No abstract

Electron probe microanalysis is a method for evaluation of concentrations of elements at the subcellular level, effective in studies of an individual cell in culture or suspension. Intracellular concentrations of cytoplasmic ions (K+, Na+, Cl-) most rapidly and markedly reacting to changes is an adequate criterion for evaluating the traumatism of manipulations used in cell technologies. Using electron probe microanalysis it will be possible to develop special procedures (for example, therapeutic cloning) for reprogramming or cryopreservation, when intactness of intracellular balance of element serves as the criterion of cell status.

Intracellular concentrations of potassium and phosphorus were determined by Electron Probe Microanalysis in mouse mature oocytes and zygotes. The oocytes were characterized by insignificant variations in the concentrations of these elements in the cytoplasm: 60 ± 4 and 103 ± 6 mM, respectively. In zygotes, on the contrary, significant variations were observed: 64 ± 16 and 84 ± 14 mM, respectively. Changes in the potassium homeostasis during the first cell cycle have been discussed.

Oocytes, as well as one-and two-cell embryos, of the mouse are characterized by a low intracellular concentration of potassium [1][2]. This may be accounted for by either a low Na/K-ATPase activity or an intense passive transport of the ä + cation from embryonic cells. Cell division cycles in early embryos have been demonstrated to be synchronized with the activity of a specific potassium channel in the plasma membrane [3].The channel designated 240 pS ä + has a number of characteristic features [4]. Its periodicity is determined by a cytoplasmic oscillator indirectly interacting with the chromosomal cycle through a cyclin-dependent kinase (cdk1). Mitogen-activated protein kinase (MAP kinase), which is active in oocytes during metaphase II of meiosis and the mitosis of the first cell cycle, is possibly a mediator between cdk1 and the 240 pS ä + channel. The inhibition of the channel in the early S phase and during the S-G 2 transition is apparently determined by the activation of tyrosine kinase.These temporal pattern of the function of the 240 pS ä + channel allow us to assume cyclic oscillations of the potassium concentration in the entire cytoplasm of the zygote. However, it is still unknown whether or not the preparation for the first division of cleavage is connected with changes in potassium homeostasis. This question cannot be answered unless there is an adequate method for measuring the potassium concentration in an individual embryonic cell. The development of electron probe microanalysis provided the necessary approaches to solving this problem [5,6]. Therefore, the purpose of this study was to determine the potassium concentration in the cytoplasm of zygotes and oocytes at the stage of metaphase II of meiosis with the use of electron probe microanalysis.Mice from the NMRI stock were used for the experiments. Mature oocytes and early embryos were obtained from six-to eight-week-old females. Estruscycle synchronization and superovulation were caused by subcutaneous injections of follicle-stimulating gonadotropin from the blood serum of pregnant horses followed by intraperitoneal injections of human chorionic gonadotropin. Oocytes washed from the oviduct were cleared of cumulus cells with the use of 0.1% hyaluronidase solution in Witten medium. One-cell embryos (zygotes) were washed from the oviduct on the first day of pregnancy. The obtained cells were washed with physiological medium three times and used in experiments immediately after that.Simultaneously with the potassium concentration, we analyzed the cytoplasmic concentration of phosphorus contained in important biological molecules involved in cell metabolism. The concentrations of elements (K and P) in the cytoplasm were determined in a cell section obtained as described in [7]. An oocyte or zygote was frozen in liquid propane and lyophilized in vacuum at 200 K. Then, the freeze-dried sample was embedded in epoxy resin, which was polymerized at 330 K. Sections (2 µ m thick) of the freeze-dried embedded cells were examined under a JSM-U3 scanning ...