Na+/K+ ATPase α1 and β3 subunits are localized to the basolateral membrane of trophectoderm cells in human blastocysts.

study questionIs there a relation between specific Na+/K+ ATPase isoform expression and localization in human blastocysts and the developmental behavior of the embryo? SUMMARY ANSWER: Na+/K+ ATPase α1, β1 and β3 are the main isoforms expressed in human blastocysts and no association was found between the expression level of their respective mRNAs and the rate of blastocyst expansion. WHAT IS KNOWN ALREADY: In mouse embryos, Na+/K+ ATPase α1 and β1 are expressed in the basolateral membrane of trophectoderm (TE) cells and are believed to be involved in blastocoel formation (cavitation). STUDY DESIGN, SIZE, DURATION: A total of 20 surplus embryos from 11 patients who underwent IVF and embryo transfer at a university hospital between 2009 and 2018 were analyzed. PARTICIPANTS/MATERIALS, SETTING,

methodsAfter freezing and thawing Day 5 human blastocysts, their developmental behavior was observed for 24 h using time-lapse imaging, and the expression of Na+/K+ ATPase isoforms was examined using quantitative RT-PCR (RT-qPCR). The expressed isoforms were then localized in blastocysts using fluorescent immunostaining. MAIN RESULTS AND THE ROLE OF CHANCE: RT-qPCR results demonstrated the expression of Na+/K+ ATPase α1, β1 and β3 isoforms in human blastocysts. Isoforms α1 and β3 were localized to the basolateral membrane of TE cells, and β1 was localized between TE cells. A high level of β3 mRNA expression correlated with easier hatching (P = 0.0261). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The expression of mRNA and the localization of proteins of interest were verified, but we have not been able to perform functional analysis. WIDER IMPLICATIONS OF THE

findingsOf the various Na+/K+ ATPase isoforms, expression levels of the α1, β1 and β3 mRNAs were clearly higher than other isoforms in human blastocysts. Since α1 and β3 were localized to the basolateral membrane via fluorescent immunostaining, we believe that these subunits contribute to the dilation of the blastocoel. The β1 isoform is localized between TE cells and may be involved in tight junction formation, as previously reported in mouse embryos. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the JSPS KAKENHI (https://www.jsps.go.jp/english/index.html), grant number 17K11215. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no conflicts of interest.