Biomedical subjects

V N Parfenov

Publications and source records attributed to V N Parfenov.

At least 19 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

[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.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

[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.

Animals↗

PubMed2002

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.

Animals↗

PubMed2002

[Distribution of RNA polymerase II in nuclei of early murine embryos].

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.

Animals↗

PubMed2002

[Extrachromosomal structures containing small nuclear RNP and coilin in the late vitellogenic oocytes of hibernating grass frogs].

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.

Animals↗

PubMed2001

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.

Animals↗

PubMed2001

[Immunoelectron study of RNA polymerase II distribution in human oocyte nuclei].

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.

Adult↗

PubMed2000

Nuclear distribution of RNA polymerase II in human oocytes from antral follicles: dynamics relative to the transcriptional state and association with splicing factors.

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 analyzed relative to the transcriptional state of the oocyte 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 showed that (1) antibodies directed against two forms of Pol II have similar pattern of intranuclear distribution, (2) both Pol II and splicing factors progressively accumulate in IGCs with decrease in the transcriptional activity of the oocyte nucleus, (3) both Pol II and splicing factors localize to PFs, and (4) Pol II is present in the NLBs at all transcriptional states of the oocyte nucleus. These studies confirm earlier proposals that PFs represent a nuclear domain in which RNA transcription/processing are spatially coupled. 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.

Adult↗

PubMed2000

[Intranuclear structures containing RNA maturation factors in early vitellogenic oocytes of Rana temporaria].

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.

Animals↗

PubMed2000

[Pre-mRNA splicing factors in nuclei of two-cells murine embryos].

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.

Animals↗

PubMed1999

[Characteristic of structural and functional organization of two-cell mouse embryos exposed to inhibitors of cell proliferation].

Within the framework of studying the "2-cell block in vitro" phenomenon, two variants of inhibitory-induced stoppage of development at a two-cell stage were produced and analysed. Mimosine arrested the cleavage on the G1/S interface, and genistein at G2 stage of the second cell cycle. In the experimentally blocked embryos a detailed study was made of the ultrastructural organization of blastomeres and intracellular localization of mitochondria vitally stained with rhodamine 123. The light and electron microscope observations testify to the viability of the embryos within a 22-24 hour exposure to inhibitors. Adhesive contacts between blastomeres were seen to slack after the treatment with both the inhibitors, resp., but in particular after genistein treatment. At the ultrastructural level no significant destructive modifications in blastomere organization were noticed. The cytoplasm of the control and treated embryo cells displayed diffusely distributed sheets of intermediate filaments, morphologically looking immature mitochondria and numerous aggregated lipid inclusions. The nuclear morphology was similar in both cases. Mitochondria of the treated embryo cells kept their ability to accumulate rhodamine 123, which testifies to their functional activity. However, the character of mitochondrial intracellular distribution was seen to change from diffuse to clustered. Numerous mitochondria clusters were concentrated mainly in the perinuclear area of blastomeres. As in the control ones, in the treated embryos the position of the nuclei was visualized by ring-like concentrations of mitochondria in the central part of blastomeres; in mimosine-treated cells the "rings" were thickened and contained mitochondria clusters. In genistein-treated embryos, mitochondria form numerous tiny clusters uniformly distributed in the cytoplasm; the perinuclear "rings" are still present, though less distinct than in the control embryos. Thus, it may be concluded that although the inhibitory treatment of two-cell embryos truly modified the mitochondrial distribution in these, the eventual pattern of such changes differed considerably from that characteristic of embryos in the state of "2-cell block in vitro". These results support the view on the unique character of morphofunctional modifications that occur in the latter embryos.

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PubMed1998

Dynamics of distribution of splicing components relative to the transcriptional state of human oocytes from antral follicles.

The distribution of two splicing components (snRNP and SC-35) and coilin were studied by immunogold/electron microscopy in human oocytes from antral follicles at different levels of transcriptional activity (i.e., active, intermediate, and inactive). The results showed a decrease of snRNPs and SC-35 in the karyoplasm as the oocytes progress from a transcriptionally active to the inactive state. The main areas of accumulation of both these splicing components in all stages of oocytes appeared to be the interchromatin granule clusters (IGCs). Within the IGCs, the two splicing components seemed to be spatially segregated, with the snRNPs predominantly bound to the fibrillar region, whereas the SC-35 factors are being enriched in the granular zone. The p80 coilin was found only in the nucleolus-like body (NLB), which is present in all three stages of oocytes; no coiled bodies were evident. These data are consistent with the notion that splicing occurs in the karyoplasm and that the splicing components are mobilized from a storage site (IGCs) to the site of action.

Adult↗

PubMed1998

[Splicing factors in oocyte nuclei from human antral follicles].

