Stem cells giving rise to extraembryonic tissues.

The review is devoted to characterization of stem cells involved in the formation of extraembryonic tissues during the early development of mammalian embryos. Here we present our results of characterization of stem cells from the trophoblast and extraembryonic endoderm of voles and comparative analysis of these cells and the corresponding mouse cells and discuss possible signal pathways maintaining these cells in undifferentiated state.

[Modifications in major satellite methylation in the nucleus of a two-cell mouse embryo dependent on developmental conditions].

The study of the degree of DNA methylation in the nucleus, in particular of the major satellite in two-cell mouse embryos developing in the maternal organism, in standard cultural media M16 used for cultivation of mouse embryos and M2 media used for manipulations with embryos in the air was conducted. Two-cell embryos nucleus aged 44-46 hours after chorionic hormone injection were investigated. The revealed results are evidence for the dependence of the major satellite Ts methylation level of the developmental conditions of embryos.

FGF4 independent derivation of trophoblast stem cells from the common vole.

The derivation of stable multipotent trophoblast stem (TS) cell lines from preimplantation, and early postimplantation mouse embryos has been reported previously. FGF4, and its receptor FGFR2, have been identified as embryonic signaling factors responsible for the maintenance of the undifferentiated state of multipotent TS cells. Here we report the derivation of stable TS-like cell lines from the vole M.

[Localization of satellite DNA and associated protein in respect to nucleolar precursor bodies in one- and two-cell mouse embryos].

Nucleolar precursor bodies (NPB) are characteristic structures in the nuclei of one- and two cell mouse embryos. The alignment of centromeric (CEN) and pericentromeric (periCEN) chromosome regions to the chromatin layer surrounding NPB is known. Mus musculus 4 satellite DNA (satDNA) types are known to be located in CEN region--mouse minor satellite (MiSat) and mouse satellite 3 (MS3); and periCEN region--mouse major satellite (MaSat) and mouse satellite (MS4).

[The study of preimplantation development of mice embryos labelled with green fluorescent protein gene (EGFp)].

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.

Evidence for the existence of satellite DNA-containing connection between metaphase chromosomes.

Physical connections between mitotic chromosomes have been reported previously. It was assumed that the interchromosome connection was based on the DNA-protein thread. However, the data about DNA sequences and protein component in the thread is fragmentary.

[Theoretical and applied aspects of epigenetic reprogramming in mammalian development].

Epigenetic reprogramming implies changes in germ and somatic cells of an embryo, which are the consequences of gene activity regulation by means of DNA methylation, histone modification, and altered chromatin compaction. This suggests that epigenetic changes in mammalian cell nucleus occur during gametogenesis and toti-potent zygote formation. Epigenetic changes proceed during morphological and inductive interactions between cleaving blastomeres and subsequent interactions between the inner cell contents and trophoectoderm, as well as when the germinal layers (blastophyllums) and their derivatives appear, i.

High resolution mapping of ribosomal DNA in early mouse embryos by fluorescence in situ hybridization.

The nucleolar precursor bodies (NPBs) are numerous discrete entities present in the nuclei of early mammalian embryos, which structurally support active rRNA genes. However, whether all rRNA genes, including those not transcribed, are spatially associated with NPBs, and moreover what is the general arrangement of ribosomal DNA (rDNA) in early mouse embryos, still remain unanswered questions. In our study, we examined the localization of rDNA in transcriptionally silent (one-cell and early two-cell) and transcriptionally active (late two-cell) mouse embryos by highly sensitive fluorescence in situ hybridization with probes complementary to mouse rDNA repeats.

[Localization of chromosomal nucleus organizing regions in one-cell mouse embryos and oocytes by fluorescence in situ hybridization].

In mouse zygotes, ribosomal genes (rDNA) are transcriptionally silent and so-called "nucleolar precursor bodies" are present instead of typical nucleoli. However, the functional significance of these structures remains obscure. Specifically, it remains unknown whether structural association between the nucleolar precursor bodies and rDNA are maintained when rDNA synthesis is switched off.

Persistence of human mitochondrial DNA throughout the development to the blastocyst of mouse zygotes microinjected with human mitochondria.

The conditions for transfer of human mitochondria into fertilised mouse ova were elaborated. Species-specific primers were designed to discriminate human mitochondrial DNA (mtDNA) and the endogenous mtDNA in the preimplantation embryos. Human mitochondria isolated from the HepG2 cell line were microinjected into murine zygotes, and the latter cultured for 96 h to the blastocyst stage.

[Cytogenetic analysis of sister chromosome sets in the second polar body and in pronuclei of unicellular mouse embryos. I. Frequency and origin of aneuploidy in embryos heterozygous for the reciprocal chromosome translocation T[14;15]6Ca].

We carried out a cytogenetic study of ovulating oocytes and unicellular embryos, heterozygous by reciprocal chromosomal translocation T[14;15]6Ca. Okadaic acid was used to induce premature condensation of the interphase chromosomes in the embryos, and the number of G1 chromosomes was counted in the second polar body and pronuclei. It was shown that cytogenetic analysis of the sister chromosomal sets adequately determines the frequency of chromosomal segregation errors during oocyte meioses I and II.

