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

Abstract

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/KATPase ␤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 downregulation 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.

Preimplantation embryo programming: transcription, epigenetics, and culture environment

“…In addition, enzyme activity increases just prior to blastocyst formation in all mammalian species examined to date (Watson 1992, Watson & Barcroft 2001. The expression of all the principal Na/K-ATPase isozymes has been defined as well as the functions of Na/K-ATPase a1 and b1 subunits in supporting blastocyst formation in the mouse , MacPhee et al 2000, Barcroft et al 2004, Madan et al 2007). In addition, aquaporins and their role in facilitating blastocyst formation in the mouse have been investigated (Barcroft et al 2003, Offenberg & Thomsen 2005.…”

“…A more direct approach is to alter the levels of mRNA (either under express or overexpress) and look for a phenotypic effect. RNA interference (RNAi) has become a well-established technique to study gene function in several species (Madan et al 2007). In domestic species, however, the use of RNA interference technology in domestic species is still in its infancy with only a handful of papers published on the subject (Tesfaye et al 2007).…”

Binding of FGF2 to FGFR2 in an autocrine mode in trophectoderm cells is indispensable for mouse blastocyst formation through PKC-p38 pathway

“…4,5 The Na-K ATPase is also a critical mediator of blastocyst formation as it establishes a transtrophectoderm ionic gradient that directs fluid movement across the epithelium of the TE. 4,6,7 Aquaporins (AQP3 and AQP9) in the TE membrane facilitate the movement of fluids across the TE from the 'outside' to the 'inside' of the blastocyst cavity, along the ionic gradient established by the Na-K ATPase localized on the baso-lateral membrane. 4,8 After segregation of the early lineages is complete, the blastocyst emerges from the zona pellucida to invade the maternal uterine endometrium, where it implants.…”

“…The Na-K ATPase plays an important role in the establishment of a trans-trophectoderm ionic gradient that directs fluid movement across the TE epithelium during expanded blastocyst formation. 7,39 During the process of Na C transport, Na C crosses the apical membrane via ENaC into TEs and is extruded into the blastocoels by Na-K ATPase on the basal side of the membrane. 7,39,40 Thus, the activities of ENaCa and Na-K ATPase must be highly coordinated to accommodate variations in Na C transport during expanded blastocyst formation.…”

“…7,39 During the process of Na C transport, Na C crosses the apical membrane via ENaC into TEs and is extruded into the blastocoels by Na-K ATPase on the basal side of the membrane. 7,39,40 Thus, the activities of ENaCa and Na-K ATPase must be highly coordinated to accommodate variations in Na C transport during expanded blastocyst formation. In this study, the knockdown of FGF2 or FGFR2 in the TE was accompanied by the down-regulation of the expression of Na-K ATPase and ENaCa in TEs.…”