Irina O. Bogolyubova

Localization of poly(A)(+) RNA and mRNA export factors in interchromatin granule clusters of two-cell mouse embryos.

Interchromatin granule clusters (IGCs), also known as nuclear speckles, splicing factor compartments, or SC35-domains, are one of the most universal nuclear organelles of the cell. We have used two-cell mouse embryos as an experimental system to study the possible association of poly(A)+ RNA and factors involved in RNA export (Tip-associated protein [TAP] and heterogenous nuclear ribonucleoprotein A/B [hnRNP A/B]) with IGCs. Poly(A)+ RNA was localized by microinjections of 2′-O-Me(U)22 probes conjugated with tetramethylrhodamine. RNA export proteins were detected by immunofluorescence confocal laser microscopy and immunogold-labeling electron microscopy. We found that poly(A)+ RNA was located in IGCs regardless of the transcriptional state of the nuclei. hnRNP A/B was also found to characterize IGCs of the embryo nuclei with various levels of transcription activity. In transcriptionally active embryo nuclei, TAP was detected in the vicinity of IGCs rather than inside them; however, when transcription was inhibited by drugs, TAP was localized to IGCs. Our data support the idea that IGCs not only serve as splicing factor reservoirs, but also take part in mRNA retention and export.

FRET analysis of interactions between actin and exon-exon-junction complex proteins in early mouse embryos

Spatial interactions between nuclear actin and some proteins of the exon-exon junction complex (EJC) have been demonstrated in the nuclei of early mouse embryos by using Förster resonance energy transfer (FRET). FRET has been registered for the pairs actin-Y14, actin–Aly/REF and actin–NXF1/TAP in nucleoplasmic, irregularly shaped zones and in nucleolus precursor bodies (NPBs). We suggest that FRET signals in the nucleoplasm correspond to the bordering zone of transcriptionally active chromatin. Detection of FRET in NPBs indicates that the list of NPB functions is broader than has been assumed to date. The FRET pattern, typical for transcriptionally active embryos, is retained after treatment with DRB (a transcription inhibitor). Spatial interactions between actin and Y14 have been found to occur in an RNA-dependent manner. Our data thus suggest that nuclear actin is directly involved in mRNA export in early mouse embryos.

Immunocytochemical study of YB-1 nuclear distribution in different cell types

In the present work we studied the distribution of YB-1 in the nuclei of mouse hepatocytes, early embryos and human skin fibroblasts with the use of light and electron microscopy. To reveal YB-1, we applied rat polyclonal antibody against the C-terminal fragment of YB-1 molecule and rabbit polyclonal antibody against full-length YB-1 molecule. YB-1 distribution patterns varied significantly in different cell types. YB-1 was found to be colocalized with RNA polymerase I in mouse hepatocytes and embryos. Besides, YB-1 was revealed in a population of Cajal bodies in 2-cell mouse embryos but not in other cells studied.

F-actin distribution pattern in the nuclei of early mouse embryos

Nuclear actin is the essential component of gene expression. Here we show that the pattern of F- actin distribution in the nuclei of early mouse embryos depends on the experimental conditions and does not represent nonspecific cell reaction for the experimental influence.

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.

An Immunocytochemical Study of Interchromatin Granule Clusters in Early Mouse Embryos

Interchromatin granule clusters (IGCs) are universal nuclear domains. Their molecular composition and functions were studied in detail in somatic cells. Here, we studied IGCs in the nuclei of early mouse embryos during zygotic gene activation (ZGA). We found that the size of IGCs gradually increases during realization of ZGA events. Using immunocytochemical approaches, we showed that the molecular composition of IGCs is also modified in mouse embryos. The hyperphosphorylated form of RNA polymerase II and the transcription factor TFIID have been revealed in IGCs before the end of ZGA. Association of these factors with IGCs became more noticeable during ZGA realization. Our data suggest that IGCs in early mouse embryos have some functional peculiarities connected most probably with IGC formation de novo. We believe that IGCs in early mouse embryos not only are storage sites of splicing factors but also may be involved in mRNA metabolism and represent the multifunctional nuclear domains.

Localization of mRNA export factors in early mouse embryos

Abstract Background: Mammalian early embryogenesis is characterized by drastic alterations of gene expression patterns due to a course of metabolic and structural changes known as the zygotic gene activation. This

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.