[Actin and mRNA export factors distribution in the nucleus of preovulatory mouse oocytes].

Nuclear actin is a part of the chromatin remodeling complex involved in transcriptional and associated with nascent mRNA, providing them an ability to export from the nucleus. It is currently proved that many factors involved in mRNA editing and mRNA export are localized in IGCs. Using confocal laser scanning microscopy and immunogols labeling technique, we identified actin and A1 hnRNP protein in the nuclei of oocytes from mouse preovulatory follicles. We have demonstrated that nuclear actin is detected in association with the chromatin and IGCs. Components of mRNA export complex including proteins A1 and NXF1/TAP are localized in IGCs together with actin. In accordance with the concept suggesting that IGCs participate in RNA retention in the nucleus we discuss that mRNA transcription-export complex including actin, A1 hnRNP protein and NXF1/TAP, along with mRNA and transcription factors is formed in IGCs.

[Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes of the insect Tenebrio molitor].

The nucleus ofvitellogenic oocytes of the yellow mealworm, Tenebrio molitor, contains a karyosphere that consists of the condensed chromatin embedded in an extrachromosomal fibrogranular material. Numerous nuclear bodies located freely in the nucleoplasm are also observed. Amongst these bodies, counterparts of nuclear speckles (= interchromatin granule clusters, IGCs) can be identified by the presence of the marker protein SC35. Microinjections of fluorescently tagged methyloligoribonucleotide probes 2'-O-Me(U)22, complementary to poly(A) tails of RNAs, revealed poly(A)+ RNA in the vast majority of IGCs. We found that all T. molitor oocyte IGCs contain heterogeneous ribonucleoprotein (hnRNP) core protein Al that localizes to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and a part of nucleoplasmic IGCs also contain the adapter protein Aly that is known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was observed to colocalize with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner whereas it is RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data suggest on a role of the nucleoplasmic IGCs in mRNA biogenesis and retention in a road to nuclear export.

[Interchromatin granule clusters of mouse preovulatory oocytes, organization, molecular composition and possible functions].

At the diplotene stage of meiotic prophase, the nucleus of mouse preovulatory oocytes contains multiple interchromatin granule clusters (IGC). These nuclear compartments are universal and evolutionary conserved and enriched in pre-mRNA splicing factors. Nowadays, IGCs are believed to play an important role in gene expression events and contain different molecular components that allow coupling of many processes from transcription to mRNA export. We obtained the data on the distributions of poly(A)+RNA, hnRNPS A/B, and NXF1/TAP factor of mRNA export. These factors were found to associate with IGCs of mouse preovulatory oocytes. In the present study, we have demonstrated for the first time the dynamics of large IGCs after specific phosphorilation of SR-proteins with okadaic acid, an inhibitor of protein phosphatases. Using electron microscopy, conventional fluorescent and confocal microscopies, as well as microinjections of olygonucleotide probes in mouse oocytes, some features of structural organization and molecular compositions of IGCs in the nuclei of mouse oocyte from antral follicles were established. Possible roles of IGCs in pre-mRNA metabolism and the participation of these structures in mRNA export are discussed.

[Nucleolus transformation in oocytes of mouse antral follicles. Revealing of coilin and RNA polymerase I complex components].

This study is the continuation of our previous investigation of the nucleolus transformation in growing oocytes from mouse multilayer follicles (Pochukalina, Parfenov, 2006). Here in the present research we have examined the features of organization and molecular composition of nucleolus like body, or postnucleolus, in two groups of oocytes with different chromatin configuration from mouse antral follicles. Using light and electron immunocytochemistry, we have defined the dynamics of ribosomal RNA synthesis and processing molecular component distribution in postnucleolus. Considerable changes in RNA polymerase I distribution and its colocalization with coilin at the periphery of postnucleolus were revealed. Putative role of coilin in formation of complexes with ribosomal RNA synthesis/processing components is discussed.

[Synthesis of nucleic acids and localization of atrial natriuretic peptide in the crayfish haemocytes].

