Identification and dynamics of Cajal bodies in relation to karyosphere formation in scorpionfly oocytes
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- Batalova, F.M., Stepanova, I.S., Skovorodkin, I.N. et al. Chromosoma (2005) 113: 428. doi:10.1007/s00412-004-0328-y
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In oocyte nuclei of the scorpionfly, Panorpa communis, we have recently defined a population of nuclear bodies (NBs) that contain some components of Cajal bodies (CBs). In the present study, we used several criteria [presence of coilin, U7 snRNA, RNA polymerase II (pol II) and specific ultrastructure] to identify these NBs as CBs. The essential evidence for CB identification came from experiments with microinjection of fluorescein-tagged U7 snRNA. Consistent with the U7 data, we found pol II and pre-mRNA splicing factor, SC35, in Panorpa oocyte CBs. We show here that the dynamics of CBs differs from that in somatic cells and correlates with the level of oocyte chromosome condensation. We also found that the significant increase of CB size is accompanied by condensation of the chromosomes in the karyosphere, which is indicative of a decline in transcription. Using immunogold microscopy we determined that pol II and coilin are shared by CBs and the granular material associated with condensed chromosomes in the Panorpa karyosphere. The colocalization of pol II, U7 snRNA and splicing factors with CBs at the inactive stage of late oogenesis suggests that the latter may serve as storage domains for components that were earlier engaged in RNA transcription and processing.
It is now well established that the extrachromosomal part of the nucleus of different cells is highly ordered and contains nuclear bodies (NBs), the term used to designate extrachromosomal nuclear organelles regardless of their content and possible functions (Brasch and Ochs 1992). Among NBs, Cajal bodies (CBs) are the best-studied nuclear organelles (for a review see Matera 1999; Gall 2000). Despite the wide spectrum of investigations on the structure and molecular composition of CBs, their identification is not a simple problem, particularly in the oocyte. In our opinion, to qualify as a CB, an NB should contain at least some marker components, such as coilin and the U7 small nuclear ribonucleoprotein (snRNP). (For additional arguments for considering U7 as an adequate marker of CBs, see below.)
It should be mentioned that until recently the functions of CBs were more conjectural than established. Only recently have intensive studies on molecular characterization of CBs been performed to elucidate possible functions of these structures (Matera 1999; Dundr and Misteli 2001). Two more or less alternative suggestions seem to be the most attractive: (1) CBs might be involved in transport and maturation of snRNPs (Bauer and Gall 1997; Carvalho et al. 1999; Sleeman and Lamond 1999; Darzacq et al. 2002; Jády et al. 2003; Gall et al. 2004); (2) CBs might be the initial sites for assembly of the RNA transcription and processing machinery (Gall et al. 1999; Morgan et al. 2000; Doyle et al. 2002). As conserved structures, CBs are found in nuclei of various cell types in evolutionarily diverse organisms, from plants to invertebrates to mammals.
Our previous studies revealed some features of CB dynamics in insect and mammalian oocytes. In oocytes, CBs are the most prominent and numerous structures in partially or fully inactivated nuclei when chromosome condensation and karyosphere formation occur (Batalova et al. 2000; Bogolyubov et al. 2000; Parfenov et al. 2003). In contrast, in somatic cells, CBs are usually formed when RNA synthesis actively occurs (Ferreira et al. 1994; Rebelo et al. 1996; Ogg and Lamond 2002). On the other hand, it has been shown that quiescent endothelial cells exhibit larger amount of CBs per nucleus (Alliegro and Alliegro 1998), so CBs are not formed only when RNA synthesis actively occurs. The reasons and significance of such differences in behavior of CBs remain unclear.
In the present study we used Panorpa communis oocytes as an experimental model, because the meroistic polytrophic ovarioles have some undoubted advantages. (1) Each P. communis ovariole contains a set of oocytes in different stages of development with different levels of chromosome condensation, reflecting levels of transcriptional activity ranging from active to inert. (2) A single oocyte nucleus of P. communis contains numerous well resolvable NBs 2–30 μm in diameter. (3) The absence of nucleoli and rRNA synthesis in P. communis oocytes, as demonstrated in our in situ hybridization experiments (Batalova et al. 2000), facilitates the analysis of other extrachromosomal structures. (4) The microanatomy of P. communis female gonads and the general morphology of oocyte nuclei are well established for this species (Gruzova 1962; Ramamurty 1963; Simiczyjew 1996).
