Special Nuclear Structures in the Germinal Vesicle of the Common Frog with Emphasis on the So-Called Karyosphere Capsule.

The karyosphere (karyosome) is a structure that forms in the oocyte nucleus-germinal vesicle (GV)-at the diplotene stage of meiotic prophase due to the assembly of all chromosomes in a limited portion of the GV. In some organisms, the karyosphere has an extrachromosomal external capsule, the marker protein of which is nuclear F-actin. Despite many years of theories about the formation of the karyosphere capsule (KC) in the GV of the common frog Rana temporaria, we present data that cast doubt on its existence, at least in this species. Specific extrachromosomal strands, which had been considered the main elements of the frog's KC, do not form a continuous layer around the karyosphere and, according to immunogold labeling, do not contain structural proteins, such as actin and lamin B. At the same time, F-actin is indeed noticeably concentrated around the karyosphere, creating the illusion of a capsule at the light microscopy/fluorescence level. The barrier-to-autointegration factor (BAF) and one of its functional partners-LEMD2, an inner nuclear membrane protein-are not localized in the strands, suggesting that the strands are not functional counterparts of the nuclear envelope. The presence of characteristic strands in the GV of R. temporaria late oocytes may reflect an excess of SMC1 involved in the structural maintenance of diplotene oocyte chromosomes at the karyosphere stage, since SMC1 has been shown to be the most abundant protein in the strands. Other characteristic microstructures-the so-called annuli, very similar in ultrastructure to the nuclear pore complexes-do not contain nucleoporins Nup35 and Nup93, and, therefore, they cannot be considered autonomous pore complexes, as previously thought. Taken together, our data indicate that traditional ideas about the existence of the R. temporaria KC as a special structural compartment of the GV are to be revisited.

Nuclear Distribution of the Chromatin-Remodeling Protein ATRX in Mouse Early Embryos during Normal Development and Developmental Arrest In Vitro.

The chromatin-remodeling protein ATRX, which is currently recognized as one of the key genome caretakers, plays an important role in oogenesis and early embryogenesis in mammals. ATRX distribution in the nuclei of mouse embryos developing in vivo and in vitro, including when the embryos are arrested at the two-cell stage-the so-called two-cell block in vitro-was studied using immunofluorescent labeling and FISH. In normally developing two- and four-cell embryos, ATRX was found to be closely colocalized with pericentromeric DNA sequences detected with a probe to the mouse major satellite DNA. The association of ATRX with pericentromeric heterochromatin is mediated by nuclear actin and reduced after the treatment of embryos with latrunculin B. When culturing embryos in vitro, the distribution pattern of ATRX changes, leading to a decrease in the association of this protein with major satellite DNA especially under the two-cell block in vitro. Taken together, our data suggest that the intranuclear distribution of ATRX reflects the viability of mouse embryos and their probability of successful preimplantation development.

The dynamics of DAXX protein distribution in the nucleus of mouse early embryos.

Nuclear distribution of Death-associated protein 6 (Daxx) was studied using fluorescent and electron microscopy in mouse embryos at different stages of development in vivo, from zygote to morula. Daxx was found in association with transcriptionally silent chromatin predominantly with a heterochromatin rim surrounding the nucleolus precursor bodies (NPBs) at all stages studied. At the zygote stage, Daxx was detected only at the periphery of NPBs both in male and female pronuclei. At the late two-cell stage, Daxx was localized not only in the heterochromatin rim at the periphery of NPBs but also in heterochromatin zones not associated with NPBs. At the morula stage, a diffuse distribution of Daxx prevailed. Scarce Daxx-positive zones were detected only in some embryos at the nucleolar periphery. Thus, Daxx is noticeably redistributed during mouse embryo cleavage, and the most conspicuous areas of Daxx concentration are observed at the end of two-cell stage. Daxx is found colocalized with the chromatin-remodeling protein ATRX exclusively in two-cell embryos, but the heterochromatin areas containing either Daxx or ATRX individually are also observed at this stage. However, most zones containing both Daxx and ATRX demonstrated a low FRET-efficiency. This suggest that two molecules are not approached sufficiently close for molecular interactions to occur. Our data suggests that Daxx may function without cooperation with ATRX at least at some stages of early mouse development.

