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The discovery of cell-free DNA in blastocoele fluid opens new perspectives for the development of preimplantation genetic diagnosis of human chromosomal and genetic diseases. In this review we analyzed the results of the first studies, which made it possible to evaluate the effectiveness of the application of a new source of biological material and showed a high degree of agreement between the results of molecular karyotyping with cell-free DNA and blastocyst cells. The results suggest the possibility of developing a noninvasive method of preimplantation genetic diagnosis, which may open a new round of progress in the field of assisted reproductive technologies and the genetics of early stages of human ontogenesis.

The procedure of obtainment of chimeric blastocysts of mice by laser nanosurgery methods without using any other techniques is described. To perform the experiments, a special laser micromanipulator was invented. The murine embryonic stem cells (ESC), which were transformed with pEF-GFP vector, encoding the green fluorescent protein, were used in the experiments. ESC were introduced into the perivitelline space of murine embryos at the stage of 8 cells using the laser micromanipulator. The operated embryos were cultured in vitro until the stage of emergence from zona pellucida. The fluorescence and its precise localization were registered using a confocal microscope. It was shown for the first time that the inclusions of ESC introduced with the lased micromanipulator were found not only in the inner cell mass (ICM) but also in the trophectoderm of the chimeric blastocyst. The technology of nanosurgical operations at early stage preimplanted mammalian embryos using laser techniques opens great opportunities not only for solution of fundamental tasks of experimental embryology of mammals but also for obtainment of chimeric and transgenic animals with predetermined genotype.

The effect of beta-endorphin on 2-, 4- and 8-cell embryo development in vitro was studied. It is shown, that hormone has no effect on 2-cell embryos development, but it has enhanced viability of 4- and 8-cell mouse embryos. The number ofblastocyst formation increases in presence of 0.1 microM beta-endorphin in embryo cultured medium but the number of blastocyst with abnormal structure decreases. The effect of hormone on the change of intracellular concentration of Ca2+ ion in 2-, 4- and 8-cell mouse embryo has been studied with the help of fluorescent microscopy. The effect of adenylate cyclase, and phospholipase activity blockers and opioid blocker naloxone on the change of intracellular concentration of Ca2+ ion in early mouse embryo in the presence of beta-endorphin have been also studied. It is shown that 2-cell embryo has opioid and nonopioid beta-endorphin receptors, whereas 4- and 8-cell mouse embryos have only nonopoioid beta-endorphin receptors. It is also shown that the effect of beta-endorphin in the early mouse embryo through a nonopioid receptors occurs with the participation of intracellular Ca2+ and adenylate cyclase signaling system.

In this work, preimplantation mouse embryos were used as a model to study the distribution of nuclear actin in a comparative aspect. To reveal actin, different polyclonal antibodies raised against the C-terminal domains of actin were applied. An antibody raised against the C-terminal marks nuclear actin more intensively as compared to the cytoplasm, especially in areas of condensed chromatin. The fluorescence of the nuclei was less apparent when an antibody against N-terminus is applied. This antibody revealed actin associated with low condensed chromatin. In 2-cell but not in elder embryos, some Cajal bodies were also stained with this antibody. However, the pattern of actin association with transcriptions sites does not depend on the type of anti-actin antibodies used in this study. We suggest that the distinctions of staining patterns revealed using different anti-actin antibodies may be explained by different capacity of the antibodies for staining, but not by their selective detection of different functional forms of nuclear actin in mouse embryos.

For the first time using genome-wide Infinium HumanMethylation27 BeadChip Array (Illumina, USA) DNA methylation pattern was determined in the human cytotrophoblast and extraembryonic mesoderm. These tissues are the derivatives of trophectoderm and inner cell mass of blastocyst and have substantial differences in dynamics of epigenetic genome reprogramming during early stages of differentiation. The genome of the extraembryonic mesoderm cells has been shown to be more methylated compared to the cytotrophoblast similarly to other mammalian species. Differences in methylation pattern of single CpG-dinucleotides and dinucleotides localized within promoter CpG-islands have been found. It has been shown that the majority of single CpG-dinucleotides in both tissues were methylated whereas promoter CpG-island sites were not. Comparative analysis revealed 202 differentially methylated genes in extraembryonic mesoderm and 40 genes in cytotrophoblast. These genes are responsible for diverse biological processes. However, in the extraembryonic mesoderm the main functional groups included genes responsible for DNA binding and transcriptional factors' activity whereas in the cytotrophoblast--for transport and protein and cytokine secretion.

