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.     

Localization of interchromatin granule cluster and Cajal body components in oocyte nuclear bodies of the hemipterans

An oocyte nucleus contains different extrachromosomal nuclear domains collectively called nuclear bodies (NBs). In the present work we revealed, using immunogold labeling electron microscopy, some marker components of interchromatin granule clusters (IGCs) and Cajal bodies (CBs) in morphologically heterogeneous oocyte NBs studied in three hemipteran species: Notostira elongata, Capsodes gothicus (Miridae) and Velia caprai (Veliidae). Both IGC and CB counterparts were revealed in oocyte nuclei of the studied species but morphological and biochemical criteria were found to be not sufficient to determine carefully the define type of oocyte NBs. We found that the molecular markers of the CBs (coilin and non-phosphorylated RNA polymerase II) and IGCs (SC35 protein) may be localized in the same NB. Anti-SC35 antibody may decorate not only a granular material representing "true" interchromatin granules but also masks some fibrillar parts of complex NBs. Our first observations on the hemipteran oocyte NBs confirm the high complexity and heterogeneity of insect oocyte IGCs and CBs in comparison with those in mammalian somatic cells and amphibian oocytes.     

Cajal bodies in insect oocytes. I. Identification and immunocytochemical characteristics of Cajal bodies in vitellogenic oocytes of the yellow mealworm beetle

In vitellogenic oocytes of Tenebrio molitor (inactive stage), numerous fibrogranular nuclear bodies (NBs) are present. Using immunofluorescent microscopy, these NBs were shown to contain pre-mRNA splicing factors (small nuclear [sn] RNPs and SR-protein, SC35) as well as RNA polymerase II. A limited set of NBs also contained coilin, a marker protein for Cajal bodies (CBs). We suggest that in T. molitor oocytes, coilin-containing NBs, which also contain splicing factors and RNA polymerase II, seem to represent CBs. In the species studied, no morphological features of CBs were established as compared with other NBs, which do not contain coilin. Microinjectons in oocytes of myc-tagged coilin mRNA, followed by revealing newly translated protein with antibody specific for this tag, have shown targeting of myc-coilin with CBs. The own and literary data on the morphology and molecular composition of CBs are discussed in terms of searching for criteria for CB identification in cells of different origin, and at active and inactive stages.     

Interchromatin granule clusters in vitellogenic oocytes of the flesh fly, Sarcophaga sp

Insect oocyte nuclei contain different extrachromosomal nuclear bodies including Cajal bodies and interchromatin granule clusters (IGCs). In the present study, we describe IGC equivalents in the vitellogenic oocytes of the flesh fly, Sarcophaga sp. These structures were found to consist of 20-40-nm granules and also include the fibrillar areas of high and low electron density. Immunogold labeling electron microscopy revealed IGC marker protein SC35, Sm proteins, and trimethylguanosine cap of small nuclear (sn) RNAs in these bodies. Antibody against the non-phosphorylated RNA polymerase II selectively labeled the fibrillar areas of low electron density located inside the IGCs.     

Universal nuclear domains of somatic and germ cells: some lessons from oocyte interchromatin granule cluster and Cajal body structure and molecular composition

It is now clear that two prominent nuclear domains, interchromatin granule clusters (IGCs) and Cajal bodies (CBs), contribute to the highly ordered organization of the extrachromosomal space of the cell nucleus. These functional domains represent structurally stable but highly dynamic nuclear organelles enriched in factors that are required for different nuclear activities, especially RNA biogenesis. IGCs are considered to be the main sites for storage, assembly, and/or recycling of the essential spliceosome components. CBs are involved in the biogenesis of several classes of small RNPs as well as the modification of newly assembled small nuclear RNA. We have summarized data on the molecular composition, structure, and functional roles of IGCs and CBs in the nuclei of mammalian somatic cells and oocytes of some animals with a special focus on insects. We have focused on similarities and differences between the IGCs and CBs of oocytes and the well‐studied CBs and IGCs of cultured mammalian somatic cells. We have shown the heterogeneous character of oocyte IGCs and CBs, both in structure and molecular content. We have also demonstrated the unique capacity of oocytes to form close structural interactions between IGC and CB components. We proposed to consider these joint structures as integrated entities, sharing the features of both IGCs and CBs.     

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.     

Re-localization of nuclear DNA helicase II during the growth period of bovine oocytes

Nuclear DNA helicase II (NDH II) is the bovine homolog of human RNA helicase A. The aim of this study was to compare NDH II localization between somatic cells (bovine embryonal fibroblasts) and female germ cells (oocytes), with the main focus on the dynamic changes in the redistribution of NDH II during the growth phase of the bovine oocytes. The fine granular staining of NDH II was spread in the whole nucleoplasm of fibroblasts, excluding the reticulated nucleoli. In contrast, the large reticulated nucleoli of the growing oocytes isolated from early antral follicles exhibited strong positivity for NDH II together with the immunostaining signals of upstream binding factor (UBF) and RNA polymerase I subunit (PAF53), documenting the high synthetic activity of these nucleoli. At the time of termination of oocyte growth, NDH II was preferentially located at the nucleolar periphery together with proteins of fibrillar centres. In fully grown oocytes, NDH II was still present in the thin periphery shell around the compact nucleolar core. The semiquantitative RT-PCR revealed that the average signal of NDH II mRNA in fully grown oocytes was only at 40% level in comparison with growing oocytes. Western blot analysis further confirmed that a 140 kD NDH II protein was abundant in growing oocytes, while the signal was substantially weaker in fully grown oocytes. The significant decrease in NDH II gene expression and in NDH II mRNA translation correlates with a termination of the oocyte growth. Altogether, the results demonstrate that NDH II expression parallels the activity of ribosomal RNA biosynthesis in the bovine growing oocytes.     

Cajal bodies (coiled bodies) in the nuclei of the house cricket (Acheta domesticus) oocytes

The morphology and fine structure of Cajal bodies (coiled bodies, CB) in the germinal vesicles (oocyte nuclei) of the house cricket, Acheta domesticus have been analyzed. It is shown that in the studied species CBs arise as early as in the youngest previtellogenic oocytes, and are located next to or within aggregations of multiple nucleoli. Surprisingly, two morphological types of CBs have been found in the analyzed specimens. On the basis of EM studies we suggest that they represent subsequent developmental stages of CB morphogenesis.     

Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes in the yellow mealworm Tenebrio molitor

The nucleus of vitellogenic oocytes of the yellow mealworm Tenebrio molitor contains a karyosphere that consists of 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 2??-O-Me(U)₂₂ methyl oligoribonucleotide probes, 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 A1 localized to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and some nucleoplasmic IGCs also contain the adapter protein Aly known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was colocalized with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner, whereas it was RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data provide evidence for the implication of nucleoplasmic IGCs in mRNA biogenesis and retention on the path to nuclear export.