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

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

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Nuclear Bodies of Stage 6 Oocytes of Rana temporaria Contain Nucleolar and Coiled Body Proteins VLADIMIR N. PARFENOV,* DONNA S. DAVIS,† GALINA N. POCHUKALINA,* CLARE E. SAMPLE,†,‡ AND KURUGANTI G. MURTI†,‡,1 *Institute of Cytology, Russian Academy of Science, St. Petersburg, Russia; †Department of Virology and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105-2794; and ‡Department of Pathology, University of Tennessee, Memphis, Memphis, Tennessee 38163

INTRODUCTION The genetically inactive stage 6 oocyte nuclei of Rana temporaria contain 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. AntisnRNP 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. q 1996 Academic Press, Inc.

1 To whom reprint requests should be addressed at Department of Virology and Molecular Biology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, Tennessee 38105-2794. Fax: (901)-523-2622.

It is becoming increasingly clear that the eukaryotic nucleus is a highly organized structure in which the various nuclear functions are compartmentalized [1]. In addition to the nucleoli and chromosomes, the nucleus contains several other structures, the best studied of which are the coiled bodies (CBs). Although CBs were discovered in the early part of this century [2], their significance is just being realized (for a review see reference 3). The CBs are highly conserved structures present in plants and animals. They are spherical, measure 0.1 to 1.0 mm in diameter, and contain densely packed coiled fibers. The major protein of CBs is p80coilin as recognized by staining with autoimmune patient sera that specifically stain CBs. The CBs also contain snRNPs which are components of splicing machinery and fibrillarin, a nucleolar protein. Despite the wealth of information on the structure and composition of CBs, two key questions concerning these structures remain to be answered: where do CBs originate and what is their function? Since CBs often associate with the nucleolus, it has been hypothesized that CBs originate from the nucleolus and that they function in rRNA synthesis, maturation, or transport. However, the association of CBs with the nucleolus may also reflect the fusion of the former with the latter. Additionally, CBs do not contain rRNA, rDNA, nucleolar proteins such as RNA polymerase I, B23, or nucleolin [4]. The facts that CBs contain snRNPs and at least one other splicing factor and that their number increases with increase in gene expression led to the proposal that they play some role connected with the participation of splicing snRNPs in gene expression [3]. However, the absence of the factor SC-35 which is required for the initial steps in splicing [5, 6] and nascent RNAs in CBs [7–9] raises doubts concerning this function as well. Thus the origin and function of CBs remain enigmatic. In an attempt to understand the origin of CBs, we have searched for coilin-containing structures in the genetically inert stages in the developing oocytes (stage