In this study, preimplantation mouse embryos were used as a new model for investigation of actin distribution in the nuclei and identification of functional forms of intranuclear actin. Combination of direct detection of actin by fluorescent-conjugated falloidin and DNase I with the method of indirect immunofluorescence was applied as an integrated approach to study localization of actin in the nuclei of two-cell mouse embryos. Monomeric actin and two oligomeric forms of actin were detected in the nuclei, and each of these forms demonstrated its own pattern of distribution. Oligomeric actin recognized by antibodies to C-terminal domain of actin was associated with condensed chromatin as well as with metaphase chromosomes and chromatin of second polar body. Monomeric actin and another oligomeric form recognized by antibodies to N-terminal domain were revealed in the area of dispersed chromatin localization.

Embryonic stem cells are able to give rise after differentiation to derivatives of three germinal layers (ectoderm, endoderm, and mesoderm) and to functional gametes. This property of cells is referred to as pluripotency. The pluripotent status of preimplantation embryo cells and embryonic stem cells is maintained by a complicated system of molecular signaling pathways and transcription factors. The key regulators in this system are the transcription factors OCT4 and NANOG. The role and place of these factors in the pluripotency-sustaining system and their interaction with other factors are considered in the review. Data are presented on the structure, chromosomal location, expression, and regulation of the Oct4 and Nanog genes in mammals.

The effect of antisense oligonucleotides specific to mRNA of the proapoptotic gene harakiri (Hrk) on the development of mouse SAMP1 (senescence-accelerated mouse prone) and (C57BL/6J x DBA/2J)F1 preimplantation embryos cultured in vitro was investigated. The SAMP1 mice are characterized by genetically determined decrease of fertility along with the highly frequent perturbations of embryonic development. Reproduction indices of the (C57BL/6J x DBA/2J) hybrids lie within the normal range. Because of this, preimplantation abnormalities in this line were induced by the action of proapoptotic agent bleomycine. It was demonstrated that antisense inhibition of the Hrk expression had no effect on the frequency of genetically determined abnormalities of early embryonic development in SAMP1 mice. In case of induced abnormalities, addition of oligonucleotides specific to mRNA of proapoptotic Hrk gene influenced the number of abnormalities, and at the same time, improved the quality of survived embryos via increasing the blastocyst hatching.

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.

One of the crucial problems of developmental biology is the study of mechanisms of regulation of gene expression in early embryogenesis. Here we studied dynamics of mosaic appearance of a marker fluorescent protein in in vitro developing mice embryo derived from zygotes after microinjections to male pronuclei of cloned DNA fragment carrying EGFP under control of different promoters. Main attention was paid to initial stages of development, when structural rearrangements and reprogramming of both parental genomes, activation of zygotic genes, and control of development by embryo genome take place.

We studied the influence of combined microinjection of a gene engineering construct and site-specific endonuclease Sal in the pronucleus on preimplantation development of (CBA x C57BL)F1 mouse embryos in vitro. The rate of survival of the embryos was estimated according to their capacity to develop until the blastocyst stage and hatch from zona pellucida. The results obtained suggest that the microinjection of exogenous DNA jointly with endonuclease SalI at concentrations from 0.1 to 0.01 U/microl decreased reliably the rate of survival, as compared to the control (p < 0.05 and p < 0.01, respectively). However, a decrease of endonuclease SalI concentration in the injection mixture to 0.01 U/microl enhanced the capacity of mouse embryos to develop until the blastocyst stage and hatch from zona pellucida, as compared to the embryos microinjected with exogenous DNA and endonuclease SalI at a higher concentration.

We studied the distribution of mRNAs involved in dorsoventral differentiation of Xenopus laevis embryos (Xbra, chordin, Xnot, Xvent1, Xvent2, and Xwnt11) among polyribosomes (the translated form of mRNA) and informosomes (the nontranslated form of mRNA). It was shown using molecular hybridization that all of the studied templates are in the informosomes until the midgastrula stage, thus suggesting regulation at the level of translation. At the midgastrula stage (stage 11), translation begins on mRNAs of chordin, Xnot2, Xvent1, and Xbra, although mRNAs of Xvent1 and Xbra are partially located in the informosomes. The matrices of Xwnt11 are localized predominantly in the informosomes at the midgastrula stage, while those of Xvent2 are not seen in the polyribosomes until the end of gastrulation. We propose that the fate of different mRNAs is determined by different mechanisms of r-translational regulation in different cell lineages. It cannot be excluded that the Xvent2 transcript is not involved in translation and fulfills its morphogenetic functions in the form of RNA or RNP.

