Analysis of the effects of blue light on morphofunctional status of in vitro cultured blastocysts from mice carrying gene of enhanced green fluorescent protein (EGFP).

We studied the effect of blue light (440-490 nm) on the development of late blastocysts of mice carrying the gene of enhanced green fluorescent protein (EGFP). Exposure to blue light for 20 min reduced adhesive properties of blastocysts and their capacity to form primary colonies consisting of the cells of inner cell mass, trophoblast, and extraembryonic endoderm. The negative effects of blue light manifested in morphological changes in the primary colonies and impairment of differentiation and migration of cells of the trophoblast and extraembryonic endoderm. The problems of cell-cell interaction and inductive influences of the inner cell mass on other cell subpopulations are discussed. EGFP blastocysts were proposed as the model for evaluation of the mechanisms underlying the effects of blue light as the major negative factor of visible light used in in vitro experiments on mammalian embryos.

[A role of trophoblastic cells in regulation of mouse blastocyst survival in vitro after microinjection and osmotic stress].

We have evaluated the morphology of the mouse preimplantation embryos at developmental stages from morula to late-blastocyst after two different impacts: microinjection of modified Witten's medium and osmotic stress in physiological osmolarity (310 mOsM), in 5% glucose (560 mOsM) at high concentration of NaCl (614 mOsM). Results of our research showed that these stresses caused similar changes in embryo morphology: volume was reduced followed by its recovery in culture medium (osmolality was less than a physiological value, 260 MOsM). The ability of embryos to recover the volume and morphology up to the initial level depends on a stage of embryo development and consequently competence of TB cells. In this study it was revealed that a key role in regulation of volume homeostasis after microinjection and after short-time (30-60 min) osmotic stress belongs to TB cells. Both physical effects induce the further embryo development in vitro up to the formation of primary colonies of embryonic and trophoblastic cells. These data could be used to develop the morphological criteria for a prediction of blastocyst-stage embryonic implantation potential.

[The process of heme synthesis in bone marrow mesenchymal stem cells cultured under fibroblast growth factor bFGF and hypoxic conditions].

It was demonstrated that fibroblast growth factor bFGF influences the process of heme synthesis, the proliferation activity and viability of bone marrow mesenchymal stem cells in culture under hypoxic conditions. The addition of fibroblast growth factor bFGF (7 ng/ml) to the medium under above conditions led to the accumulation of aminolevulinic acid--an early porphyrin and heme precursor, an increase in CD 71 expression--a transferrin receptor, and also a decrease in porphyrin pigments and heme contents--a late precursor and end products of heme synthesis, respectively. It was found that cultivation of the cells under hypoxic conditions and bFGF is an optimum to maintain high viability and proliferation capacity of the mesenchymal stem cells.

[Comparative estimation of the EGFP effects on the development of embryos obtained by reciprocal crossing of C57BL/6-Tgn(ACTbEGFP01Osb/J and C57BL/6 mice].

The effect of enhanced green fluorescent protein (EGFP) on the in vitro viability of early embryos of C57BL/6--Tgn(ACTbEGFP010sb/J mice has been studied. The number of viable ova in hemizygous females (-/egfp) has been shown to decrease. Irrespective of the EGFP level, it has no deleterious effect on the early development of embryos obtained by reciprocal crossing of hemizygous (-/egfp) and wild-type (-/-) mice.

[Effect of LIF factor on lipid bilayer].

It has been found that the recombinant protein LIF induces jumps of current in bilayer lipid membranes, which indicates the formation of ionic channels. Some properties of these channels (dependence on voltage, sing of potential, ionic strength of solution, and lipid composition) were studied. A difference between the effects of protein LIF of eukaryotic and prokaryotic origin was shown.

[The influence of LIF (leukemia inhibitory factor) on the functional status of mouse line R1 embryonic stem cells].

The influence of cytokine LIF (Leukemia Inhibitory Factor) on the viability, and proliferation of mouse R1 line embryonic stem cells (ESC) and their distribution by cell cycle stages has been investigated. LIF (5-20 ng/ml) increased growth of colonies and maintained high proliferative and pluripotent properties of R1. LIF was also involved into the inhibition of spontaneous cell differentiation and apoptotic cell death; it also decreased the rations of S/G2+M cell cycle and doubling-time of cell population.

[Effect of the stem cell factor on the morphology and functional state of mouse embryonic stem cells].

The effect of the stem cell factor on the state of membranes and functional activity of mouse embryonic stem cells cultivated in LIF (Leukemia Inhibitory Factor) cytokine-free and LIF-containing media has been studied. It was shown that the stem cell factor induces changes in the viscosity of membrane lipid bilayer and increases the respiration rate, the ATP level, and the proliferation activity of embryonic stem cells. An intricate character of the LIF-dependent modification of biological effects of the stem cell factor was revealed.

[Influence of "zero" magnetic field on the growth of embryonic cells and primary embryos of mouse in vitro].