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6 as per Dumont, see reference 14) of the frog Rana temporaria. The rationale for choosing the stage 6 oocytes is as follows. Two previous studies have shown that when rRNA synthesis/processing is inhibited by drugs [10] or when cells of hibernating animals (at low metabolic activity) are examined [11, 12], the CBs integrate with the nucleolus. Based on these studies, we felt that in oocytes at a low level of gene expression it would be possible to observe the interrelationships between CBs and nucleoli. To clearly visualize the coilin-containing structures in the oocyte, we extracted the oocytes with a buffer that removes most of the soluble structures but preserves the nucleoskeleton [13]. The procedure reduced the density and complexity of the nuclear contents while preserving the coilin-containing structures. The results showed that stage 6 oocytes contain nuclear bodies that have at least five structurally different zones that label with nucleolusspecific as well as CB-specific antibodies (anti-fibrillarin, anti-B23, anti-snRNP, and anti-coilin). Extensive searching in the genetically active stage 3 oocytes failed to reveal these bodies. These bodies, however, share certain features with the ‘‘prenucleolar bodies’’ in nuclei assembled in vitro with Xenopus extract, sphere organelles in the active stage of oogenesis of several amphibians, and binnenko¨rper of insect germinal vesicles (reviewed in ref. 27) but differ from each of these in certain important respects. We designate them as multicomponent bodies (MCBs) to reflect their mixed composition. The occurrence of these bodies demonstrates the close interrelationship between CBs and nucleoli. MATERIALS AND METHODS Oocytes. Oocytes from the ovary of the grass frog R. temporaria were used. Mature females were collected from their hibernation sites in the region of Pskov (Northwest Russia). Yolk oocytes measuring Ç1600 mm in diameter (stage 6, reference 14) were used. Antibodies. Anti-coilin antibody (R288 rabbit antiserum to p80 coilin) was a kind gift of Dr. E. K. L. Chan, The Scripps Research Institute (LaJolla, CA). B23 antibody (chicken) was provided by Dr. Scott Kaufmann (Johns Hopkins University, Baltimore, MD). Mab Y12 against the Sm epitope was the courtesy of Dr. J. Steitz (Yale University, New Haven, CT). Anti-fibrillarin was obtained from Dr. Yong Ren, Baylor College of Medicine (Houston, TX). The gold-conjugated secondary antibodies were bought from Amersham (Lisle, IL). Extraction of oocytes and electron microscopy/immunogold labeling. Separated individual oocytes were extracted with cytoskeletal (CSK) buffer (100 mM NaCl, 300 mM sucrose, 10 mM Pipes, pH 6.8, 3 mM MgCl2, 0.5% Triton X-100, and 1.2 mM phenylmethylsulfonyl fluoride) for 20 min at 07C. The oocytes were incubated with CSKAS buffer [CSK buffer containing 250 mM (NH4)2SO4 in place of NaCl] for 20 min at 07C. The extracted oocytes were then treated with CSK buffer containing 100 mg/ml DNase for 20 min at room temperature and rinsed with the CSK buffer. All steps of extraction were performed according to published procedures [13]. Extraction was performed both on oocytes surrounded by follicular cells and on oocytes from which the follicular cells were removed by treatment for 2 h with 0.2% collagenase solution followed by manual removal

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with forceps. In both cases the results of extraction were identical. Immunogold labeling and electron microscopy were done as described previously with some modifications [15]. Briefly, the extracted oocytes were fixed in 3.7% formaldehyde and 0.1% glutaraldehyde in phosphate-buffered saline (PBS) for 1 h. The samples were dehydrated and embedded in LR white resin. The blocks were sectioned on a Sorvall ultramicrotome and sections were picked up on nickel grids. The grids were floated on PBS containing 0.02 M glycine and Tris-buffered saline (TBS) containing 0.5% fish gelatin (15 min each) to minimize nonspecific binding of the antibodies. The grids were floated on primary and secondary antibodies diluted 20- to 50-fold with TBS containing 1% fish gelatin for 1.5 h at 377C. The grids were rinsed, stained with 4% aqueous uranyl acetate, and viewed in a JEOL 1200 EXII electron microscope operated at 80 kV. Appropriate controls were maintained by omitting the primary antibody from the procedure or by using an irrelevant primary antibody; neither of these controls showed significant labeling.

RESULTS AND DISCUSSION

Ultrastructure of nuclear bodies in stage 6 oocytes. Synthetically, the stage 6 oocytes are characterized by insignificant levels of gene expression [16–18]. Morphologically, the stage 6 nuclei contain highly condensed chromosomes surrounded by numerous predominantly fibrillar nucleoli and other nuclear bodies. We have described the structure of these nuclear components earlier and have identified nuclear bodies containing coiled threads typical of CBs [19]. In the present study, we have extracted the stage 6 oocytes with a buffer that removes most of the DNA and soluble proteins [13], thus exposing the karyoskeleton. The karyoskeletons thus obtained revealed the nuclear bodies described above but with greater structural clarity. These bodies measure 2 to 8 mm and have a unique structure with multiple morphologically recognizable zones. We refer to them as MCBs. These bodies are distinct from the amplified nucleoli readily identifiable in this extracted material. The nucleoli are frequently vacuolated and appear to be composed exclusively of fibrillar material (6-nm-thick fibrils). An example of one such nucleolus is shown in Fig. 1A. Figure 1B shows a portion of the nucleolus that had been labeled with the nucleolar marker B23 by the immunogold labeling method. An electron micrograph of an MCB is shown in Fig. 1C. Each MCB contained at least five morphologically recognizable zones. Zone I consisted of electron-dense, high-contrast fibrils measuring about 30 nm in diameter, zone II contained fine filaments with a diameter of about 6 nm typically seen in the inactive nucleoli of this stage (compare with Fig. 1B), and zone III consisted of meshwork of amorphous material of lower electron density. Zones I and III of MCBs are reminiscent of the dense fibrillar component (DFC) and fibrillar centers (FC) typically seen in the eukaryotic nucleolus [24], respectively. The striking feature, however, of the MCBs are two other zones that contain packed coiled threads and granules typical of coiled bodies. One of

