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 alpha and beta isoforms during mouse preimplantation development, only the alpha 1 and beta 1 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/KATPase beta 1 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 (TM) Na/K- ATPase beta 1 subunit oligos or nonspecific Stealth (TM) 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 beta 1 subunit siRNA oligos developed to the blastocyst stage as compared with 73% for control-injected and 91% for non-injected controls. Na/K-ATPase beta 1 subunit down-regulation was validated by employing reverse transcription-PCR and whole-mount immunofluorescence methods to demonstrate that Na/K-ATPase beta 1 subunit mRNAs and protein were not detectable in beta 1 subunit siRNA-microinjected embryos. Aggregation chimera experiments between beta 1 subunit siRNA-microinjected embryos and controls demonstrated that blockade of blastocyst formation was reversible. The distribution of Na/K-ATPase beta 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 beta 1 subunit siRNA-microinjected embryos. Our results demonstrate that the beta 1 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.