Cytological research on the argentophilic nucleolus-organizer regions of the metaphase chromosomes in parthenogenetic mouse embryos]

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.     

Localization of satellite DNA and associated proteins in respect to nucleolar precursor bodies in one- and two-cell mouse embryos

Nucleolar precursor bodies (NPB) are discrete entities in zygotic pronuclei and in the nuclei of two-cell mouse embryos. Centromeric (CEN) and pericentromeric (periCEN) chromosome regions are associated with the chromatin layer surrounding NPB. Four types of satellite DNA (satDNA) are currently known in Mus musculus, including mouse minor satellite 4 (MiSat), mouse satellite 3 (MS3) in the CEN region, mouse major satellite (MaSat), and mouse satellite (MS4) in the periCEN region. We determined the localization of these four types of mouse satDNA and associated proteins (RNA-helicase p68, SMC3, Rad21 subunits of the cohesin complex and SYCP3 subunit of the synaptonemal complex) in respect to NPB. Partially flattened nuclei of the one- and two-cell intact embryos and embryos treated with okadaic acid (OA) were used. It was found that different satDNA are localized in different regions at the NPB surface: periCEN MaSat occupied almost the whole NPB surface; CEN MiSat, MS3 and periCEN MS4 were located more peripherally. All four satDNA did not cover the entire NPB area, which indicates the presence of other DNA sequences involved in the association with NPB periphery. Among the proteins probed, RNA-helicase p68 and components of multiprotein cohesin and synaptonemal complexes (SCs) showed the most prominent colocolization with NPB. Our results support the idea that NPB are chromocenter precursors.     

Factors determining the frequency and pathways of the parthenogenetic development of ethanol-activated mouse oocytes

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.     

Characteristics of the ultrastructural organization of metaphase chromosomes in mouse embryos in the early cleavage stages

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.     

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.