It was shown that 35.3% ejaculated rabbit spermatozoa washed from seminal plasma are capable of interacting with heterogeneous DNA. The major part (85.2%) of bound DNA was located in the post-acrosomal part of the sperm head. After incubation with plasmid pRK31acZ the spermatozoa transferred it in the oocytes during in vivo fertilization, as shown by expression of reporter gene lacZ in 19.3% of preimplantation embryos. Additional treatment of the mobile spermatozoa with DMSO and heat shock raised the efficiency of exogenous DNA incorporation in the spermatozoa, as expressed in the percentage of embryos containing bacterial beta-galactosidase (61.7%). It is proposed to use the capture of heterogeneous DNA by the spermatozoa and its transfer in the oocytes for production of transgenic animals and in studies of regulation of gene expression at the early stages of embryogenesis.

The hybridization kinetics of nuclear RNAs of loach embryos labelled with [3H]uridine for 1 hour with DNA excess shows that during embryogenesis (from the blastula to the gastrula stage) the number of newly formed RNA molecules transcribed from repeating DNA sequences in considerably reduced. This occurs both in the RNA fraction extracted from the nuclei with phenol pH 7.7 and having a low sedimentation coefficient and a low degree of polyadenylation, and in the RNA fraction extracted with phenol pH 9.0 having a higher sedimentation coefficient and a higher degree of polyadenylation.

Phagocytosis was studied in three populations of trophoblast cells of the grey vole: primary and secondary giant cells, and spongiotrophoblast. In the two former, the cell growth is accomplished by endomitotic polyploidization only, whereas the spongiotrophoblast cells divide mitotically for a long time. The phagocytosis of these differs from that in giant cells. No more than 1--2 erythrocytes were phagocyted by the spongioblast diploid cells, while giant cells were able to phagocyte over several dozens of erythrocytes. The spongioblast cells are haemophagous and phagocyte only anucleate blood cells -- erythrocytes; unlike, giant cells, when ingrowing within the uterus mucose, are capable of taking up and digesting, in addition to erythrocytes, leukocytes, decidual and endothelial cells.

Radioimmunological method was applied to the study of progesteron and 17 beta-estradiol profiles in the peripheral blood plasma of pregnant mice from the time of the zygote formation (the 1st day of gestation) to the blastocyst implantation (the 5th day of gestation). The peak of a progesteron concentration in the plasma and the onset of reduction in the 17 beta-estradiol concentration were noted in the evening of the 3rd day of pregnancy. From this period there was a fall in the 17 beta-estradiol and progesterone content in the blood plasma. In the evening of the 3rd day of pregnancy the 14-18-cell embryos passed from the oviduct into the uterine cornu. The role played by the embryo in changes of the maternal hormonal status is discussed.

The cell cycle structure in the cells of loach embryos at the early blastula stage (5 h of development at 21 degrees) is markedly altered under the influence of injection of homologous low molecular weight nuclear RNA and, as a result, the number of cells in G2-phase. The DNA amount in the embryo increases by 20%. At the midblastula stage (7 h) no increase in the number of cells in G2-phase was found.

The dynamics of protein synthesis in the loach embryos has been studied by means of autoradiography at the stages of cleavage, blastula and gastrula. During the synchronous cleavage divisions, nuclear proteins are mainly synthesized. From the early blastula stage until the early gastrula stage, the intensity of nuclear protein synthesis increases 2.5 times whereas the intensity of cytoplasmic and total protein synthesis is low and relatively constant. After the onset of gastrulation the intensity of nuclear and cytoplasmic protein synthesis increases 3-4 times and at the late gastrula stage it decreases twice as compared with that at the midgastrula stage. During blastulation, no regional differences in the intensity of nuclear and cytoplasmic protein synthesis were found. With the onset of gastrulation, a vegeto-animal gradient of labeled aminoacid incorporation into nuclear and cytoplasmic proteins appears. During gastrulation, reliable differences were found between the intensity of labeled aminoacid incorporation into proteins of the cells of intact and dissociated blastoderms. During this period, the intensity of protein synthesis in embryonic shield is higher than that in the extraembryonic part of blastoderm.