Three types of cells circulate in haemolymph of the crayfish Astacus astacus: agranular haemocytes (HCs I), small-granule haemocytes (HCs II) and large-granule haemocytes (HCs III). Their proliferation, differentiation and function remain poorly understood. By means of light and electron microscopic autoradiography using [3H]-thymidine, we have revealed that only HCs I are capable of DNA synthesis and mitosis whereas HCs II and HCs III are replicatively inactive. To determine whether the HCs I are proliferating progenitor cells for the granular HCs, we have analyzed autographs of HC population in 1, 2, 7 and 21 days after a single [3H]-thymidine administration. Contrary to the expectation, we have failed to find labeled HCs II and HCs III. These findings raise doubts on the capacity of the HCs I to differentiate into two other types of HCs. By autoradiography using 3H-uridine, it has been detected that intensity of the RNA synthesis was the greatest in HCs I and less by a factor of two and four in HCs II and HCs III, respectively. Additionally, by EM immunocytochemistry, ANP-like immunoreactivity was revealed in the large granules of the HCs III. We assume that availability of ANP in secretory granules extends the possible functions of the crayfish HCs and suggests their participation in regulation of water-salt balance and immune responses.

[Cellular aspects of hypertrophic cardiomyopathy pathogenesis: the role of cardiomyocyte polyploidy and activation of the proliferating cell nuclear antigen in the myocardium].

Mechanisms of hypertrophy development in hypertrophic obstructive cardiomyopathy (HOCM) have not been enough investigated. In our study, there have been examined patients with severe HOCM at different ages, including children, and patients with essential arterial hypertension (EAH). There was found, that HOCM in children compared to adults was characterized by considerable interventricular septum (IVS) hypertrophy and it was accompanied by the acceleration of cardiomyocyte polyploidy. The average ploidy level of cardiomyocytes in children with HOCM was higher than analogous indices in adults. The average ploidy level of nuclei, the part of PCNA-positive nuclei and polyploidic nuclei of cardiomyocytes in aduls with HOCM were authentically higher than in patients with EAH. Activation of the nuclear antigen in stromal cells was detected only in patients with HOCM. Our findings provide evidence of an important role of cardiomyocyte polyploidy and activation of the proliferating cell nuclear antigen in development of the myocardial hypertrophy in patients with HOCM.

[Cajal bodies in insect oocytes. II. New data on the molecular composition of cajal bodies in oocytes of the house cricket Acheta domesticus with special reference to interactions between cajal bodies and interchromatin granule clusters].

Cajal bodies (CBs) in oocytes of the house cricket Acheta domesticus are large, perfectly spherical nuclear organelles with a complex internal structure. These consist of a fibrillar coilin-containing matrix and a central cavity with a prominent fibrogranular body inside; the latter has been referred to as an "internal" interchromatin granule cluster (IGC). Within the matrix of CBs we detected transcriptional co-activators CBP/p300 and TATA-box binding protein (TBP). No RNA polymerase II was revealed in CBs of both normal and actynomycin D treated oocytes. In the nucleoplasm of A. domesticus oocytes, besides CBs, free IGCs were observed. In oocytes treated with actynomycin D, the amount of "free" IGCs in the nucleoplasm increase significantly, granular and fibrillar components of IGCs were seen segregated, and RNA polymerase II and CBP/p300 were found to be accumulated in fibrillar zones of IGCs.

Dynamics of satellite binding protein CENP-B and telomere binding protein TRF2/MTBP in the nuclei of mouse spermatogenic line.

The location of centromeric protein CENP-B and telomeric protein TRF2/MTBP in the mouse spermatogenic line has been studied using indirect immunofluorescent and immunoelectron microscopy. CENP-B localized to the heterochromatic parts of the nuclei at meiotic stages. A clearly distinct chromocenter forms in the nucleus at stages 3-4 of spermatid maturation; CENP-B localizes in it and in the area adjacent to the future acrosome. CENP-B localization in the subacrosomal area and in the chromocenters' periphery demonstrates that centromeres are organized in two groups in mouse spermatozoa, unlike human centromeres. TRF2/MTBP concentrates around the forming chromocenter at spermiogenesis early stages. The TRF2/MTBP main signal migrates into the area of acrosomal membrane at the course of spermatozoon maturation. TRF2/MTBP never localizes inside the synaptonemal complex but can be found in the areas where the synaptonemal complex attaches to the nuclear envelope. At the pachytene and diplotene stages when chromosomes separate from the nuclear envelope, some amount of the protein remains bound to the nuclear membrane while the other part reveals itself in chromosomes. TRF2/MTBP accumulates in the future acrosome from the very beginning of its formation. In the mature spermatozoon TRF2/MTBP decorates the acrosomal membrane as well as spreads in condensed chromatin.

[The nucleolus in oocytes of multylayer mouse follicles: topography of fibrillarin, RNA polymerase I and coilin].