In the present study we examined the molecular composition and dynamics of P. communis oocyte CBs in correlation with chromosome condensation and karyosphere formation. We report here additional evidence for CB identification obtained from experiments with microinjection of fluorescently tagged U7 snRNA in P. communis oocytes. It is well known that CBs are highly enriched in U7 snRNA. For instance, in the amphibian germinal vesicle, about 85% of the U7 snRNA was found in CBs, whereas the rest was in the karyoplasm (Wu et al. 1996). U7 snRNA was also readily demonstrable by in situ hybridization in CBs of cultured mammalian cells (Frey and Matera 1995). Therefore, U7 snRNA may be considered as a specific marker for CB identification.
Finally, we examined the topological relationship between CBs and the granular material associated with condensed chromosomes in the P. communis karyosphere. Using immunoelectron microscopy we identified proteins that are shared by CBs and this granular material.
Materials and methods
The specimens of the scorpionfly, P. communis L. (Mecoptera: Panorpidae), were collected in June and July in the village of Toksovo (Leningrad Region, Russia) and Stary Peterhof (St. Petersburg, Russia). Ovaries were isolated in OR2 medium (Wallace et al. 1973) or in Ringer’s solution for insects (7.5 g/l NaCl, 0.35 g/l KCl, 0.21 g/l CaCl2).
For routine electron microscopy, single ovarioles were fixed in 2.5% glutaraldehyde (Polyscience) in 0.05 M cacodylate buffer, pH 7.4, for 1.5 h, then postfixed in 1% OsO4 in the same buffer for 1 h. After dehydration in an ascending series of ethanol and acetone, the specimens were embedded in Epon 812 (Fluka). Semithin sections of 0.5–1 μm in thickness were stained with 1% methylene blue in 1% borax. Ultrathin sections were cut with a Reichert Jung ultracut microtome, mounted on nickel grids, contrasted by uranyl acetate and lead citrate, and examined in a JEM-100C or JEM-7A electron microscope at 80 kV.
Antibodies used for immunocytochemistry. (mAb, monoclonal antibody)
SR protein, non-snRNA splicing factor of pre-mRNA
Fu and Maniatis (1990)
Unphosphorylated C-terminal domain of RNA polymerase II
Thompson et al. (1989)
mAb anti-DNA (030)
Chemicon International, USA
Rabbit polyclonal serum R288
Carboxy-terminal fragment (14 kDa) of human p80-coilin
Andrade et al. (1991)
For indirect immunofluorescent cytochemistry, squashed preparations of ovarioles were prepared according to Hulsebos et al. (1984). Briefly, single ovarioles were placed in 5 μl OR2 medium on a siliconized microscope coverslip, slightly squashed on a gelatin-coated slide, and briefly frozen in liquid nitrogen. After the coverslip was removed with a razor blade, the slide was fixed in 4% paraformaldehyde in 96% ethanol for 30 min, rinsed in 70% ethanol, and stored in 1×PBS before immunostaining. The antibody treatment was performed according to Wu et al. (1991). Briefly, preparations were incubated in 10% fetal serum (Gibco) in 1×PBS for 10 min to prevent nonspecific antibody binding. The incubation in first antibody solution (Table 1) was carried out overnight in a moist chamber at 4°C. After rinsing in 1×PBS, the preparations were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (diluted 1:200) for 1.5 h at room temperature. After rinsing in 1×PBS, the preparations were mounted in 50% glycerol containing 1 mg/ml p-phenylendiamine (Sigma) and 0.5 μg/ml 4′,6-diamidimo-2-phenylindole and analyzed in an Axioskop (Karl Zeiss) fluorescent microscope.
To study nuclear targeting of U7 snRNA in oocytes, we injected fluorescein-labeled U7 into the ooplasm. The RNA consisted of capped 60 nucleotide transcripts of the wild-type Xenopus U7 snRNA gene transcribed in vitro with fluorescein-UTP (Wu et al. 1996). A 96 nucleotide fluorescein-tagged pBluescript transcript that does not correspond to any known cellular RNA was used as a control. Constructs were kindly provided by J.G. Gall (Carnegie Institution, Baltimore, USA). Before microinjection, dried-down samples were reconstituted by adding sterile distilled water. The injections were carried out by using glass capillaries and an Eppendorf 5242 microinjector. Approximately 5 nl of RNA solution was injected into each oocyte. After injection, the ovarioles were kept in Ringer’s solution for approximately 12 h in a moist chamber at 4°C. Then squashed preparations of the ovarioles were prepared as described. The preparations were stained for 1 min with TO-PRO-3; 1:1000 (Molecular Probes) to reveal DNA, mounted in Vectashield medium (Vector Laboratories) and imaged with a Leica TCS SL confocal microscope.
