THE KARYOSPHERE CAPSULE IN OOCYTES OF HIBERNATING FROGS RANA TEMPORARIA CONTAINS ACTIN, LAMINS AND SMALL NUCLEAR RNP.

The nuclei of late vitellogenic oocytes of hibernating frogs Rana temporaria were studied. During the studied period of oogenesis, chromosomes are significantly inactivated and surrounded by a fibrillar karyosphere capsule. Formation of the karyosphere capsule in the grass frog oocytes was previously investigated in detail at the light and electron microscopic levels, but the molecular composition of the capsule still remains uncertain. Immunofluorescent staining of whole-mount preparations of oocyte nuclei revealed the following proteins in the karyosphere capsule: actin, lamins A, C and B, and the Sm proteins of snRNPs. A putative role of these proteins in formation of the karyosphere capsule is discussed.

[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.

[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.

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.

[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.

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.

[Splicing factors in oocyte nuclei from human antral follicles]. / Faktory splaisinga v iadrakh ootsitov iz antral'nykh follikulov cheloveka.

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.

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.

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.

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.

[The formation of the karyosphere in the oogenesis of insects and amphibians]. / Formirovanie kariosfery v oogeneze nekotorykh nasekomykh i amfibii.

This review deals with the authors' own and literary data on the ultrastructural and cytochemical organization of insect and amphibian oocyte nuclei, with special attention being paid to the karyosphere and its capsule. The evidence provided is supplemented with data on isolated karyospheres in Rana temporaria oocytes. A conclusion is made that the karyosphere is a complex structure which contains all chromosomes in the limited space of the oocyte nucleus, and that these chromosomes are, as a rule, in the process of inactivation. It is inferred that the karyosphere capsule commonly appears in gigantic oocyte nuclei (more than 100 micron in diameter) containing extrachromosomal DNA. The analogy of capsule organization in different invertebrate and vertebrate species is discussed, in addition to the involvement of presumably homologous nuclear structures (e.g. derivatives of synaptonemal complexes and nuclear envelope) in capsule formation. It is assumed that the karyosphere capsule is a specially organized part of the nuclear matrix. The capsule provides nuclear compartmentalization and chromosome localization in the germinal vesicle. Studies of this sort open up new possibilities to further investigation of intranuclear morphogenesis.

[The organization of the karyosphere and capsule before oocyte maturation in the common frog]. / Organizatsiia kariosferi s kapsuloi pered sozrevaniem ootsitov travianoi liagushki.

The oocytes of the 6th stages, according to Dumont (1972), taken from R. temporaria in nature in April were studied. The semithin sections of Araldit-embedded material, as well as preparations of karyospheres with capsules isolated from nuclei using Callan's method (Callan et al., 1987), were analysed. Ultrastructural investigations were carried out both on intact nuclei and on nuclei extracted in 2M NaCl. The April oocytes differ from the winter ones previously analysed by Gruzova and Parfenov (1977), in the distribution of capsule components: the fibrous material surrounds, as previously, the central part of karyosphere, whereas the pseudomembranes penetrate into the chromosome tangle. The fibrous material reveals stability (resistance) to 2M NaCl treatment. Another feature of the April oocytes is the development of small intracellular bodies (0.3-2 microns). These bodies are composed of coiled threads 40-50 nm in diameter and connected with the nucleoli. The morphogenesis of the capsule fibrous component is discussed in connection with the double nature of the capsule material. Participation of actin filaments in the formation of the fibrillar part of the capsule is discussed. A similarity of the bodies composed of coiled threads in oocyte nuclei is drawn with the "coiled bodies" of somatic cell (Lamond, Carmo-Fonseca, 1993).

[Characteristics of karyosphere formation in the lake frog. Light microscopy data]. / Osobennosti formirovaniia kariosfery ozernoi liagushki. Svetomikroskopicheskie dannye.

Morphological peculiarities of the oocyte nuclear organization were examined in R. ridibunda during winter and spring (February-March). Numerous nucleoli were seen to be assembled around regressive lampbrush chromosomes in the centre of the nucleus, and a central body was formed to which the chromosomes were attached. As result, a structural complex is constituted that involves a karyosphere and a capsule. Nucleoli are characterized by segregation and intensive fragmentation of their material. In result, a considerable part of nucleolar DNA is eliminated in the form of ring and polymorphous structures (micronucleoli). Besides the membranous component of nucleoli (nucleolar threads or tails) is lost. Towards the end of this period, nucleoli with complicated morphology become spherical again. The formation of the central body is started from the appearance of some small optically-light protein structures 5-20 nm in diameter (central body precursors-CBP). CBP are closely surrounded with ring micronucleoli to make intimate contact with the chromosomes and nucleolar threads. CBP commonly lie in one region of the nucleus not far from each other. The formation of a definitive central body obviously occurs due to a fusion of some small CBP. A conclusion is made of the nucleolar origin of the ring and polymorphous structures and of their essential role in the central body formation. The participation of chromosomal and eliminated nucleolar DNA in this process is discussed.