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.

Glomerulosomes: morphologically distinct nuclear organelles of unknown nature.

The nucleus of some representatives of the genus Pelomyxa (Amoebozoa, Archamoebae, Pelobiontida) contains specific bodies (membrane-less organelles). They may be either embedded in the nucleolar mass or detached from the nucleolus. We termed these nuclear bodies the glomerulosomes for their characteristic ultrastructural appearance. The glomerulosomes are distinct nuclear bodies, about 1 µm in diameter. The morphological and diagnostic unit of a glomerulosome is an electron-dense thread/string, about 30-40 nm in thickness. These threads are not direct continuation of the nucleolar material. The threads create the unique geometric appearance of the glomerulosome by being organized into precisely parallel rows/cords. Each cord of the threads can curve at different angles within the glomerulosome body, but the threads themselves are not coiled. Nowadays, the glomerulosomes have been discovered in P. palustris, P. stagnalis, P. paradoxa, and Pelomyxa sp. Despite the unique appearance of glomerulosomes, their existence may be a more common phenomenon in eukaryotic cells than just a specific feature of the nucleus of elected pelomyxes.

New data on spermatogenic cyst formation and cellular composition of the testis in a marine gastropod, Littorina saxatilis.

Knowledge of the testis structure is important for gastropod taxonomy and phylogeny, particularly for the comparative analysis of sympatric Littorina species. Observing fresh tissue and squashing fixed tissue with gradually increasing pressure, we have recently described a peculiar type of cystic spermatogenesis, rare in mollusks. It has not been documented in most mollusks until now. The testis of adult males consists of numerous lobules filled with multicellular cysts containing germline cells at different stages of differentiation. Each cyst is formed by one cyst cell of somatic origin. Here, we provide evidence for the existence of two ways of cyst formation in Littorina saxatilis. One of them begins with a goniablast cyst formation; it somewhat resembles cyst formation in Drosophila testes. The second way begins with capture of a free spermatogonium by the polyploid cyst cell which is capable to move along the gonad tissues. This way of cyst formation has not been described previously. Our data expand the understanding of the diversity of spermatogenesis types in invertebrates.

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.

Karyosphere (Karyosome): A Peculiar Structure of the Oocyte Nucleus.

The karyosphere, aka the karyosome, is a meiosis-specific structure that represents a "knot" of condensed chromosomes joined together in a limited volume of the oocyte nucleus. The karyosphere is an evolutionarily conserved but morphologically rather "multifaceted" structure. It forms at the diplotene stage of meiotic prophase in many animals, from hydra and Drosophila to human. Karyosphere formation is generally linked with transcriptional silencing of the genome. It is believed that karyosphere/karyosome is a prerequisite for proper completion of meiotic divisions and further development. Here, a brief review on the karyosphere features in some invertebrates and vertebrates is provided. Special emphasis is made on terminology, since current discrepancies in this field may lead to confusions. In particular, it is proposed to distinguish the karyosphere with a capsule and the karyosome (a karyosphere devoid of a capsule). The "inverted" karyospheres are also considered, in which the chromosomes situate externally to an extrachromosomal structure (e.g., in human oocytes).

The exon junction complex factor Y14 is dynamic in the nucleus of the beetle Tribolium castaneum during late oogenesis.

BACKGROUND: The oocyte chromosomes of the red flour beetle, Tribolium castaneum, are gathered into a knot, forming a karyosphere at the diplotene stage of meiotic prophase. Chromatin rearrangement, which is a characteristic feature of oocyte maturation, is well documented. The T. castaneum karyosphere is surrounded by a complex extrachromosomal structure termed the karyosphere capsule. The capsule contains the vast majority of oocyte RNA. We have previously shown using a BrUTP assay that oocyte chromosomes in T. castaneum maintain residual transcription up to the very end of oocyte maturation. Karyosphere transcription requires evidently not only transcription factors but also mRNA processing factors, including the components of the exon junction complex with its core component, the splicing factor Y14. We employed a gene engineering approach with injection of mRNA derived from the Myc-tagged Y14 plasmid-based construct in order to monitor the newly synthesized fusion protein in the oocyte nuclei. RESULTS: Our preliminary data have been presented as a brief correspondence elsewhere. Here, we provide a full-length article including immunoelectron-microscopy localization data on Y14-Myc distribution in the nucleus of previtellogenic and vitellogenic oocytes. The injections of the fusion protein Y14-Myc mRNA into the oocytes showed a dynamic pattern of the protein distribution. At the previtellogenic stage, there are two main locations for the protein: SC35 domains (the analogues of interchromatin granule clusters or nuclear speckles) and the karyosphere capsule. At the vitellogenic stage, SC35 domains were devoid of labels, and Y14-Myc was found in the perichromatin region of the karyosphere, presumably at the places of residual transcription. We show that karyosphere formation is accompanied by the movement of a nuclear protein while the residual transcription occurs during genome inactivation. CONCLUSIONS: Our data indicate that the karyosphere capsule, being a destination site for a protein involved in mRNA splicing and export, is not only a specializes part of nuclear matrix separating the karyosphere from the products of chromosome activity, as believed previously, but represents a special nuclear compartment involved in the processes of gene expression in the case the karyosphere retains residual transcription activity.

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.

An early post-traumatic reaction of lymph-heart striated muscle fibers in adult frog Rana temporaria during the first postoperative week: An electron microscopic and autoradiographic study.

