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.     

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.     

Amplification of ribosomal genes and formation of extrachromosomal nucleoli in oocytes of starfish Henricia hayashi (Asteroidea: Echinasteridae)

DNA and RNA specific dyes, Ag-NOR staining and in situ hybridization were used for studying the nucleolar apparatus in the growing oocytes of Henricia hayashi (Asteroidea: Echinasteridae). A plasmid containing ribosomal genes of Drosophila melanogaster (Kolchinsky et al., 1980) labelled with 3H by nick-translation served as an rDNA probe. Multiple extrachromosomal nucleoli are formed by the cascade type as a result of growth and subsequent fragmentation of the chromosomal (primary) rDNA body and its derivative extrachromosomal (secondary) rDNA bodies. Ribosomal genes were shown in all nucleolar structures. Argentophilia of the primary and secondary DNA bodies appears to be due to the dense packing of the rDNA-containing material. Ag(+) NORs were detected in the extrachromosomal multiple nucleoli and NOR complexes. Amplification of rDNA is a highly probable conclusion from the existing data.     

Structure of the germinal vesicle during oogenesis in leech Glossiphonia heteroclita (Annelida, Hirudinea, Rhynchobdellida)

Oogenesis in the glossiphoniid leech Glossiphonia heteroclita (Hirudinea, Rhynchobdellida) is nutrimental, i.e., the growing oocyte is supported by specialized germline cells, the nurse cells. The main function of the nurse cells is to provide oocytes with cell organelles and RNAs (mainly rRNA). However, in studied leech species, irrespective of the nutrimental mode of oogenesis, the germinal vesicle (GV = oocyte nucleus) seems to be very active in rRNA production. As shown in the present study, during early previtellogenesis in the GV the meiotic chromosomes and prominent primary nucleoli occur. In late previtellogenesis the chromosomes condense and occupy a limited space of nucleoplasm in close vicinity to primary nucleolus, forming a karyosome. At the onset of vitellogenesis several prominent extrachromosomal DNA bodies appear in close association with the karyosome. At the same time, the primary nucleolus is no longer visible in the GV. As vitellogenesis proceeds the extrachromosomal DNA bodies undergo fragmentation and numerous spherical, RNA- and AgNOR-positive inclusions occur in the nucleoplasm. They are regarded as multiple nucleoli. Finally, in late oogenesis numerous accessory nuclei are formed in close proximity to the nuclear envelope. They usually contain one dense body, morphologically similar to multiple nucleoli. The amplification of rDNA genes, the occurrence of extrachromosomal DNA bodies, as well as the presence of multiple nucleoli and accessory nuclei are described for the first time in the phylum Annelida.     

Intranuclear actin microfilaments in the oocytes of the common frog

For identification and distribution of actin microfilaments in hand-isolated nuclei of R. temporaria oocytes (stage 6, according to Dumont, 1972) different methods were used: heavy meromyosin decoration, antiactin immunofluorescence with monoclonal antibodies, staining with rhodamine phalloidin, and electrophoresis in polyacrylamide gel. The nuclei of R. temporaria oocytes contain a considerable quantities of actin microfilaments which form intranuclear meshwork. Microfilaments are connected with the nucleoli, nucleolar RNP-complexes and nuclear envelope. Immunofluorescence with antiactin monoclonal antibodies reveals a strong staining of microfilaments and nucleoli. A slight staining of nucleoli is observed after the treatment of nuclei with rhodamine phalloidin. A specific role of intranuclear microfilaments in direct transport of nucleolar material from the nucleus into the oocyte cytoplasm, in stabilization of the karyosphere (the late diplotene oocyte complex of chromosomes with numerous nucleoli) is discussed in addition to its keeping in a definite region of the nucleus. A supposition is drawn on the functional significance of the connection between microfilaments and nuclear matrix. Based on our own and literature data, a conclusion is drawn, that the intranuclear filament actin may be one of the leading components in morpho-functional organization of the nucleus as the whole.     

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.     