By electron microscopy, conventional fluorescence and confocal microscopy, some features of structural and molecular organization of the nucleolus in oocyte nucleus from mouse multilayer follicles were examined. The examined nucleolus lacks almost all characteristic nucleolar components, such as fibrillar centers, dense fibrillar and granular components. This nucleolus consists exclusively of a homogenous filamentous material and is penetrated by numerous interstices. Besides, a striking association of the nucleolus with coilin, a marker of Cajal bodies, was observed. We could map the coilin accumulation in three different areas: around, in the periphery, or inside the nucleolus. Additionally, we examined a topological relationship between coilin and two key proteins of nucleolar transcription-processing machinery, RNA polymerase I and fibrillarin. RNA polymerase I rather than fibrillarin was found to be colocalized with coilin. Finally, we propose that data on dynamics of coilin relation with the nucleolus may elucidate a possible role of coilin in functional relationship between the nucleolus and Cajal bodies.

Nuclear structure in early mouse embryos: A comparative ultrastructural and immunocytochemical study with special emphasis on the "2-cell block in vitro".

The embryos from many outbred and inbred strains of mice are arrested at the late 2-cell stage when cultured in vitro in simple culture media. This phenomenon is referred to as the "2-cell block in vitro". The ultrastructural morphology of the nuclei of the blocked embryos is not yet well described. In the present paper we documented the results of a comparative study on the nuclei of mouse embryos, both normally developing and arrested at the 2-cell stage. The blocked embryos maintain the morphological integrity of their nuclei. Main nuclear domains (nucleolus precursor bodies, interchromatin granule clusters, perichromatin granules, and perichromatin fibrils), typical for the control embryos, are observed in the blocked ones. A number and morphological characteristics of these nuclear substructures are not changed significantly in the blocked embryos. At the same time, RNA polymerase II and pre-mRNA splicing factors are redistributed in the nucleus of the blocked embryos. Although something goes to show that nuclear organization of the blocked embryos differ from that of the control, we could not reveal in the blocked embryos distinct signs of degeneration which might characterize aged or dying cells. Our data confirm a peculiar functional state of the 2-cell blocked embryos.

Identification and dynamics of Cajal bodies in relation to karyosphere formation in scorpionfly oocytes.

In oocyte nuclei of the scorpionfly, Panorpa communis, we have recently defined a population of nuclear bodies (NBs) that contain some components of Cajal bodies (CBs). In the present study, we used several criteria [presence of coilin, U7 snRNA, RNA polymerase II (pol II) and specific ultrastructure] to identify these NBs as CBs. The essential evidence for CB identification came from experiments with microinjection of fluorescein-tagged U7 snRNA. Consistent with the U7 data, we found pol II and pre-mRNA splicing factor, SC35, in Panorpa oocyte CBs. We show here that the dynamics of CBs differs from that in somatic cells and correlates with the level of oocyte chromosome condensation. We also found that the significant increase of CB size is accompanied by condensation of the chromosomes in the karyosphere, which is indicative of a decline in transcription. Using immunogold microscopy we determined that pol II and coilin are shared by CBs and the granular material associated with condensed chromosomes in the Panorpa karyosphere. The colocalization of pol II, U7 snRNA and splicing factors with CBs at the inactive stage of late oogenesis suggests that the latter may serve as storage domains for components that were earlier engaged in RNA transcription and processing.

[Karyosphere and extrachromosomal nuclear bodies in oocytes of the scorpionfly, Panorpa communis].

Oocyte nuclear structures were studied for the scorpionfly Panorpa communis at different stages of oocyte growth, from pachytene to the first meiotic division. Using immunofluorescent and immunogold microscopy, we analyzed the nuclear distribution of RNA polymerase II, splicing factors and coilin. These factors were revealed in close association with perichromatin fibrils and, later, with some elements of the karyosphere and extrachromosomal nuclear bodies (NBs). Besides, it was shown that large amounts of P. communis oocyte NBs represent Cajal bodies (CBs) and contain CB marker protein, coilin, as well as RNA polymerase II, and in some cases an essential splicing factor, SC35. The presence of SC35 is commonly not characteristic of CBs in somatic cells. CB dynamics was traced in inactivated oocyte nuclei, during a gradual condensation of chromosomes and their final assembling into the karyosphere. It has been shown that coilin, RNA polymerase II and SC35 protein are common compounds shared by CBs and some granular material associated with these condensed chromosomes. CB remnants were demonstrated in the ooplasm after the breakdown of nuclear envelope before the first meiotic division. In inactivated oocyte nuclei, CBs serve presumably as storage compartments for some inactive components essential for gene expression.

Atrial natriuretic peptide in the granular cells of the snail heart.

