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.     

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.     

Scattering of the silver-stained proteins of nucleolar organizer regions in Ishikawa cells by actinomycin D.

Scattering of the silver-stained proteins of nucleolar organizer regions (Ag-NOR proteins) was produced by actinomycin D in Ishikawa cells. Scattering of Ag-NOR proteins was found only in cells treated with actinomycin D and various other agents had no effect. Scattering was dose-dependent up to 10(-2) micrograms/ml of actinomycin D, but it was not found at higher concentrations that caused marked inhibition of total DNA and RNA synthesis. Actinomycin D (10(-2) micrograms/ml) caused the following changes: (i) nucleolar segregation and (ii) emergence of dense fibrillar bodies in the nucleoplasm. Ag-NOR proteins were observed on the fibrillar centers and surrounding fibrillar components in control nucleoli, on the fibrillar and amorphous zones in segregated nucleoli, and on the dense fibrillar bodies emerging in the nucleoplasm. The scattering of Ag-NOR proteins was due to the argyrophilic nature of the dense fibrillar bodies. Actinomycin D (10(-1) micrograms/ml) also caused similar morphological alterations in the nucleolus and nucleoplasm, but Ag-NOR proteins were observed only on nucleolar remnants.     

Cytochemical and ultrastructural studies of ribonucleoprotein containing structures in oocytes of Acheta domesticus

Summary Two distinct types of ribonucleoprotein containing structures are found in oocytes of the house cricket, Acheta domesticus, a large secondary or accessory nucleolus and many small primary nucleoli. The secondary nucleolus increases in size during oocyte development and is similar in appearance to the nucleolus of somatic cells. The primary nucleoli are intimately associated with a large, extrachromosomal DNA containing body. The DNA body is no longer visible in nuclei of late diplotene stage cells when the primary nucleoli are dispersed within the nucleoplasm. Both types of nucleoli contain cytochemically detectable RNA and acid protein, little or no DNA and basic protein, and particulate structures similar to but smaller than cytoplasmic ribosomes.The authors acknowledge the technical assistance of Miss Celeste Malinoski and Mrs. Marcia Andrews. This work was supported by a U.S.P.H.S. grant, number GM-16440-01 and grants number L-16 and J-1 from the Health Research Services Foundation.     

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.     

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.     

LOCALIZATION OF RIBOSOMAL DNA WITHIN OOCYTES OF THE HOUSE CRICKET, ACHETA DOMESTICUS (ORTHOPTERA: GRYLLIDAE)

A large DNA-containing body is present in addition to the chromosomes in oocytes of the house cricket Acheta domesticus. Large masses of nucleolar material accumulate at the periphery of the DNA body during the diplotene stage of meiotic prophase I. RNA-DNA hybridization analysis demonstrates that the genes which code for 18S and 28S ribosomal RNA are amplified in the ovary. In situ hybridization indicates that the amplified genes are localized within the DNA body of early prophase cells. As the cells proceed through diplotene the DNA which hybridizes with ribosomal RNA is gradually incorporated into the developing nucleolar mass.     

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.     

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.     

Specific interference of metalaxyl with endogenous RNA polymerase activity in isolated nuclei fromPhytophthora megasperma f. sp.medicaginis

The systemic fungicide metalaxyl preferentially inhibits [³H]uridine incorporation into RNA by mycelium ofPhytophthora megasperma f. sp.medicaginis. Even at high concentrations of metalaxyl inhibition is not complete but circa 80%. Neither uptake of [³H]uridine nor its conversion into UTP is inhibited, indicating that interference with RNA synthesis takes place. Synthesis of RNA that lacks poly(A) sequences is more affected than that of poly(A)⁺ RNA. Metalaxyl has no effect on the activity of RNA polymerases present in mycelial extracts fromPhytophthora nor on that of polymerases I and II that have been partially purified with a procedure involving precipitation with polyethyleneimine, selective elution of RNA polymerases from the polyethyleneimine precipitate, ammonium sulfate fractionation, and DEAE-Sephadex chromatography. RNA polymerase II in mycelial extracts is half-maximally inhibited by α-amanitin at concentrations below 0.01 ¼g/ml. Both metalaxyl and α-amanitin inhibit endogenous RNA polymerase activity of isolated nuclei ofPhytophthora. According to their sensitivity to metalaxyl and α-amanitin, three types of endogenous activity can be distinguished: (a) an α-amanitin-sensitive type, the activity of which is stimulated by ammonium sulfate; (b) an α-amanitin-insensitive but metalaxyl-sensitive type; and (c) a type insensitive to both metalaxyl andα-amanitin. The first type of activity is characteristic of RNA polymerase II; the identity of the latter two remains to be elucidated. Metalaxyl andα-amanitin do not have any effect on free nuclear polymerases when assayed at a concentration of 50 mM ammonium sulfate with poly[d(A-T)] as exogeneously added template in the presence of actinomycin D to inhibit endogenous RNA polymerase activity. At 250 mM ammonium sulfate the free polymerase activity becomes α-amanitin sensitive but remains metalaxyl insensitive. Metalaxyl apparently inhibits RNA synthesis by specific interference with template-bound andα-amanitin-insensitive RNA polymerase activity. Endogenous polymerase activity of nuclei isolated from a metalaxyl-resistant mutant ofP. megasperma f. sp.medicaginis is not inhibited by metalaxyl, indicating that interference with RNA synthesis is the primary action of metalaxyl and that modification of the target site may lead to resistance.