Asymmetric DNA methylation between sister chromatids of metaphase chromosomes in mouse embryos upon bisphenol A action.

Earlier we showed that asymmetric methylation of sister chromatids (AMSC) was a specific characteristic of differentiation potency, and supposed that AMSC could be a useful marker of environmental impact connected with differentiation and/or dedifferentiation. Here we investigated the level of AMSC in chromosomes and the nuclei methylation in mouse preimplantation and postimplantation embryos, in comparison with the undifferentiated cells of mouse embryonal carcinoma cell line F9, and human differentiated HEK293 cells upon BPA influence. We found that exposure of mouse preimplantation embryos to BPA caused a significant decrease in the level of AMSC in chromosomes and the nuclei methylation. The BPA exposure of potentially differentiating F9 cells had no any influence on DNA methylation in nuclei but significantly decreased the number of AMSC. The level of DNA methylation and AMSC in HEK293 cells were not also changed. These data indicate that BPA exerts significant influence on differentiating and potentially differentiable cells. The most sensitive BPA targets are preimplantation embryos and stem cells.

[THE INFLUENCE OF THE PREPARATION PRETREATMENT ON IN SITU DETECTION OF 5-METHYLCYTOSINE IN METAPHASE CHROMOSOMES AND IN INTERPHASE NUCLEI].

Qualitative and quantitate analysis of DNA methylation in situ at the level of cells, chromosomes and chromosomal domains is extremely important for the diagnosis and treatment of various diseases, the study of ageing and the consequences of environmental impacts. An important question arises, whether the revealed in situ methylation pattern reflects DNA methylation per se and (or) availability of the DNA for antibodies, which in turn depends on the peculiarities of chromatin structure and chromosome condensation. These events can lead to an incorrect evaluation of the actual pattern of DNA methylation. To avoid this shortcoming as far as possible, we have modified the most widely used method of revealing 5-methylcytosine in situ with monoclonal antibodies. Here we have shown that the detection of DNA methylation staining of chromosomes including C-heterochromatin, chromosomal arms and sister chromatids is drastically dependent on pretreatment of chromosomal preparations for immunocytochemical study using fluorescent antibodies. Using undifferentiated stem cells of mouse embryonal carcinoma line F9, it has been found that change in preparations storage results in a sharp fluorescence decrease up to complete disappearance of the signal in centromeric heterochromatin. With the help of the method described in the work, we have first revealed the asymmetry of sister chromatids methylation in metaphase chromosomes of F9 cell and lymphocytes of human periphery blood. This may lead to asymmetry of transcriptional signature of daughter cells after division. The proposed here modification of 5-methylcytosine detection in situ provides a more complete characterization of methylation of chromosomes and chromosomal domains, compared to previously published methods.

[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. The methylation level of the nucleus DNA was shown to increase with a deterioration of environmental conditions. It was reported, that in the case of cultivation in M2 media not suitable for long cultivation, the DNA Ts methylation level, MaSat in particular, was higher compared to other embryo groups. Accordingly, not only a significant number of genes but also repeats of satellite DNA are involved in epigenetic regulation.

[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). We determined the localization of 4 types of mouse satDNA CEN and periCEN regions and associated proteins: RNA-helicase p68, SMC3, Rad21 subunits of the cohesin complex and SYCP3 subunit of the synaptonemal complex (SC). Partially flattened nuclei of the one- and two-cell embryos and embryos treated with ocadaic acids (OA) were used. Different satDNA fragments revealed distinct domains at the surface of NPB: periCEN MaSat was always localized in NPB more internally covering almost entire surface of NPB while CEN MiSat, MS3 and periCEN MS4 showed more peripheral localization. All 4 satDNA did not cover the entire areas of the NPB, indicating the presence of other DNA sequence involved in its formation. RNA-helicase p68 and components of multiprotein cohesin and synaptonemal complexes are the necessary components of NPB. Our results support the opinion that NPB serve as a precursor of chromocenters.

[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. We demonstrated on the mouse cultured cell line and prematurely condensed chromosomes that: (a) all four mouse satellite DNA fragments (major and minor satellite, mouse satellite 3 (MS3) and mouse satellite 4 (MS4)) were involved in the thread formation; (b) MS4 was involved in the thread to the least extent among all the other fragments; (c) telomere was never a member of the thread; (d) the thread was synthesized at a late G(2) phase; (e) RNA helicase p68 and CENP-B were among the protein components of the interchromosome connection. It was shown by FACS analysis that in mouse and human cell lines: (1) the flow karyotype spectrums were never free from chromosome aggregates; (2) chromosome association did not depend on the chromosome length and each chromosome was free to associate with the other.

[Localization of chromosomal nucleus organizing regions in one-cell mouse embryos and oocytes by fluorescence in situ hybridization]. / Vyiavlenie iadryshkoobrazuiushchikh raionov khromosom v odnokletochnykh zarodyshakh myshi s pomoshch'iu fluorestsentnoi gibridizatsii in situ.