Amphibian oocytes and sphere organelles: are the U snRNA genes amplified?

The sphere organelles (spheres) ofXenopus and other amphibian oocytes are known to contain small nuclear ribonucleoprotein particles (snRNPs) and have been suggested to play a role in snRNP complex assembly. Coupled with the similarities that exist between spheres and nucleoli and the quantitative and kinetic aspects of snRNA synthesis in theXenopus oocyte, we have investigated whether or not the U snRNA encoding genes are amplified inXenopus oogenesis, the spheres being possible sites for the location of such extrachromosomal gene copies. By applying a number of quantitative nucleic acid hybridization procedures to both total and fractionated oocyte and somatic DNA, employing both homologous and heterologous U snRNA gene probes and suitable amplification and non-amplification control probes, we show that the U snRNA genes do not undergo any major amplification inXenopus oogenesis. Therefore, the analogy between the sphere organelles and nucleoli appears to be limited. The role of the spheres and their relationship to other snRNP containing structures, specifically B snurposomes, and the sphere organizer loci remains obscure.by A. Spradling     

Absence of rDNA amplification in the uninucleolate oocyte of the cockroach Blattella germanica (Oorthoptera: Blattidae)

Amplification of the genes coding for ribosomal RNA oocurs in the oocytes of a wide variety of organisms. In oocytes of various species of crickets (Orthoptera: Gryllidae) the amplified DNA is contained in a large extrachromosomal DNA body. Multiple nucleoli form about the periphery of the DNA body during the diplotene stage of meiosis I. In contrast to the general pattern of orthopteran oocytes, oocytes of the cockroach Blattella germanica demonstrate a single large nucleolus instead of many nucleoli. In order to determine whether the genes coding for rRNA are amplified in the oocytes of B. germanica, the relative amount of rDNA in oocytes was compared with the rDNA content of spermatocytes and somatic cells. An extrachromosomal DNA body similar to that present in crickets is not present in B. germanica. A satellite DNA band which contains nucleotide sequences complementary to rRNA accounts for approximately 3-5% of the total DNA in somatic and in male and female gametogenic tissues. Female cells contain approximately twice as much rDNA as do male cells. An XX-XO sex-determining mechanism is operative in B. germanica. In situ hybridization with rRNA indicates that the nucleolar organizer is located on one end of the X chromosome and that oocytes do not contain more than twice the amount of rDNA found in spermato cytes. The data indicate that rDNA is not amplified in the uninucleolate oocyte of B germanica.     

Mutational analysis of p80 coilin indicates a functional interaction between coiled bodies and the nucleolus

Coiled bodies are conserved subnuclear domains found in both plant and animal cells. They contain a subset of splicing snRNPs and several nucleolar antigens, including Nopp140 and fibrillarin. In addition, autoimmune patient sera have identified a coiled body specific protein, called p80 coilin. In this study we show that p80 coilin is ubiquitously expressed in human tissues. The full-length human p80 coilin protein correctly localizes in coiled bodies when exogenously expressed in HeLa cells using a transient transfection assay. Mutational analysis identifies separate domains in the p80 coilin protein that differentially affect its subnuclear localization. The data show that p80 coilin has a nuclear localization signal, but this is not sufficient to target the protein to coiled bodies. The results indicate that localization in coiled bodies is not determined by a simple motif analogous to the NLS motifs involved in nuclear import. A specific carboxy-terminal deletion in p80 coilin results in the formation of pseudo-coiled bodies that are unable to recruit splicing snRNPs. This causes a loss of endogenous coiled bodies. A separate class of mutant coilin proteins are shown to localize in fibrillar structures that surround nucleoli. These mutants also lead to loss of endogenous coiled bodies, produce a dramatic disruption of nucleolar architecture and cause a specific segregation of nucleolar antigens. The structural change in nucleoli is accompanied by the loss of RNA polymerase I activity. These data indicate that p80 coilin plays an important role in subnuclear organization and suggest that there may be a functional interaction between coiled bodies and nucleoli.     

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.     

