Regulatory volume decrease in response to hypotonic stress is typical of the oocytes and early mouse embryos. Changes in the kinetics of osmotic reaction can be used as a marker of the modulating effect of the incubation medium on transmembrane transport in embryonic cells. Quantitative laser scanning microtomography (QLSM) was used to measure oocyte volume. In this paper, it is shown that addition of 5 μM glycine, taurine, or GABA, as well as ATP to Dulbecco's medium abolished the regulatory volume decrease in mature mouse oocytes.

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The structure of bacterial film formed at the inner surface of the recirculation reactor tube, is studied. The surface relief of the biofilm was visualized by scanning electron microscopy. The effect of electrochemically activated water solution on the film formed from planktonic lactobacteria or E. coli was studied. Treatment with electrochemically activated water solution destroys cells and polymeric matrix of the biofilm.

Osmolarity of Dulbecco's medium at which the volume of two-cell mouse embryo remained similar to that of intact embryo was determined. The method is based on comparison of kinetic curves describing the volume of embryonic cell in solutions of different osmolarity. The blastomere volume was measured by quantitative laser microtomography after fixed osmotic stress intervals. It was found that Dulbecco's saline with 125 mM NaCl solution is an isotonic solution for two-cell mouse embryo. This concentration corresponds to 290 mOsm, which is lower than osmolarity (~310 mOsm) of media routinely used for culturing of differentiated cells or biological fluids, e.g. blood plasma.

In discussing the causes of the compensatory response to hypoosmotic stress (regulatory volume decrease, RVD), the induced transport of osmolytes from the cell through the volume sensitive organic osmolyte anion channel (VSOAC) is considered first [1]. A Сl -channel with similar pharmacological prop erties, coupled to nonspecific release of amino acids, was found on zygote and oocyte membranes [2,3]. The interest to the involvement of glycine in osmotic regulation is caused by the fact that a specific trans porter, GLYT1, was found on the membrane of mouse zygote and two cell embryo [4]. This active Na + (Cl -) dependent transport of glycine was later found in mature oocytes at the stage of MII development [5]. GLYT1 provides transmembrane transport of glycine in both directions [6], which suggests the possibility of its involvement in RVD. However, the role of glycine in the compensatory response of an embryonic cell to hypotonic stress remains poorly understood [7], and only single papers are devoted to RVD regulation in the mature oocytes, the immediate precursor of early embryogenesis [3].This study shows that glycine present at a physio logical concentration (10 mM) in the extracellular space blocks the development of RVD in the oocyte. The effect is similar to the non osmotic effect of ATP, an inhibitor of VSOAC. A decrease in the concentra tion of the amino acid to the submillimolar level does not recover the compensatory reaction. This fact sug gests that oocytes have mechanisms of glycine medi ated regulation of the osmotic response.Isolated mouse MII oocytes were prepared as described in [8]. The volume of a single oocyte (Fig. 1) was measured by quantitative laser scanning micros copy (QLSM). The procedure of sample preparation for QLSM and the details of measurement of spatial characteristics were published earlier [9, 10].The obtained kinetics of the change in the cell vol ume in the oocyte in the course of hypoosmotic stress is shown in Fig. 2.In the absence of glycine, RVD is triggered in response to hypoosmotic stress, when the initial swell ing is accompanied by an adaptive response, as a result of which the cell tends to recover the intact volume (Fig. 2). Earlier, the authors of [5] demonstrated the presence of GLYT1 on the membrane of mouse oocyte MII. This glycine transporter is reversible, i.e., it per forms active transmembrane transport of amino acids in both directions, which is due to the gradient of sodium ions on the cell membrane. Hypothetically, the driving force in the case of the oocyte may be a rel atively high level of Na + , which was detected, for example, in the frog egg [11]. Unfortunately, it is not yet possible to repeat such measurements in the mouse oocyte. As a result, GLYT1, which pumps glycine from the cytoplasm to the extracellular space, may be involved in RVD. In this case, the intracellular con centration of glycine does not necessarily change, because the decrease in the cell volume compensates for the on mass decrease in the content of the amino acid.Note that th...

Mouse single cell embryos exhibit robust Regulatory Volume Decrease (RVD). In what manner the very early mammalian embryo following zygote stage is appreciably altered by the anisotonic extracellular solution is, as yet, totally unclear. Little attention was paid to this direction since there was no way to deter mine the blastomere volume. This work has served to quantitatively investigate the osmotic response of bicel lular mouse embryos employing Laser Scanning Microtomography (LSM) followed with three dimensional reconstruction (3 DR). We have shown that bicellular mouse embryos in hypotonic Dulbecco's experience RVD. Embryonic cells subjected to hyposmolar exhibit rapid osmotic swelling followed by gradual shrinking back toward their original volume. The van 't Hoff law defines swelling phase with the effective hydraulic con ductivity of 0.3 micron ⋅ min -1 ⋅ atm -1 . Water release during RVD in bicellular mouse embryos is abolished by Cytochalasin B (Cyto B) and the volume recovery is insensitive to ouabain treatment.

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