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. Here, we studied for the first time the rDNA topology in one-cell mouse embryos and MII oocytes using fluorescence in situ hybridization and mouse rDNA probes. Our data suggest that in the pronuclei of one-cell embryos, rDNAs form rather compact clusters, whose number does not exceed that of nucleolus organizing chromosomes characteristic for the haploid set of mouse chromosomes. In zygotic pronuclei, not all nucleolar precursor bodies are associated with rDNA and not all rDNA repeats are attached to the nucleolar precursor bodies. Altogether, these data favor the idea that spatial interactions of nucleolus organizing chromosomes and nucleolar precursor bodies are not obligatory. We assume that associations between nucleolar precursor bodies and nucleolus organizing chromosomal regions are mediated by centromeric heterochromatin. The total numbers of silver stained nucleolus organizing chromosomes in CBA and C57BL mice are different. rDNA genes are unequally distributed among nucleolus organizing chromosomes and nucleolus organizing regions of sister chromatids.

[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]. / Tsitogeneticheskii analiz sestrinskikh naborov khromosom vo vtorom poliarnom tel'tse i v pronukleusakh odnokletochnykh zarodyshei myshei. I. Chastota i proiskhozhdenie aneuploidii u zarodyshei, geterozigotnykh po retsiproknoi khromosomnoi translokatsii 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. Trisomy and monosomy were observed in 36.2% embryos, while 2.2% featured tetrasomy or double monosomy. Errors of the first meiotic division caused aneuploidy in 28.5% embryos; trisomy and monosomy resulted from the homologs non-disjunction and chromatid presegregation in 17.6 and 10.9%, respectively. Numeral chromosomal aberrations in 4.1% of the embryos resulted from abnormal chromosomal segregation during oocyte meiosis II, while paternal chromosomal aberrations were found in 5.8% embryos. The main advantage of the proposed method is not only the higher accuracy in estimating the meiotic error frequency, but also the possibility to trace the origin of aneuploidy in mammalian embryos.

DNA-strand breaks in chromosomes of early mouse embryos as detected by in situ nick translation and gap filling.

The nick translation and gap filling procedures, without external addition of nicking enzymes, were performed in situ on fixed chromosomes of mouse preimplantation and postimplantation embryos and of bone marrow in order to detect possible DNA single-strand breaks (nicks and (or) gaps). All chromosome preparations were made using the same technique. Nick translation of chromosomal DNA with DNA polymerase I (Pol I) or gap filling with the Klenow fragment of Pol I in the presence of biotinylated-dUTP, demonstrated a regular absence of label on chromosomes of postimplantation embryos and bone marrow. No difference in sensitivity was found between the holoenzyme and the Klenow fragment. In preimplantation embryos, the chromosome reactivity in nick translation was highest at the blastocyst stage and varied according to cleavage divisions of the zygote.

[Single-stranded DNA breaks in the chromosomes of early mouse embryos]. / Odnotsepochechnye razryvy DNK v khromosomakh rannikh zarodyshei myshei.

The occurrence and distribution pattern of spontaneous single-strand breaks (nicks) and/or gaps of mouse chromosomal DNA were studied with the help of nick-translation procedure omitting exogenous nucleases. The holoenzyme and a Klenow's fragment were used at a concentration of 0.I. U/20 microl of reaction mixture, resp. Bio-dUTP and streptavidin-alkaline phosphatase were used for labeling and detection. Chromosomes of postimplantation embryos and bone marrow were not stained. Chromosomes of all preimplantation stages of development were homogeneously stained with prominent dots of various size and intensities of grayish. DNA Pol I and the Klenow enzyme demonstrated a similar pattern of labelling. The centromeric heterochromatin was not labeled. The label was localized asymmetrically exclusive of NOR and telomeric regions.

Single-strand DNA breaks in nuclei of early mouse embryos detected by in situ nick translation.

The nick translation procedure without external addition of nicking enzymes was performed in situ on fixed nuclei of mouse preimplantation, and postimplantation embryos, as well as bone marrow in order to detect possible DNA single-strand breaks. All preparations of nuclei were made using the same technique. Nick translation of nuclear DNA with DNA polymerase I in the presence of biotinylated-dUTP demonstrated a characteristic absence of label on nuclei of postimplantation embryos and bone marrow. The nuclear reactivity varied according to the cleavage divisions of the zygote, being highest at the four-cell stage.

[Cytological research on the argentophilic nucleolus-organizer regions of the metaphase chromosomes in parthenogenetic mouse embryos]. / Tsitologicheskoe issledovanie argentofil'nykh iadryshkoobrazuiushchikh raionov metafaznykh khromosom u partenogeneticheskikh zarodyshei myshei.