The present investigation reveals that a 250-fold screening of the geomagnetic field ("zero" geomagnetic fields, 200 nT) is a biologically active factor that adversely affects embryonic cells and the processes of early embryogenesis as a whole. In particular, the cultivation of primary embryonic fibroblasts in "zero" geomagnetic fields causes reduces the capacity for adhesion and proliferation, changes the monolayer morphology and increases cell death. In a more highly organized experimental model, two-celled mouse embryos, the exposure to the "zero" field results in an increase of plasma membrane permeability for dyes, a reorganization of the cytoskeleton because of alpha-actin redistribution, and the disturbance of the spatial orientation of blastomeres. As a result, the development of two-celled mouse embryos stops, and they do not reach the stage of blastocyst. These data show the significant role of geomagnetic fields in the normal growth of embryonic cells in vitro and the regulation of mammalian embryogenesis.

[Increased survival of preimplantation mouse embryos in medium with recombinant cytokine LIF].

We studied the effects of cytokine LIF on in vitro development of 2-cell mouse embryos to the late blastocyst stage. LIF at 10 ng/ml enhanced the blastocyst formation and hatching from zona pellucida. When blastocysts were cultivated in a medium with LIF for a longer time, the trophoblast adhesive properties and proliferative activity were enhanced. In the presence of this cytokine, the trophoblast cells were attached to the substrate surface and fulfill the function of a sublayer for growth of the inner cell mass colonies with a high activity of endogenous alkaline phosphatase. Expression of LIF was detected in the oviduct and uterus epithelial tissues from day 1 until day 4 of pregnancy, thus suggesting its involvement in early development. According to the data of cultivation, cytokine LIF enhanced the adhesive properties and functional activity of the trophoblast cells, which is essential for implantation of blastocysts in the uterus.

[Stimulating effect of recombinant cytokine LIF on the mouse blastocysts during implantation].

We studied the effect of recombinant cytokine LIF (Leukemia inhibitory factor) on the isolated mouse embryos at the stages of middle and late blastocyst. We showed that this agent is necessary at the formation stage of normal trophoblast after the leaving of blastocysts from z. pellucida in vitro. This cytokine (10 ng/ml) results in the intensification of adhesion and proliferative activity of trophoblast cells. It is important for intercellular interactions with endometrium and for invasion of embryos into the uterus. The recombinant LIF has not remarkable influence upon cells of intracellular mass.

["Chain" karyotypic evolution of embryonic stem cell line R1 in vitro].

Cytogenetic anomaly frequencies were analysed in three sublines of ES R1 line in its five clonal sublines, obtained from two cell colonies after transformation of ES R1 cells by plasmid with gene lif. Cell transformation did not increase cytogenic anomalies, however, the initial sublines of ES R1 line, as well as its transformed clonal descendants bore a redundant quantity of the chromosome 8 material within the structure of various Robertsonian translocations even in cells with diploid chromosome quantity (2n = 40). In the initial sublines ES R1 and its clonal descendants a common Rb (8; 15) was revealed. It was supposed that selection for the increase in ES cell sensitivity to cytokines (in particular, LIF) under cultural conditions was accompanied by an increase in chromosomal copies, carrying genes of mapk andjak/stat, through which downstream effectors of cytokine signals for preservation of cell pluripotention and propagation are realized. Genes of chromatid separation and chromosome segregation control (for example, separase gene Esp1 in chromosome 15) may be passively involved in this process, thus promoting acceleration of karyotype evolution in ES cells.

Effect of leukemia-inhibiting factor on bilayer lipid membrane.

We studied the effect of leukemia-inhibiting factor on bilayer lipid membranes. Leukemia-inhibiting factor in a concentration of 10 ng/ml nonspecifically increased membrane permeability for ions. Leukemia-inhibiting factor acts as a surface-active substance on bilayer lipid membranes.

[The simulation of the cooperative effect of development in a culture of early mouse embryos after irradiation with electromagnetic waves in the millimeter range]. / Imitatsiia kooperativnogo éffekta razvitiia v kul'ture rannikh zarodyshei myshi posle oblucheniia élektromagnitnymi volnami millimetrovogo diapazona.

We have found that two-cell mouse embryos cultured in vitro can be stimulated by electromagnetic irradiation in the millimeter range. After 30 min of exposure, they acquire the ability to develop in culture on their own and can reach the stage of blastocyst in a relatively large volume of Whitten cultural medium (150 microliters) without serum or growth factors. It is proposed that millimeter range electromagnetic waves activate metabolic processes and specifically the synthesis of factors controlling early embryonic development in culture.

[Effect of millimeter waves on the early development of the mouse and sea urchin embryo]. / Deistvie millimetrovykh voln na rannee razvitie zarodyshei myshei i morskikh ezhei.

The action of nonthermal electromagnetic radiation (EMR) of the millimeter range on the early development of murine and sea urchin embryos was investigated. An MRTA-01E-03 generator with a frequency of 54-78 GHz and radiation intensity of 0.06 mWt/cm2 was used. The embryos were irradiated during 30 min at the stage of two blastomeres. The number of murine embryos that reached the blastocyst stage increased (up to 97.3% in comparison with 87.5% in control). The total time of cultivation up to the blastocyst stage was also shorter (72 h) than in control (96 h). The irradiation had effect on the development of sea urchin embryos only if embryos with a weakened viability were tested. The results indicate that millimeter electromagnetic radiation has a stimulating effect on the early development of embryos, increasing the resistance of embryos to unfavorable environmental conditions.

