Abstract
Studies have shown that some electrolytes, including Na+ and K+, play important roles in embryonic development. However, these studies evaluated these electrolytes by using inhibitors or knockout mice, with no mention on the changes in the intracellular electrolyte concentrations during embryogenesis. In this study, we used the electrolyte indicators CoroNa Green AM and ION Potassium Green-2 AM to directly visualise intracellular concentrations of Na+ and K+, respectively, at each embryonic developmental stage in mouse embryos. We directly observed intracellular electrolyte concentrations at the morula, blastocyst, and hatching stages. Our results revealed dynamic changes in intracellular electrolyte concentrations; we found that the intracellular Na+ concentration decreased, while K+ concentration increased during blastocoel formation. The degree of change in intensity in response to ouabain, an inhibitor of Na+/K+ ATPase, was considered to correspond to the degree of Na+/K+ ATPase activity at each developmental stage. Additionally, after the blastocyst stage, trophectoderm cells in direct contact with the blastocoel showed higher K+ concentrations than in direct contact with inner cell mass, indicating that Na+/K+ ATPase activity differs depending on the location in the trophectoderm. This is the first study to use CoroNa Green AM and ION Potassium Green-2 AM in mouse embryos and visualise electrolytes during embryonic development. The changes in electrolyte concentration observed in this study were consistent with the activity of Na+/K+ ATPase reported previously, and it was possible to image more detailed electrolyte behaviour in embryo cells. This method can be used to improve the understanding of cell physiology and is useful for future embryonic development studies.
Highlights
Pre-implantation development in mouse embryo involves sequential division of a fertilised egg into blastomeres
The ouabain + ION Potassium Green-2 AM group was significantly affected compared to the control (p = 0.0040), CoroNa Green AM (p = 0.041), and ouabain + CoroNa Green AM (p = 0.0192) groups (Fig 4), indicating that ION Potassium Green-2 AM was more cytotoxic than CoroNa Green AM, in the ouabain combination group. (p = 1.000, control vs. CoroNa Green and ouabain + CoroNa Green groups; p = 0.129, control vs. ION Potassium Green) (Fig 4)
Changes in the intensities of Na+ and K+ from morula to hatching demonstrated that the intracellular Na+ concentration decreased while K+ concentration increased during blastocoel formation
Summary
Pre-implantation development in mouse embryo involves sequential division of a fertilised egg into blastomeres. Many studies examining blastocoel formation have reported that water movement, along with the Na+ concentration gradient, Na+/H+ exchangers, and Na+ channel, play key roles in Na+ influx at the apical membrane [6]. Because blastocoel formation is the most dynamic process in embryo development, it is important to elucidate electrolyte behaviours at these stages. Concentration in mammals varies between 5–15 mM depending on the cell type, whereas the extracellular Na+ concentration is much higher at approximately 120–150 mM [10] This concentration gradient is established by the activity of ion channels/transporters and pumps and is required to maintain the resting membrane potential [10]. We used these electrolyte indicators to visualise Na+ and K+ concentrations at each developmental stage in mouse embryos, to gain insights into the cytophysiology of embryonic development. Immunostaining of Na+/K+ ATPase was performed to elucidate the relationship between Na+/K+ ATPase expression and activity at each developmental stage
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