Silver staining technique visualizing argentophilic nucleolus organizer regions (Ag-NORs) was used for studying parthenogenetic mouse embryos produced by artificial activation of oocytes in Ca(2+)-Mg(2+)-free medium. Ag-NOR-containing chromosomes were detected in metaphases of parthenogenetic embryos during six successive cleavage divisions starting with the two-cell stage. The frequency of metaphases with varying AG-NOR number in diploid parthenogenones was similar to that in the control (fertilized) embryos. Average number of metaphase Ag-NOR chromosomes (calculated per diploid chromosome set) in haploid parthenogenones exceeded that in the control; in some cases all NORs were stained by silver. This is evidence that latent ribosomal cistrons in some chromosomes can be activated.

[Parthenogenetic development in response to the treatment of mouse oocytes with a weak alkali. Experiments with methylamine]. / Partenogeneticheskoe razvitie v otvet na obrabotku iaitsekletok myshi slaboi shcheloch'iu. Opyty s metilaminom.

Ovulated mouse oocytes were incubated in methylamine-containing medium M16 for different periods of time. Methylamine appeared to activate oocytes, and most of them developed by haploid parthenogenesis. Methylamine action depended on its concentration and time of incubation. The data obtained suggest that the increase of intracellular pH unblocks meiosis and activates mammalian oocytes [correction of cocytes].

[Characteristics of the ultrastructural organization of metaphase chromosomes in mouse embryos in the early cleavage stages]. / Osobennosti ul'trastrukturnoi organizatsii metafaznykh khromosom zarodyshei myshi na rannikh stadiiakh drobleniia.

The structural organization of the mouse metaphase chromosomes in the early embryonic development (I-IV cleavages) was studied using serial ultrathin section. It was shown that in the first cleavage the metaphase chromosomes consist of DNP fibrils 20-25 nm in diameter, which are distributed nonuniformly along the chromosomes. It was suggested that parts of chromosomal arms formed by tightly packing DNP fibrils may correspond to the G-bands revealed by the routine Giemsa staining. In metaphase chromosomes of 8-16-cell embryos DNP fibrils form chromonema--thick threads about 90 nm in diameter. The chromonemata are evenly organized along chromosomal arms. The centromeric heterochromatin always consists of DNP fibrils tightly arranged in a block having no chromonemal level of organization. In all the cells studied chromosomes form structural contacts (associations) by their centromeric heterochromatin regions.

[Parthenogenetic development of murine ova activated by heat shock]. / Partenogeneticheskoe razvitie iaitsekletok myshei, aktivirovannykh teplovym shokom.

The effects of heating oviducts up to 37-42 degrees on the ovulated mouse eggs have been studied. The heating of oviducts at 39.5 degrees for 7 min resulted in 85% activation. The subsequent increase in temperature did not raise the incidence of activation but led to the formation of micronuclei and other pathological changes in the pronuclei. The heating of oviducts at 39.5 degrees for 14 min demonstrated marked changes in heat resistance, which were dependent on the postovulatory age of eggs. The freshly ovulated eggs were characterized by a low resistance and were not activated by the heat shock. If the oviducts were first heated and then cooled, and again heated, most eggs were activated and their in vitro development was the best of all experimental series. The mechanisms of egg activation by heating are discussed.

[Parthenogenesis in mammals]. / Partenogenez mlekopitaiushchikh.

A review of studies dealing with spontaneous and induced parthenogenesis in mammals. The main methods of artificial egg activation, ways of their development and causes of mortality of the parthenogenetic mouse embryos are considered. The possibilities of using parthenogenesis for solving urgent problems of mammalian developmental biology are estimated and prospects of further studies in this field are outlined.

[Factors determining the frequency and pathways of the parthenogenetic development of ethanol-activated mouse oocytes]. / Izuchenie faktorov, opredeliaiushchikh chastotu i puti partenogeneticheskogo razvitiia iaitsekletok myshi, aktivirovannykh étanolom.

Ethanol activates the eggs inside the mother upon intraperitoneal, rather than intragastric, injection. The eggs are also activated and engaged into parthenogenetic development when the eggs or the whole oviducts with the ovulated eggs are placed in a culture medium with ethanol. The intensity of the activating effect of ethanol in vitro and ways of parthenogenetic development depend both on the ethanol concentration and temperature. At a temperature below 17 degrees ethanol did not activate the mouse eggs. There is a temperature optimum for each ethanol concentration studied (from 2 to 6.6%), at and ways of parthenogenetic development depend both on the ethanol concentration and the ability of parthenogenetic embryos to develop until the blastocyst stage are determined by the efficiency of activation and depend on the selection of optimal conditions for the action of ethanol. Cytochalasin B or D did not enhance the activating action of ethanol on the mouse eggs. The mechanisms of ethanol action on the eggs are discussed.