[The effect of the mutant genes Ta, Ra and Sd on early embryogenesis in mice]. / Vliianie mutantnykh genov Ta, Ra, Sd na rannii émbriogenez myshei.

We studied early embryonic mortality of mice from mutant stocks Tabby (Ta, X-chromosome) and RaSd (RaSd/++, chromosome 2) maintained in the heterozygous state in F1 CBA x C57B1/6 hybrid. Tabby and RaSd mice were reciprocally crossed with F1 mice and examined for the morphological status of embryos washed from the oviduct on the third day of pregnancy, when the stage of eight blastomeres is normally attained. Mortality was evaluated from the number of embryos which did not reach the expected stage by this time. The results have shown that 2-4 cell embryos, which have received gene Ta with the X-chromosome of the female parent, differed from embryos with F1 genotype at the same stage of development by their increased mortality rate, whereas among embryos obtained from RaSd, the mortality was mainly observed before cleavage. Death of embryos receiving the mutant gene from hemizygous Ta males or heterozygous RaSd/++ males was not significantly different from the mortality of embryos without these mutations.

[Study of cryoprotective properties of antifreeze glycoproteins from the white sea cod Gadus morhua on low temperature freezing of mouse embryos]. / Issledovanie kriozashchitnykh svoistv antifriznykh glikoproteinov iz belomorskoi treski Gadus morhua pri nizkotemperaturnom zamorazhivanii émbrionov myshi.

The effect of antifreeze glycoproteins on morphological preservation and viability of mouse embryos cryopreserved in liquid nitrogen was studied. Cryomicroscopic observations indicated that as distinct from the cryoprotector dimethylsulfoxide antifreeze glycoproteins cause phenomena typical of vitrification (absence of visible crystallization, transparency of medium). The embryos frozen in the presence of freeze glycoproteins had after thawing a higher morphological preservation (87.5%) than those frozen with 1M and 3M dimethylsulfoxide (53.8 and 71.1%, respectively). However, they were nonviable and did not develop in culture. A high viability after cryoconservation was achieved only when antifreeze glycoproteins (20 mg/ml) were used together with dimethylsulfoxide (3M). In this case, the number of viable embryos, which developed in culture to the stage of compact morula and blastocyst, was 65.0%, i.e. two and four times higher than in the presence of 3 M and 1 M dimethylsulfoxide, respectively.

[The use of antifreeze glycoproteins in the freezing in liquid nitrogen of early mouse embryos]. / Ispol'zovanie antifriznykh glikoproteinov pri zamorazhivanii v zhidkom azote rannikh zarodyshei myshei.

The cryoprotective properties of antifreeze glycoproteins (AFGPs) isolated from White Sea cod (Gadus morhua) blood were analyzed. Improved preservation of morphological characteristics was observed in two-cell mouse embryos frozen in the presence of AFGP but without other cryoprotectors. However, subsequent development after thawing was achieved only with embryos frozen in a medium containing both AFGP and dimethyl sulfoxide. The maximum increase in the number of surviving embryos (by a factor of approximately two, as compared with the control) was observed at AFGP and DMSO concentrations of 20 mg/ml and 25%, respectively.

[Analysis of the reasons and mechanisms determining the occurrence of individual development in animals, beginning from the zygote (role of the cytoskeleton)]. / K analizu prichin i mekhanizmov, opredeliaiushchikh vozniknovenie individual'nogo razvitiia u zhivotnykh, nachinaia s zigoty (rol' tsitoskeleta).

Results of studies are presented revealing the role of motor activity (the energetic rule of skeletal muscles) as a factor determining the beginning of initiation of the organism individual development from the moment of zygote formation. The blockage of the cytoskeleton function (cytochalasin B, verapamil) was shown to result in the arrest of individual development and death of embryos. Mechanisms and causes of ambiguous action of these compounds are analysed. A biphasic pattern of the reaction of embryo cells is established. During the first short phase the function of microfilaments located near the membrane is blocked and further development of embryos is stopped. During the second, later and longer phase the function of cytoplasm microfilaments is blocked. The existing concepts as to the causes determining the individual development are discussed.

[Effect of electromagnetic SHF-radiation on the morphofunctional status of early mouse embryos]. / Deistvie élektromagnitnogo SVCh-uzlucheniia na morfofunktsional'noe sostoianie rannikh zarodyshei myshei.

A differential effect of 915 MHz microwave radiation with 500 Hz amplitude modulation of 1 ms pulses on the morphofunctional state of mouse 8-cell embryos has been shown. Then development is not disturbed by irradiation for 15 to 20 min. This effect is suggested to correlate with some changes in the properties of cell membranes as a result of possible local increases of temperature.