Coilin Shuttles between the Nucleus and Cytoplasm In Xenopus Oocytes

Coiled bodies are discrete nuclear organelles often identified by the marker protein p80-coilin. Because coilin is not detected in the cytoplasm by immunofluorescence and Western blotting, it has been considered an exclusively nuclear protein. In the Xenopus germinal vesicle (GV), most coilin actually resides in the nucleoplasm, although it is highly concentrated in 50-100 coiled bodies. When affinity-purified anti-coilin antibodies were injected into the cytoplasm of oocytes, they could be detected in coiled bodies within 2-3 h. Coiled bodies were intensely labeled after 18 h, whereas other nuclear organelles remained negative. Because the nuclear envelope does not allow passive diffusion of immunoglobulins, this observation suggests that anti-coilin antibodies are imported into the nucleus as an antigen-antibody complex with coilin. Newly synthesized coilin is not required, because cycloheximide had no effect on nuclear import and subsequent targeting of the antibodies. Additional experiments with myc-tagged coilin and myc-tagged pyruvate kinase confirmed that coilin is a shuttling protein. The shuttling of Nopp140, NO38/B23, and nucleolin was easily demonstrated by the targeting of their respective antibodies to the nucleoli, whereas anti-SC35 did not enter the germinal vesicle. We suggest that coilin, perhaps in association with Nopp140, may function as part of a transport system between the cytoplasm and the coiled bodies.     

Regulated specific proteolysis of the Cajal body marker protein coilin

Cajal bodies (CB) are subnuclear domains that contain various proteins with diverse functions including the CB marker protein coilin. In this study, we investigate the proteolytic activity of calpain on coilin. Here, we report a 28-kDa cleaved coilin fragment detected by two coilin antibodies that is cell cycle regulated, with levels that are consistently reduced during mitosis. We further show that an in vitro calpain assay with full-length or C-terminal coilin recombinant protein releases the same size cleaved fragment. Furthermore, addition of exogenous RNA to purified coilin induces proteolysis by calpain. We also report that the relative levels of this cleaved coilin fragment are susceptible to changes induced by various cell stressors, and that coilin localization is affected by inhibition or knockdown of calpain both under normal and stressed conditions. Collectively, our data suggest that coilin is subjected to regulated specific proteolysis by calpain, and this processing may play a role in the regulation of coilin activity and CB formation.     

Nuclear Bodies of Stage 6 Oocytes ofRana temporariaContain Nucleolar and Coiled Body Proteins

The genetically inactive stage 6 oocyte nuclei ofRana temporariacontain 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.     

Nucleoli and perinuclear bodies in trophocytes of Panorpa communis contain factors of pre-mRNA splicing

The distribution of pre-mRNA splicing factors and protein coilin was examined in trophocyte nuclei (TN) in polytrophic ovarioles of Panorpa communis. In situ hybridization, using antisense U1 and U6 snRNA 3H-riboprobes, showed that TN were labeled evenly. Immunostaining at light and electron microscopic levels revealed in some TN nucleolar structures containing small nuclear RNP (snRNP) and protein coilin characteristic of the Cajal bodies/coiled bodies (CB). No free CBs were found in TN. These data showed that CB in TN are present only in the nucleoli. One of characteristic features of P. communis trophocytes is the presence of several types of perinuclear bodies (PB) in the cytoplasm. We distinguish between three types of PBs. PB-1 consist of spherical bodies (10-20 microns) with vacuoles composed of closely packed fibrils. PB-2 are irregularly shaped bodies (0.3-2.0 microns) consisting of a fibro-granular material. PB-2 are located near the nuclear envelope and contact the nucleoplasm material through nuclear pores. PB-1 and PB-2 join together to form a complex PB of the third type. All types of PB are not surrounded with a membrane and sometimes have mitochondria on their surface. The immunogold technique at the ultrastructural level revealed snRNP in PB-2. These results have enabled us to make a conclusion that PB-2 may be storage sites of snRNPs required for a future development of the embryo.     

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.