The paired female gonads of the scorpionfly, P. communis, consist of meroistic polytrophic ovarioles. The general morphology of P. communis ovarioles is similar to that in other mecopteran groups with meroistic ovarioles (Simiczyjew 2003). Each ovariole includes the germarium with young oocytes at the beginning stages of first meiotic prophase (including pachytene) and the vitellarium, where the growing oocytes, in a protracted diplotene stage, occur within 10–12 follicles.
Before karyosphere formation, at the pachytene stage, chromatin is dispersed through the nucleus. No NBs were observed at this stage.
It should be noted that in P. communis, NBs vary significantly in their structure and size. Generally, NBs consist of fibrillar and granular material. The granular material consists predominantly of large granules about 30–50 nm in diameter (Fig. 2c).The fibrillar material displays different levels of packing within NBs, e.g., tightly packed material in spherical portions of NBs and more loosely packed material in the extensions from these spheres (Fig. 2b, c). Portions of the fibrillar material are often observed in tight connection with patches of chromatin (Fig. 2b).
Immunogold labeling of previtellogenic oocytes with anti-SC35 antibody shows labeling of both the small fibrillar NBs and granular material located in close proximity to chromatin (Fig. 2d).
At this stage, labeling with anti-coilin antibody shows that NBs of different morphological types, including larger NBs (Fig. 2e) and chromosome-associated granular material (Fig. 2f), contain coilin, the marker protein for CBs.
In some recent studies the occurrence of coilin in a nuclear structure has been suggested as an essential and sufficient criterion to define this NB as a CB (Grande et al. 1997; Schul et al. 1998; Morgan et al. 2000; Deryusheva and Gall 2004). Based on this criterion we designate all coilin-containing NBs in P. communis oocytes as CBs regardless of their different morphology and size (see below).
In late previtellogenic oocytes, the karyosphere is a more compact structure consisting of condensed chromatin blocks and granular material. Also, it may include ring-shaped structures of unknown nature (Fig. 3e). The number of NBs reaches a maximum at this stage (Fig. 9c). In addition, a new population of NBs (1 or 2–3 per nucleus) appears at this stage. The latter NBs are very large (up to 30 μm in diameter), quite spherical in shape (Fig. 3c), and are often vacuolated. At the ultrastructural level, these NBs consist of fibrils of low electron density, which are organized in characteristic “coiled threads”. These NBs retain the same characteristic ultrastructure into the later stages of vitellogenesis (Fig. 7f, g). It should be noted that such threads may be seen clearly only in Epon embedded material after routine fixation with glutaraldehyde and osmium tetroxide. In LR White embedded material after paraformaldehyde fixation for immunoelectron microscopy the fine structure of these NBs is poorly preserved.
The largest CBs as well as a number of smaller ones, give a positive reaction with anti-SC35 and with anti-RNA polymerase II (anti-pol II) antibodies (Fig. 4b). Immunogold labeling of these bodies revealed that coilin, SC35 protein and pol II are present in the granular material (Fig. 6a, c, d). As at the previous stage, the granular material exists as a part of complex CBs or it may occupy extended areas in the karyosphere.
Anti-SC35 antibody used for indirect immunofluorescent microscopy revealed discrete fluorescence of the karyosphere at this stage (Fig. 7c). This pattern of fluorescence suggests that anti-SC35 staining may be related to the patches of granular material in the karyosphere. Immunogold labeling with anti-SC35 antibody confirmed this assumption. The results are shown in Fig. 7d. The areas of granular material, consisting of 30–50 nm granules, are labeled with a density comparable to anti-SC35 labeling of the same material at the previous stage (Fig. 6c).
At mid vitellogenesis, the fully developed karyosphere contains very condensed chromatin blocks surrounded by regions of granular material (diameter of granules about 30–50 nm) as in the previous stages. It is often observed that the karyosphere displays an intimate connection with the large CB (Fig. 7e–g). At this stage, our labeling study with anti-coilin polyclonal antibody revealed that the large CB still contains a noticeable amount of coilin (Fig. 7h).