According to the current opinion, lymph-heart striated muscle represents a specialized type of skeletal muscles in frogs. Here, we studied muscle fibers in mechanically damaged lymph hearts during the first postoperative week using electron-microscopic autoradiography. We present evidence that both, the satellite cells and pre-existing muscle fibers bordering the site of injury, contribute directly to the lymph-heart muscle regeneration. Several muscle fibers located in the vicinity of the damaged area displayed features of nuclear and sarcoplasmic activation. We also observed ultrastructural changes indicating activation of a few satellite cells, namely decondensation of chromatin, enlargement of nuclei and nucleoli, appearance of free ribosomes and rough endoplasmic reticulum tubules in the cytoplasm. Electron-microscopic autoradiography showed that 4 h after single (3)H-thymidine administration on the seventh day after injury not only the activated satellite cells, but also some nuclei of myofibers bordering the injured zone are labeled. We showed that both, the myonuclei of fibers displaying the signs of degenerative/reparative processes in the sarcoplasm and the myonuclei of the fibers enriched with highly organized myofibrils, can re-enter into the S-phase. Our results indicate that the nuclei of lymph-heart myofibers can reactivate DNA synthesis during regenerative myogenesis, unlike the situation in regenerating frog skeletal muscle where myogenic cells do not synthesize DNA at the onset of myofibrillogenesis.

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.

Nuclear structures in Tribolium castaneum oocytes.

The first ultrastructural and immunomorphological characteristics of the karyosphere (karyosome) and extrachromosomal nuclear bodies in the red flour beetle, Tribolium castaneum, are presented. The karyosphere forms early in the diplotene stage of meiotic prophase by the gathering of all oocyte chromosomes in a limited nuclear volume. Using the BrUTP assay, T. castaneum oocyte chromosomes united in the karyosphere maintain their transcriptional activity until the end of oocyte growth. Hyperphosphorylated RNA polymerase II and basal transcription factors (TFIID and TFIIH) were detected in the perichromatin region of the karyosphere. The T. castaneum karyosphere has an extrachromosomal capsule that separates chromosomes from the rest of the nucleoplasm. Certain structural proteins (F-actin, lamin B) were found in the capsule. Unexpectedly, the karyosphere capsule in T. castaneum oocytes was found to be enriched in TMG-capped snRNAs, which suggests that the capsule is not only a structural support for the karyosphere, but may be involved in biogenesis of snRNPs. We also identified the counterparts of 'universal' extrachromosomal nuclear domains, Cajal bodies (CBs) and interchromatin granule clusters (IGCs). Nuclear bodies containing IGC marker protein SC35 display some features unusual for typical IGCs. SC35 domains in T. castaneum oocytes are predominantly fibrillar complex bodies that do not contain trimethyl guanosine (TMG)-capped small nuclear (sn) RNAs. Microinjections of 2'-O-methyl (U)22 probes into the oocytes allowed revealing poly(A)+ RNAs in these nuclear domains. Several proteins related to mRNA export (heterogeneous ribonucleoprotein core protein A1, export adapters Y14 and Aly and export receptor NXF1) were also detected there. We believe that unusual SC35 nuclear domains of T. castaneum oocytes are possibly involved in mRNP but not snRNP biogenesis.

Interchromatin granule clusters of the scorpionfly oocytes contain poly(A)+ RNA, heterogeneous ribonucleoproteins A/B and mRNA export factor NXF1.

IGCs (interchromatin granule clusters), or nuclear speckles, are one of the most universal subnuclear organelles of eukaryotic cells. We have used insect oocytes to study the possible association of poly(A)+ RNA and some factors involved in mRNA export with IGCs. Oogenesis of the mecopteran, Panorpa communis, used as a model object, is characterized by a strict cessation of oocyte genome transcription activity towards the end of oocyte growth. Our previous studies on P. communis oocyte nuclei have shown that oocyte IGC counterparts in this species are very unusual, both in morphology and molecular composition, compared with the typical IGCs of mammalian somatic cells traditionally used as a model system. We have now used microinjections of 2'-O-Me(U)22 probes conjugated with the fluorochrome TAMRA to localize poly(A)+ RNA in IGCs. RNA export proteins were also detected by immunofluorescent/confocal and immunogold labelling electron microscopy. We found that poly(A)+ RNA, hnRNPs A/B and NXF1 mRNA export factors are located in IGCs regardless of the transcriptional status of the nucleus. Our data support the idea of IGCs as universal and conserved nuclear domains that serve not only as splicing factor reservoirs, but also take part in mRNA retention and export.

Cajal bodies and interchromatin granule clusters in cricket oocytes: composition, dynamics and interactions.

The organization and molecular composition of complicated Cajal bodies (CBs) and interchromatin granule clusters (IGCs) in oocytes of the house cricket, Acheta domesticus, were studied using immunofluorescent/confocal and Immunogold labeling/electron microscopy. In A. domesticus oocytes, the CB consists of the fibrillar matrix and a central cavity containing a predominantly granular body with insertions of tightly packed fibrillar material. The latter structure was identified as an "internal" IGC, since it is enriched with the SC35 protein, a marker for IGCs. The IGCs located outside the CB were also identified. Microinjections of the fluorescein-tagged U7 snRNA into the ooplasm showed the targeting of the U7 to the matrix of the CB. Some other essential CB components (coilin, snRNPs, fibrillarin) were found to be colocalized in the matrix of the CB. Neither confocal nor Immunogold microscopy revealed significant amounts of RNA polymerase II (pol II) in the CB of A. domesticus oocytes. The splicing factor SC35 was detected in the matrix of the CB. In oocytes treated with DRB, the amount of IGCs in the nucleoplasm increased significantly, granular and fibrillar components of IGCs were segregated, and the fibrillar areas accumulated pol II. Additionally, IG-like granules were shown to display on the surface of the CB probably due to a shifting from the internal IGC. We believe that in A. domesticus oocytes, CBs are involved in nuclear distribution of splicing factors, but their role in pol II transport is less significant. We also suggest that the formation of complicated CBs is a result of interconnection between two different nuclear domains, CBs and IGCs.