UNLABELLED: Cardiomyocytes of vertebrates combine contractile and endocrine functions. They synthesize and secrete atrial natriuretic peptide (ANP), which is localized in their specific granules. The presence of ANP has been shown in some tissues of invertebrates, including the heart of molluscs. We have studied localization of ANP in cells of the snail heart. METHOD: The atrial and ventricular tissues of the snail Helix pomatia were studied by electron microscope immunocytochemistry, using anti-ANP antibodies. ANP-immunoreactivity has been detected in granules of granular cells located on the luminal surface of the snail myocardium. These cells are abundant in the atrium being very rare in the ventricle. Granular cells at different stages of maturation were revealed. Immature granular cells have light granules of moderate size with homogeneous tight content, while mature granular cells are huge in size and all their granules are fused together. The material of these granules loosens up and almost completely fills up the cytoplasm. No ANP-immunoreactivity was observed in muscle cells or nerve fibers. A possible origin of granular cells from the cardiac endothelial cells is discussed. The molluscan heart, similar to that of vertebrates, is a bifunctional organ. However, contrary to the heart of vertebrates, in the molluscan heart contractile and endocrine functions are separated between different types of cells.

[Distribution of RNA polymerase II in nuclei of early murine embryos]. / Raspredelenie RNK-polimerazy II v iadrakh rannikh myshinykh émbrionov.

Nuclear distribution of unphosphorylated and phosphorylated forms of RNA polymerase II in two celled mouse embryos was studied using immunoelectron microscopy. RNA polymerase II was shown to be present in the karyoplasm of mouse embryo nuclei at the early two-celled stage, that is before activation of the embryonic genome. During this period, RNA polymerase II is diffusely localized in the nuclei being not associated with any nuclear domains. After embryonic genome activation, the pattern of the nuclear distribution of RNA polymerase II is changed. At the late two-celled stage, its prominent part is deposited in pronucleoli, as well as in interchromatin granule clusters and perichromatin fibrils. At this stage, unphosphorylated RNA polymerase II is mainly associated with perichromatin fibrils. The obtained data fit well with the notion of the coordinated distribution of RNA polymerase II and splicing factors, and of their association with the same nuclear domains.

[Extrachromosomal structures containing small nuclear RNP and coilin in the late vitellogenic oocytes of hibernating grass frogs]. / Ekstrakhromosomnye struktury, soderzhashchie malye iadernye RNP i koilin, v iadrakh pozdnikh vitellogennykh ootsitov zimuiushchikh travianykh liagushek.

We have studied extrachromosomal structures in the germinal vesicle (GV) of the late vitellogenic oocytes of hibernating frogs Rana temporaria. During this period of oogenesis, chromosomes are completely inactivated to be surrounded by a fibrillar karyosphere capsule (Gruzova, Parfenov, 1993). Using immunostaining and in situ nucleic acid hybridization, we have identified three types of extrachromosomal structures: Cajal bodies (CB), nucleoli, and micronucleoli. Immunostaining of GV spreads has revealed that CB and nucleoli contain coilin, a marker protein for CB. The nucleoli were also positively stained with antibodies against Sm-epitope and trimetylguanosine cap of small nuclear (sn) RNP. According to the results of in situ nucleic acid hybridization, the nucleoli contain U6 snRNA. To further investigate a distribution of coilin in GV of the late vitellogenic oocytes of R. temporaria, we injected myc-tagged transcripts of full length human coilin (Wu et al., 1994) into the cytoplasm of oocytes and followed the pathway of the newly translated protein with an antibody specific for the tag. Immunofluorescent staining of spread GV contents demonstrated a specific staining of the nucleoli within 3 h after injection. We suggest that the newly synthesized myc-coilin may be phosphorilated and targeted to the nucleoli.

[Immunoelectron study of RNA polymerase II distribution in human oocyte nuclei]. / Immunoélektronnoe issledovanie raspredeleniia RNK-polimerazy II v iadrakh ootsitov cheloveka.

The intranuclear distribution of two (unphosphorylated and hyperphosphorylated) forms of RNA polymerase II (Pol II) was studied in human oocytes from antral follicles using immunogold labeling/electron microscopy. The distribution of Pol II was as well as to the distribution of two splicing factors (snRNPs and SC-35) in the intranuclear entities, namely, interchromatin granule clusters (IGCs), nucleolus-like bodies (NLBs), and perichromatin fibrils (PFs). The results have shown that 1) antibodies directed against two forms of Pol II have a similar pattern of intranuclear distribution 2) both Pol II and splicing factors progressively accumulate in IGCs with a decrease in the transcriptional activity of the oocyte nucleus, 3) both Pol II and splicing factors are located on PFs, and 4) Pol II is present in the NLBs at all transcriptional states of the oocyte nucleus. The accumulation of Pol II and splicing factors in IGCs, concomitant with a decrease in the transcriptional activity, suggests a coordinated mechanism for the movement of both Pol II and splicing factors from the sites of action to the sites of storage.