A cytogenetic study of G1-chromosomes in one-cell stage mouse embryo and in corresponding second polar body. Evaluation of aneuploidy originated in females heterozygous for translocation T[14;15]6Ca.

Meiotic chromosomes in ovulated oocytes and G1-chromosome sets visualized in the 2nd PB and in the pronuclei of one-cell stage embryos treated with okadaic acid were studied in female mouse heterozygous for reciprocal translocation T[14;15]6Ca. It was found that 61.5% of oocytes were haploid, 14.

The behaviour of mitochondria and cell integration during somatic hybridisation of sister blastomeres of the 2-cell mouse embryo.

The capacity of sister blastomeres of mouse embryos for induced fusion changed during the 2-cell stage. It was at low level (24%) at the early 2-cell stage, increased and reached 98.5% at the middle 2-cell stage and fell sharply to 31% at the late 2-cell stage.

Detection of aneuploidy in human oocytes and corresponding first polar bodies by fluorescent in situ hybridization.

Purpose: The purpose of the study was to investigate the reliability of the fluorescent in situ hybridization (FISH) analysis of the first polar body (IPB) for cytogenetic evaluation of human oocytes as a method of choice in preimplantation diagnosis of chromosomal aneuploidies.

Design: Human unfertilized oocytes and their extruded IPB were analyzed using the directly labeled fluorescence alpha-satellite DNA probes to chromosomes X and 18.

Results: Paired signals for chromosomes X and 18 were observed in the second meiotic prophase (MII) of unfertilized oocytes and their extruded IPB.

[The role of the maternal genotype in the realization of the embryotoxic activity of teratogens].

Against the background of the induced iron deficit ethanol (6.4 g/kg) causes aggravation of the embryolethal effect and anomalies in 15% of embryos in 14-day pregnant rats. Changes in the genome of rat males and females after the injection of the plasmid with a foreign gene at the stage of two pronuclei and the subsequent crossing with intact animals account for the increase in sensitivity of embryos to subteratogenic doses of sodium salicilate.

Spontaneous sister chromatid differentiation (SCD) and sister chromatid exchange (SCE) in mouse blastocyst chromosomes.

The phenomenon of spontaneous differentiation (without bromodeoxyuridine in the culture medium) of sister chromatids of mouse chromosomes at the blastocyst stage of embryogenesis is described. The frequency of sister chromatid exchanges in such differentiated chromosomes was calculated.

[The quantitative study of the areas of active rDNA location and of the other parameters of the interphase nucleolus detectable by silver staining during the cell differentiation of the rat trophoblast].

Different quantitative parameters of nucleolar silver staining have been studied in the cambial rat trophoblast cells on the 12th, 13th and 14th days of gestation. It has been shown that the number of Ag-positive granules in the nucleoli varied from 10 to 120. The number and the total area of silver stained granules in the nuclei increased progressively in the course of polyploidization, but was not doubled passing to the next ploidy level.

Okadaic acid induces premature chromosome condensation reflecting the cell cycle progression in one-cell stage mouse embryos.

Haploid parthenogenetic embryos as well as fertilized mouse eggs were treated in vitro with 1-10 microM okadaic acid (OA) at the one-cell stage. Cytogenetic analysis detected that OA induces nuclear envelope breakdown (NEBD) and premature condensation of interphase chromosomes in pronuclei as well as in 2nd polar body (PB) nuclei. G1-, S-, and G2-type prematurely condensed chromosomes (PCC) were found in pronuclei of embryos of different age, which reflects their progression through the first cell cycle.

[The frequency of sister chromatid exchanges in preimplantation mouse embryos].

The frequency of sister-chromatid exchanges (SCE) was analysed in spontaneously and superovulated morulae and blastocyst of CBA, C57Bl, F1 CBA x C57Bl mice in culture with bromodeoxyuridine (BrdU). The background level for SCE was found to be 4-5 times higher in early embryos than in fetal or adult tissues of the mouse. The phenomenon of spontaneous sister-chromatid differentiation (SCD) of blastocyst chromosomes without any treatment with BrdU was observed.

Visualization of second polar body chromosomes in fertilized and artificially activated mouse oocytes treated with okadaic acid.

Objective: Our objective was to develop a new reliable method for cytogenetic analysis of the chromosome set in second polar bodies (PBs) from one-cell-stage mouse embryos.

Setting: The study took place at the Reproductive Biology and Experimental Cytogenetics Laboratories.

Methods: Oocytes from F1 hybrid and T6/T6 mice were fertilized in vitro and artificially activated with ethanol.

[Cytological study of the nucleolus organizer regions in the chromosomes of CBA and C57BL mice and their hybrids].

Transcriptionally active NORs of chromosomes visualized by AgNO3 staining were studied in bone marrow and embryos (day 10 of gestation) of CBA and C57BL mice, as well as of (CBA x C57BL)F1 hybrids. These mouse strains were shown to differ by the average number of Ag-positive NORs in marrow cells; in hybrids, the number of NORs is greater than in the parent strains. During embryogenesis, the number of chromosomes carrying silver-stained NORs increases; however, no significant differences by this parameter was detected between hybrid and C57BL embryos.