunit J. C. J.L. Mol. Biol. 2004; PubMed Scopus Google Scholar). In a cell with Na/K-ATPase β1 that is by also Na/K-ATPase β1 subunit expression in these Jr., A. Mol. Biol. PubMed Scopus Google Scholar, A. Mol. Biol. PubMed Scopus Google Scholar). studies have that Na/K-ATPase β1 subunit by the as increased in Na/K-ATPase β1 subunit and blockade of increased Na/K-ATPase β1 J. Mol. Biol. 2004; PubMed Scopus Google Scholar). these studies demonstrate that the Na/K-ATPase β1 subunit is a of Na/K-ATPase α1 function and that Na/K-ATPase subunit are important of the cell and cell In recent studies have also demonstrated that the Na/K-ATPase has an important role in tight junction formation and function Jr., A. Mol. Biol. PubMed Scopus Google Scholar, J. Physiol. Physiol. 2003; PubMed Scopus Google Scholar, A. Mol. Biol. PubMed Scopus Google Scholar, Scholar). junctions are of proteins and occludin and proteins and that the to the J. M. J. 1993; PubMed Google Scholar, M. 2000; PubMed Scopus Google Scholar, M. M. A. A. J. Biol. PubMed Scopus Google Scholar, A. P. Mol. Biol. 2003; PubMed Scopus Google Scholar). of Na/K-ATPase by ouabain treatment is with aberrant distribution of tight junction-associated proteins and also tight junction as by increased to in cell Jr., A. Mol. Biol. PubMed Scopus Google Scholar, A. Mol. Biol. PubMed Scopus Google Scholar) and mouse blastocysts (17Violette M.I. Madan P. Watson A.J. Dev. Biol. 2006; 289: 406-419Crossref PubMed Scopus (57) Google Scholar). discovered that the of ZO-1 tight junction protein distribution after ouabain treatment of early mouse embryos was with in occludin and these a for understanding the increased to that displayed (17Violette M.I. Madan P. Watson A.J. Dev. Biol. 2006; 289: 406-419Crossref PubMed Scopus (57) Google Scholar). concluded that is a of tight junction in mouse blastocysts as as cell The from the present study also support this as we observed a in normal protein distribution for ZO-1 and occludin in all Na/K-ATPase β1 subunit siRNA-microinjected embryos. is our that the aberrant protein distribution we observed in these embryos are of a tight junction formation in Na/K-ATPase β1 subunit siRNA-microinjected embryos that in a in a trophectoderm tight junction that is required to allow for of the blastocyst during the of Na/K-ATPase β1 subunit siRNA-microinjected embryos to form blastocysts have from a and of Na/K-ATPase α into the trophectoderm and also to a of tight junction and of the trophectoderm tight the Na/K-ATPase tight junction likely that are of the by J.P. Mol. Biol. 16: PubMed Scopus Google Scholar) that the Na/K-ATPase α subunit is able to this subunit to the membrane and results in of and the of the of to the Na/K-ATPase β subunit of the and to the have that of tight junction and increased is linked to the of the that is localized at the of polarized J. M.H. J. Biol. 1989; PubMed Scopus Google Scholar, A. A. A. Mech. Dev. 2000; PubMed Scopus Google Scholar, 2000; PubMed Google Scholar). In of tight junction also is with and activity A. Mol. Biol. PubMed Scopus Google Scholar). of the of tight junction that is and of in tight junction function A. Mol. Biol. PubMed Scopus Google Scholar). to in our efforts to Na/K-ATPase function during early development in role in tight junction formation and function in the early In blockade of Na/K-ATPase β1 subunit expression by microinjection of Na/K-ATPase β1 in of microinjected embryos to to the blastocyst was with aberrant expression of Na/K-ATPase α1 and tight junction ZO-1 and occludin that the Na/K-ATPase β1 subunit is a potent of Na/K-ATPase α1 subunit membrane and also tight junction protein during preimplantation development. M. Kidder and for and the the of and for with

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