In control preparations, when fluorescein-tagged transcripts that do not correspond to any known RNA were injected, no strong signal was registered. In this case, oocyte nuclei demonstrated only a weak diffuse fluorescence (Fig. 8b). No fluorescence was observed in non-injected controls (not shown).
In diplotene P. communis oocytes, we found that morphologically heterogeneous NBs share common features of CBs. These NBs are (1) small bodies containing large granules ~ 30–50 nm in diameter, (2) small fibrillar bodies, (3) complex bodies containing both granular and fibrillar material, and (4) large fibrillar bodies displaying characteristic “coiled threads” similar to the core elements of CBs in mammalian somatic cells (Monneron and Bernhard 1969). The essential feature of these CBs is that they include the marker protein coilin (Andrade et al. 1991; Raška et al. 1991). This conclusion comes from both immunofluorescent and immunoelectron studies.
In the present study we found that anti-coilin serum R288 recognizes a protein of ~43 kDa in immunoblots (Fig. 1). This finding is rather unexpected. Both mammalian p80-coilin (Andrade et al. 1993) and Xenopus coilin, originally called SPH-1 (Tuma et al. 1993), are 80 kDa proteins. At the same time, in our previous work (Bogolyubov and Parfenov 2001) we demonstrated that the molecular weight of coilin is ~70 kDa in the yellow mealworm, Tenebrio molitor. We suggest that the molecular weights of insect coilin proteins may differ from species to species but they apparently should contain a conserved C-terminal domain that reacts with the serum (Bellini 2000).
U7 snRNA, which is specifically involved in the maturation of histone pre-mRNAs (Dominski and Marzluff 1999), is another component of CBs. Fluorescently labeled U7 snRNA was specifically accumulated in the NBs after injection into the ooplasm, further suggesting that the NBs should be considered CBs. RNA pol II, also identified as a CB constituent (Matera 1999; Gall 2000; Gall et al. 1999), is present in oocyte CBs in P. communis (present study) and in the mealworm, T. molitor (Bogolyubov and Parfenov 2004). Collectively, the presence of coilin and pol II, targeting of U7 snRNA, and, in some cases, a characteristic ultrustructure allow the identification of P. communis oocyte CBs.
It may well be that CBs are universal nuclear organelles in insect oocytes. They have been described in oocytes of different insects including the house cricket and mole cricket (Jörgensen 1913; Gall et al. 1995; Tsvetkov et al. 1997; Filek et al. 2002), a dragonfly (Tsvetkov et al. 1996), some beetles (Bogolyubov and Parfenov 2001; Jaglarz 2001), and the common wasp (Jabłońska and Biliński 2001; Biliński and Kloc 2002). However, no NBs containing snRNPs, SC35 protein and/or coilin were identified by immunoelectron microscopy in oocytes of the apple blossom weevil (Świątek and Jaglarz 2004).
In P. communis oocytes, the largest CBs resemble the so-called Binnenkörper, or endobodies, characteristic for oocyte nuclei of many insect species (Bier et al. 1967). Based on immunocytochemical data, the latter nuclear organelles were identified as CBs at least for the house cricket, Acheta domesticus (Gall et al. 1995; Tsvetkov et al. 1997) and the violet ground beetle, Carabus violaceus (Jaglarz 2001). Interestingly, the characteristic fine structure of Binnenkörper like that found in P. communis oocytes has not been described previously for any other insect.
In our study on CB dynamics at the subsequent stages of P. communis oocyte growth, we found that the biogenesis of CBs (appearance— enlarging or increasing in number—disappearance) correlates with the level of chromosome condensation and stage of karyosphere development. At first, sparse CBs can be observed at early diplotene, when chromosome condensation begins. Later, when all of the condensed chromosomes unite into a compact knot (karyosphere), CBs increase in number. The changes in P. communis oocyte CBs during karyosphere formation correspond to those described previously for NBs in some other insect oocytes (Gruzova 1988; Gruzova and Parfenov 1993; Bogolyubov et al. 2000; Bogolyubov and Parfenov 2001).