Three groups of oocytes in the human antral follicules were previously distinguished on the basis of nuclear structures arrangement and 3H-uridine incorporation in the oocyte nuclei revealed by ultrastructural and autoradiographic research (Parfenov et al., 1984, 1989). These groups can be regarded as consecutive states of oocyte development, i. e. active, intermediate and inactive ones. The latter is characterized by compactization of nuclear structures arranged within a limited nuclear volume. The present study concerns the distribution of splicing factors (snRNP and SC35) and p80 coilin in the nuclei of oocytes being at either of the three states. Along with transcription decreasing in oocyte nuclei, reduction of snRNP and SC35 amounts in the karyoplasm was detected. Simultaneously, accumulation of these splicing factors occurred in clusters of interchromatin granules (CIG). snRNP and SC35 are spatially segregated in CIG. snRNP are located within the fibrillar zones of CIG, while SC35 corresponds to the granular component of CIG. CIG are the only structures containing splicing factors in the nuclei of oocytes from the human antral follicules. These nuclei lack typical coiled bodies (CB). Considerable amounts of the marker protein of CB--p80 coilin are revealed in the nucleolus-like bodies (NLB) of human oocyte nuclei. Contrary to the data obtained on the oocytes from the antral follicules of other mammals (Kopecny et al., 1996a, 1996b) NLB in human oocytes do not contain snRNPs and SC35. The present study allows to make the following conclusions: a) splicing factors recruted to the sites of transcription in karyoplasm of oocytes are assembled in CIG when inactivation of transcription takes place; b) CIG in preovulated human oocytes play substantial role in the storage and preservation of splicing factors.

Adult↗

PubMed1997

[The coiled bodies and satellite microbodies of the oocyte nuclei in hibernating Rana temporaria frogs contain actin].

Immunocytochemical analysis of preparation of dispersed nuclei content in oocytes of III-IV stages of oogenesis, in terms of Dumont (1972), from hibernating grass frogs using monoclonal antibodies against actin, revealed two types of intranuclear structures containing this protein: coiled bodies (CB) and satellite microbodies (SM). Staining of these preparations with Rhodamin-phalloidin, known specifically to interact with fibrillar actin, did not reveal it in these structures. Results of our biochemical studies, using protease ESP32 specifically cutting only globular actin, are suggesting that both CB and SM contain globular actin. Gall et al. (1975) proposed that CB may be involved in assembling and sorting of small nuclear RNA for the three main RNA processing pathways: pre-mRNA splicing, pre-rRNA processing, and histone pre-mRNA 3'-end formation. Our finding of actin in CB allows a suggestion on actin involvement in the transport of RNA processing complexes from CB to some actual places where processing of RNA takes place. According to our previous data (Tsvetkov, Parfenov., 1994), SM participate in the karyosphere capsule formation. This process is preceded by SM fusion triggered presumably by actin.

Actins↗

PubMed1996

Nuclear bodies of stage 6 oocytes of Rana temporaria contain nucleolar and coiled body proteins.

The genetically inactive stage 6 oocyte nuclei of Rana temporaria contain certain nuclear bodies that label with nucleolus-specific and coiled body (CB)-specific antibodies. We designate them multicomponent bodies (MCBs) to reflect their mixed composition. Morphologically, each MCB contains five distinct zones: zone I composed of electron-dense fibrils similar to the dense fibrillar component (DFC) of the typical eukaryotic nucleoli; zone II resembled the fibrillar material of the inactive agranular nucleoli of stage 6 oocytes; zone III consisted of fine filamentous material corresponding to the fibrillar center (FC) of lower electron density seen in the typical nucleoli; and zones IV and V contained packed coiled threads typical of CBs. Of these, zone IV was seen in the interior of MCBs and contained tightly packed coiled threads (20 nm thick), while zone V occurred at the periphery and consisted of similar threads but loosely packed and electron dense. The material of both zones IV and V resembled that of CBs. To determine the composition of these zones, we extracted oocytes with a buffer that removes chromatin and most of the soluble proteins and processed them for immunogold labeling with a variety of antibodies. Anti-p80 coilin antibody predominantly labeled zone IV and, to a lesser extent, zone V. Anti-snRNP antibody also showed a similar labeling pattern. Anti-fibrillarin antibody predominantly labeled zone I and to a lesser extent zones IV and V. Anti-B23 antibody labeled all zones. These observations suggest that MCBs contain both nucleolar and CB material. We postulate that MCBs represent storage structures which provide material needed for the early stage of embryogenesis. The demonstration of MCBs further supports the close interrelationship between nucleoli and CBs.

Animals↗

PubMed1995

Nuclear actin filaments and their topological changes in frog oocytes.

Previous morphological and biochemical studies have suggested that actin and actin-containing filaments (microfilaments) exist in the eukaryotic nucleus and that they perform important nuclear functions. However, the concept is not widely accepted. In this study, we demonstrate actin and bundles of actin in the nuclei of oocytes of Rana temporaria by immunoblotting and immunogold labeling/electron microscopy. The system and methods used here provided nuclei, free from cytoplasmic contamination. Additionally, we have compared the topological distribution of intranuclear actin filaments in two structurally and functionally distinct stages (stages 3 and 6) of oogenesis. The stage 3 nuclei are extremely active in rRNA transcription and contain multiple nucleoli located at the periphery with the central part occupied by the lampbrush chromosomes. The stage 6 nuclei are transcriptionally inert and contain both nucleoli and chromosomes confined to a small area in the central part. The nuclear lysates derived from the manually isolated stage 3 and 6 nuclei and the nuclear contents obtained by manually removing the nuclear envelope of stage 6 nucleus both contained actin as demonstrated by immunoblotting with an actin-specific monoclonal antibody. When examined by immunogold electron microscopy using the anti-actin antibody, the stage 3 oocyte nuclei showed distinct intranuclear tracks composed of bundles of actin that extended from the nucleoli and chromosomes to the nuclear envelope. The stage 6 oocyte nuclei, on the other hand, showed short stretches of actin bundles in the central part mainly in association with the nucleoli; none of these bundles extended to the nuclear envelope. Taken together, the above results suggest that actin is a structural component of the oocyte nucleus and that polymerized actin undergoes dramatic topological changes correlated with changes in the distribution of nuclear components and their function.

Actins↗