Dynamics of nuclear localization sites for COX-2 in vascular endothelial cells.

We investigated the relationships among expression, activity, and spatial organization of cyclooxygenase (COX-1 and COX-2) in endothelial cells from porcine and human cerebral microvessels and from human umbilical vein. In quiescent cells, COX-1 was detected in the perinuclear zone and the cytoplasm, while COX-2 was mainly a nuclear resident possibly connected with the nuclear matrix. COX-2 immunogold labeling was situated in the nuclear envelope, at the nuclear pores, and in connection with the perichromatin regions of the nucleus, considered to be the sites of active transcription. In human endothelial cells transcriptionally activated by interleukin (IL)-1beta, the nucleus remained a major COX-2 localization site during the first 12 h of stimulation, when COX-2 expression was maximally induced. The continuous rise in prostanoid synthesis at 17-23 h of stimulation was associated with COX-2 relocation from the nucleus to the nuclear envelope and the cytoplasm. IL-1beta did not affect COX-1 expression, activity, and localization. COX-2 nuclear localization sites and trafficking between the nucleus and the cytoplasm in endothelial cells may indicate a novel function of COX-2 in regulating gene expression.

[Pre-mRNA splicing factors in nuclei of two-cells murine embryos]. / Faktory spla'isinga pre-mRNK v iadrakh dvukhkletochnykh zarodyshe'i myshe'i.

The distribution of pre-mRNA splicing factors (snRNPs and SR-protein SC35) was studied in the nuclei of early and late 2-cell mouse embryos using light and electron immunocytochemistry. The splicing factors were shown to be present in the nuclei before the beginning of embryonic transcription. In inactive nuclei of early 2-cell embryos, SR-protein SC35 is concentrated in clusters of intranuclear granules to be absent from the karyoplasm. After the beginning of embryonic transcription activation, concentration of splicing factors in the karyoplasm increases. The data obtained are in accordance with the idea that some particular nuclear domains may be storage sites for pre-mRNA splicing factors for constant recruiting of splicing factors to perichromatin fibrils.

[Intranuclear structures containing RNA maturation factors in early vitellogenic oocytes of Rana temporaria]. / Vnutriiadernye struktury, soderzhashchie faktory sozrevaniia RNK, v rannikh vitellogennykh ootsitakh travianoi liagushki.

We have examined the distribution of RNA processing factors in the germinal vesicle (GV) of the common frog Rana temporaria during early vitellogenesis by immunostaining on light- and electronmicroscopic levels and by in situ nucleic acid hybridization. Small nuclear RNPs (snRNP) and factor SC35 involved in pre-mRNA splicing occur in lampbrush chromosome loops and numerous granules 1-3 microns in size. These granules are identical to B snurposomes of Xenopus laevis and Notophtalmus viridescens described earlier (Wu et al., 1991). Some of B snurposomes are attached to homologous loops of lampbrush chromosomes. Immunofluorescent study of Cajal bodies/coiled bodies (CB) showed that sometimes CB have B snurposomes attached to their surface. In this case splicing factor SC35 is found in B snurposomes and B-like inclusions in CB matrix. In CB without attached B snurposomes splicing factor SC35 localizes throughout the whole organelle. Staining of GV spreads with antibodies against nucleolar protein NO38 revealed this protein in CB, nucleoli and micronucleoli. Using in situ nucleic acid hybridization and immunofluorescent staining we have found that on GV spreads from hibernating frogs B snurposomes contact nucleoli. Nucleoli contain snRNP. These data suggest that nucleoli may be storage sites of snRNPs during natural inactivation of RNA synthesis. During winter season in Rana temporaria GV nucleoli become compacted and a number of micronucleoli (less than 2 microns) dramatically increases. Analysis of micronucleoli showed that they contain rRNA, protein NO38, trace amount of U3 small nucleolar RNA and do not contain fibrillarin, involved as U3 in pre-rRNA processing. We suggest that decrease of rRNA synthesis during frog hibernation results in transformation of part of nucleoli in micronucleoli.