The patterns of DNA synthesis and kinetics of cell population in the rat and mouse embryos were studied by means of 3H-thymidine autoradiography and cytofluorimetry. The rat and mouse embryos during the period of cleavage consist of a heterogenous population of blastomeres. At all the stages under study, all phases of the cell cycle occur in the blastomeres: G1, S, G2 and mitosis. The embryonic cells were distributed into groups containing 2c, 3c, 4c and more DNA. The ratio of cell number in these groups differed in the mouse and rat embryos. The mouse embryos are characterized by the appearance of a considerable amount of polyploid cells in S phase at the morula stage. The stage and species specific quantitative and qualitative patterns were established for DNA synthesis and kinetics of the cell population of blastomeres.

Changes in protein synthesis in the loach (Misgurnus fossilis) blastoderms cultivated in vitro in the Holtfreter solution after their separation from the yolk at the early and late-blastula stages were assessed by biochemical and autoradiographic methods. The embryos incubated in vitro at the from blastula stage are characterized by the sharp activation of protein synthesis and the vegetal-animal gradient of protein synthesis, as well as in the control embryos; such embryos gastrulate and proceed to primary differentiation. On the contrary, in the embryos incubated from the early blastula stage the protein synthesis is inhibited and no regional differences in its intensity are noted; such embryos do not proceed to gastrulation. Possible causes of the protein synthesis activation in differentiating blastoderms and interrelationship between the character of protein synthesis and the ability of embryos for differentiation are discussed. The protein synthesis activation and the vegetal-animal gradient appearance are considered as a biochemical criterion of primary differentiation during gastrulation in fish embryos.

The concentration of dry substance (protein) and the dry weight of nuclei, cytoplasm and cells from different blastoderm regions at the early blastula and midgastrula stages were determined by interferentional microscopy. It was shown that at the early blastula stage the dry weight of cells in the basal layer is higher than that in the outer layer. Although the protein concentration in the basal layer cells appears to be somewhat higher, differences in their dry weight are due primarily to the big volume of cytoplasm of the basal layer cells. By the midgastrula stage, the total (nucleus + cytoplasm) protein concentration increases (by 17% in the basal layer cells and by 9% in the outer layer cells) due to the increase of nuclear protein concentration. At the same time dry weight of these cells markedly decreases due to the decrease of their volumes in the process of cell divisions. At the midgastrula stage the epiblast cells have the highest dry weight due to the highest protein concentration in the cytoplasm and the biggest cell volume. The results obtained are discussed with respect to the data on the pattern of accumulation of newly synthesized protein in nuclei and cytoplasm with special reference to the duration of individual cell cycle phases.

In the cells of normal embryos of zebrafish, Brachydanio rerio, the Feulgen-positive particles were found in the cytoplasm, in addition to the following abnormalities of mitoses: retardation and loss of chromosomes, disorganization of mitotic figures similar to those seen with C-mitosis, condensation and pycnotic transformation of chromatin preceding the cellular death. The dynamics of these phenomena was traced in the course of development starting from the 8 cell stage to the late blastula stage. The high frequency of the above findings, as well as a certain extent of the topological correspondence of abnormal mitoses enable the author to propose that the normal development of bony fishes is accompanied by degradation of the hereditary material in some cells of the embryo. This may constitute a step of differentiation for at least provisional structures of the embryos.

Mammalian oocytes and embryos on the early stages of development are in need of pyruvate as source of energy. The pyruvate or pyruvate and lactate combination are necessary for the processes of the maturation and fertilisation. The 8-cell stage embryos tend to change their energetical metabolism. The mouse embryos at this stage of development begin to utilise the glucose. alpha-Ketoglutarate without pyruvate, lactate or glucose is necessary for the development of cow and pig embryos after 8-cell stage.

The comparative cytological study of lipids points to species specific character of lipid metabolism in early embryogenesis of placentate mammals. The general level of lipid metabolism is genetically determined in oogenesis being expressed by different loading of the ooplasm with substances of lipoid nature. At the cleavage stage lipids play a plastic role. Their significance as functionally active complexes is manifested in the period of the blastocyst formation, which is dependent on morphological differentiation and the beginning of the trophoblast functioning as an early metabolic organ.