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J. Biol. Chem., Vol. 282, Issue 16, 12127-12134, April 20, 2007

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Na/K-ATPase 1 Subunit Expression Is Required for Blastocyst Formation and Normal Assembly of Trophectoderm Tight Junction-associated Proteins^*

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

Na/K-ATPase plays an important role in mediating blastocyst formation. Despite the expression of multiple Na/K-ATPase and isoforms during mouse preimplantation development, only the 1 and 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/K-ATPase 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 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 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 1 subunit down-regulation was validated by employing reverse transcription-PCR and whole-mount immunofluorescence methods to demonstrate that Na/K-ATPase 1 subunit mRNAs and protein were not detectable in 1 subunit siRNA-microinjected embryos. Aggregation chimera experiments between 1 subunit siRNA-microinjected embryos and controls demonstrated that blockade of blastocyst formation was reversible. The distribution of Na/K-ATPase 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 1 subunit siRNA-microinjected embryos. Our results demonstrate that the 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.

Received for publication, January 24, 2007

^* 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.

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.

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