In the oldest P. communis oocytes, at the stage before the first meiotic division, the smaller CBs are reduced dramatically in number, while the large ones reach their maximal size. These data are in good agreement with early observations of reduction or even disappearance of CBs in mammalian oocytes from preovulatory follicles (Chouinard 1975; Parfenov et al. 2003). The reduction in CB number during late meiotic prophase I, at the time of transcription decline, is consistent with reduction of CBs during somatic prophase of mitosis (Andrade et al. 1993; Ferreira et al. 1994) and the disintegration of CBs after application of transcription inhibitors (Carmo-Fonseca et al. 1992).
One of the first extrachromosomal materials appearing in the oocyte nucleus consists of 30–50 nm granules. This granular material appears in close association with chromatin. Later, it increases significantly in amount, and can be observed not only in the karyosphere but also in other parts of the nucleus. Morphologically similar material was described in the karyosphere of a weevil (Świątek 1999). This material may comprise a single body or exist as part of more complex structures. We found that this material contains SR protein SC35 and pol II. Unexpectedly, in immunogold experiments we also found labeling with an anti-coilin antibody. These data make it difficult to compare this material directly with other known nuclear substances. Superficially these granules resemble interchromatin granule clusters (IGCs), which are characteristic of mammalian somatic cells and oocytes (Kopecny et al. 1996; Parfenov et al. 1998). On the other hand, it is easy to distinguish this material in P. communis oocytes on the basis of its larger granules and the presence of coilin. Interestingly, the accumulations of large granules (up to 70 nm in diameter) together with typical IGCs were described in pig oocytes and embryos (Kopecny et al. 1996) and in the nuclei of two-cell mouse embryos (Bogolyubova and Parfenov 2000). The authors consider these large granules to be perichromatin granules (PGs) (Kopecny et al. 1996). In mouse oocytes at the active stage (1–2 layer follicles), perichromatin fibrils were shown to associate with similar large granules (40–60 nm), which become more numerous in antral follicles at the inactive stage (Parfenov et al. 2003). The authors also believe these granules to be homologs of PGs. It was found that in mouse oocytes, PGs contain the splicing factor SC35 and the transcription factor Oct-4 (Parfenov et al. 2003).
So, in P. communis oocyte nuclei, only partial homology of the granular material to the structures discussed above can be established. Because this material in P. communis contains pol II and coilin, there is some similarity to CB components.
We found SC35 protein in both CBs and the granular, coilin-containing material. This may be due to features of the inactivated oocyte nuclei we mainly dealt with. We believe that, in oocytes, inactivation of the nucleus is manifested by karyosphere formation (Gruzova and Parfenov 1993). It is interesting that SC35 was also demonstrated in CBs of the inactive oocyte nuclei of the mouse and the mealworm (Bogolyubov and Parfenov 2001; Parfenov et al. 2003). At the same time, it is well known that SC35 protein is absent from CBs of actively transcribing somatic cell nuclei (for reviews see: Matera 1999; Gall 2000). In amphibian oocytes (at the active stages), SC35 protein is absent from the CB matrix, but it is abundant in B-snurposomes associated with the CB periphery as well as those within the matrix (Gall et al. 1999). In the house cricket, SC35 protein is seen in a restricted area within oocyte CBs at the latest stages of its development (Tsvetkov et al. 1997). Thus, SC35 may occur in CBs under certain circumstances.
In transcriptionally active nuclei, CBs are suggested to represent the sites for initial assembly of pol I, II, and III transcription/processing machinery. In inactive nuclei (e.g. in diplotene oocytes in meroistic ovaries of many insects), CBs and coilin-containing granular material may serve as storage domains that accumulate inactivated components (including pol II and splicing factors) that were engaged at earlier (active) stages in RNA transcription/processing.
At the same time, CB remnants were found in the latest P. communis oocytes, after breakdown of the nuclear envelope. This fact confirms a proposition made earlier (Bogolyubov et al. 2000; Bogolyubov and Parfenov 2001; Jaglarz 2001) that insect oocyte NBs, including CBs, may represent nuclear domains accumulating some components of gene expression for early embryogenesis. Whether CBs in P. communis oocytes may play such a role remains speculative.
We are grateful to the following people for providing antibodies used in this work: E. K. L. Chan for R288 serum, K. G. Murti for the anti-DNA monoclonal antibody, J. G. Gall for mAbs 8WG16 and anti-SC35. The authors are greatly indebted to J. G. Gall for providing fluorescein-tagged RNA constructs. We also thank Yu. I. Gukina for technical assistance. This work was supported by the Russian Foundation for Basic Research (grants No. 03-04-49389 and 04-04-48080) and the Russian Science Support Foundation.