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

Pavneesh Madan,Keeley Rose,Andrew J Watson

doi:10.1074/jbc.m700696200

Abstract

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 alpha1 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 Stealthtrade mark Na/K-ATPase beta1 subunit oligos or nonspecific Stealthtrade mark 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 alpha1 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.

Highlights

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 [3,4,5,6,7], (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 affects cavitation and blastocyst formation in a number of mammalian species [5, 11,12,13,14,15], (e) that deletion of the Na/K-ATPase ␣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 regulates the formation and function of trophectoderm tight junctions [17]
When the data were plotted and analyzed, we clearly observed that injection with the Na/K-ATPase ␤1 subunit small interfering RNA (siRNA) resulted in a significant reduction in the proportion of zygotes that completed development to the blastocyst stage (Fig. 1B)
Our results indicate that the Na/K-ATPase ␤1 subunit oversees the proper localization of Na/K-ATPase ␣1 subunit to the cortical membrane regions of each blastomere and the proper distribution and assembly of tight junction-associated polypeptides (ZO-1 and occludin) to the apical membrane regions between differentiating trophectoderm cells

Summary

Introduction

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 [3,4,5,6,7], (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, 11,12,13,14,15], (e) that deletion of the Na/K-ATPase ␣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 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 results demonstrate that the ␤1 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

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Conclusion