Application of the allogenic mesenchymal stem cells in the therapy of the bladder tuberculosis.

Urogenital tuberculosis (TB) often leads to contraction of the bladder, a reduction of the urinary reservoir capacity, and, in the latest stage, to real microcystitis up to full obliteration. Bladder TB Stage 4 is unsuitable for conservative therapy, and cystectomy with subsequent enteroplasty is indicated. In this study, using a model of bladder TB in New Zealand rabbits, the therapeutic efficacy of the interstitial injection of autologous bone-derived mesenchymal stem cells (MSCs) combined with standard anti-TB treatment in the restoration of the bladder function was demonstrated. For analysis of the MSC distribution in tissues, the latter were labelled with superparamagnetic iron oxide nanoparticles. In vitro studies demonstrated the high intracellular incorporation of nanoparticles and the absence of cytotoxicity on MSC viability and proliferation. A single-dose administration of MSCs into the bladder mucosal layer significantly reduced the wall deformation and inflammation and hindered the development of fibrosis, which was proven by the subsequent histological assay. Confocal microscopy studies of the bladder cryosections confirmed the presence of superparamagnetic iron oxide nanoparticle-labelled MSCs in different bladder layers of the treated animals, thus indicating the role of stem cells in bladder regeneration.

Detection of RNA Polymerase II in Mouse Embryos During Zygotic Genome Activation Using Immunocytochemistry.

Mammalian pre-implantation embryos represent a highly dynamic experimental model for comparative studies of nuclear structure and functions in the context of gradual reactivation of transcription. Here, we present details of the methods that allow localizing RNA polymerase II in mouse pre-implantation embryos with specific antibodies, using fluorescent/confocal and electron microscopy. We stress the special aspects of immunolabeling protocols in respect to the embryonic material. We made a special emphasis on the essential steps preceding the immunocytochemical experiments. In particular, we consider the procedures of female hormonal stimulation and embryo collection. The described approaches are also applicable to study other nuclear proteins.

Nuclear distribution of the chromatin-remodeling protein ATRX in mouse early embryogenesis.

The nucleus of mammalian embryos differs by transcriptional activity at different stages of early development. Here, we studied nuclear distribution of the chromatin-remodeling protein ATRX in pre-implantation mouse embryos. Immunofluorescent staining revealed the changes of ATRX nuclear distribution at the initial stages of early mouse development. At the stage of early zygote, a diffuse ATRX distribution pattern was prevalent. During the course of zygotic genome activation (ZGA), zones of increased ATRX concentration are observed, and they are most expressed in the nuclei of late 2-cell embryos. In the morula stage, the ATRX distribution becomes diffuse again. In zygotes, the patterns of ATRX distribution differ between male and female pronuclei. At all the stages, ATRX concentrates in the DAPI-positive areas of condensed chromatin. The level of colocalization between ATRX and heterochromatin was found the highest at the late 2-cell stage. When transcription was artificially suppressed, the pattern of intranuclear ATRX distribution was mostly determined by the mechanism of inhibitor action rather than the decreased level of transcriptional activity. Thus, the obvious changes of ATRX distribution occur and partially correlate with the main stages of ZGA during mouse early development, but these changes seem to be determined by other processes of structural and functional rearrangements of blastomere nuclei.

Nuclear distribution of RNA polymerase II and mRNA processing machinery in early mammalian embryos.

Spatial distribution of components of nuclear metabolism provides a significant impact on regulation of the processes of gene expression. While distribution of the key nuclear antigens and their association with the defined nuclear domains were thoroughly traced in mammalian somatic cells, similar data for the preimplantation embryos are scanty and fragmental. However, the period of cleavage is characterized by the most drastic and dynamic nuclear reorganizations accompanying zygotic gene activation. In this minireview, we try to summarize the results of studies concerning distribution of major factors involved in RNA polymerase II-dependent transcription, pre-mRNA splicing mRNA export that have been carried out on early embryos of mammals.