Mechanisms of Intracellular Osmolarity Changes during the Cycles of Cleavage Division in Early Embryogenesis of Mice

Abstract

Previously, it was demonstrated that the success of microsurgical manipulations during nuclear transplantation into zygotes or two-cell embryos was determined by the right choice of time for transplantation (viz. the state of a maximum cell turgor) before the onset of cleavage division [1]. The sharp oscillations of osmolarity observed within a cleavage division cycles suggest considerable variations in the intracellular concentrations of osmotically active molecules in the cells of early embryos. The experiments on nuclear transplantation into mature oocytes gave additional information about changes in cell turgor [2]. It was shown that the osmolarity started to increase immediately after the moment of fertilization. In zygotes, about 3 h before the first cleavage division, it was practically two times higher than in mature oocytes and was close to the osmolarity of media (280-300 mOsM) commonly used for culture [3, 4]. The osmolarity changed also during the two-cell stage: it was 220-230 mOsM 5-6 h before the second cleavage division and then reached 280-300 mOsM by the second cleavage division ( Fig. 1 and Fig. 2). The values of intracellular osmolarity were assessed by cell turgor in media with different osmolarities.Such cyclic oscillations of the intracellular osmolarity of embryonic cells occurring synchronously with the division cycles are interesting from both theoretical and practical viewpoints. So, the question that primarily arises is what mechanisms underlie these oscillations. The intracellular potassium concentrations in the majority of cells of warm-blooded animals are known to range from 100 to 150 mM, which, as a rule, is one order of magnitude higher than the intracellular sodium concentration [5]. Therefore, potassium together with its counterions (phosphorus and/or chlorine) are likely to determine the cell osmolarity.Recently, it was shown by electron probe microanalysis that the intracellular concentrations of potassium and phosphorus varied at the early stage of embryogenesis, namely, zygote [6], which is in favor of our assumption. However, the authors of [6] did not identify clearly the phases of the cell cycle. In addition, these results were obtained with the use of outbred mouse strains, whereas the main object of cellular biology studies, including works on the nuclear transplantation, is inbred mouse strains and F1 hybrids of these strains. It would be of interest to compare the data on the oscillations of cell turgor and osmolarity with the data on changes in the intracellular concentration of potassium and its eventual counterion phosphorus during cell cycle in early embryos of F1 mice.The purpose of this study was to measure the concentration of potassium and phosphorus during particular cell-cycle phases in zygotes and two-cell embryos of F1 mice (CBA × C57BL) and to compare these data with oscillations of cell turgor.The work was performed on 2-to 2.5-month-old F1 mice (CBA × C57BL). The mice were obtained from the Stolbovaya nursery. To derive mature oocytes, superovulation w...