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FIG. 1. (A) Low-magnification electron micrograph showing a nucleolus in the stage 6 extracted oocyte. (B) A portion of the nucleolus labeled for B23; note the homogeneous, filamentous composition of the nucleolus and the presence of B23 label. (C) Electron micrograph of a nuclear body of the stage 6 oocyte. Note the five (I, II, III, IV, V) distinct zones (see text for details). Bar in this and subsequent figures equals 0.5 mm.

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FIG. 2. Immunogold labeling of MCB with anti-p80 coilin antibody. The most intensely labeled zone appears to be the zone IV, although some label (arrows) is seen over zone V. FIG. 3. Immunogold labeling of MCB with anti-snRNP antibody. Note the predominance of the label in zone IV.

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FIG. 4. Immunogold labeling of MCB with anti-fibrillarin antibody. The label is seen predominantly over zones I, IV, and V.

these zones (zone IV) is seen within the interior of the MCB as a distinct large area containing closely packed, 20-nm-thick, coiled threads and granules of the same diameter. This zone is intimately associated with zone I with material of the latter intruding into the former. The electron density of this zone is somewhat similar to that of zone II. Zone V is seen at the periphery of MCBs and it exhibited high contrast (similar contrast as zone I) coiled threads and granules. They are similar in dimension to those seen in zone IV except that they are loosely packed and possessed higher electron density. Morphologically, zone V appears as CBs fused with or emanating from MCBs. Examination of many sectioned MCBs revealed that they are variable with respect to the extent of each of the zones but all contained the typical five zones. Composition of MCBs. The morphological examination described above suggests that MCBs contain nucleolar and CB components. To determine the precise composition of the various zones in MCBs we have used the immunogold labeling/electron microscopy method using antibodies against various nucleolar and CB antigens. Since the striking feature of the MCBs are the two zones that contain coiled threads and granules (zones

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IV and V) typical of CBs, we first examined the labeling of MCBs with the anti-p80 coilin antibody. When sections of extracted oocytes were incubated with the anticoilin antibody followed by gold-conjugated secondary antibody and examined in the electron microscope, the following results were obtained. The anti-coilin antibody predominantly labeled zone IV, a zone composed of tightly coiled threads and granules (Fig. 2). A sparse labeling of zone V was frequently seen but all other zones had little or no label. Since zones IV and V contain coiled threads but at different densities of packing, it is possible that the observed labeling difference between these zones is reflective of the variations in packing densities. One of the major characteristics of CBs is that they contain snRNPs [20] which stain intensely with an antibody against the Sm epitope (mAb Y12, reference 21). To determine if MCBs also contain snRNPs, the oocyte sections were examined after immunogold labeling with mAb Y12. The results showed (Fig. 3) that mAb Y12, just like the anti-coilin antibody, predominantly labeled zone IV of the MCBs. Some labeling of zones I and V was noted but the other zones had little or no label. These observations suggest that MCBs contain snRNPs and that zone IV which contains densely

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FIG. 5. Immunogold labeling of MCB with anti-B23 antibody. Although all zones appear labeled, zone V (inset) appears to have substantial label. Arrows in the inset point to some of the label.

packed coiled threads is enriched for snRNPs. Another typical feature of CBs is that they contain fibrillarin, the protein of the dense fibrillar component (DFC) of the nucleolus [4, 10]. Since MCBs contain a fibrillar component reminiscent of that in the nucleoli (i.e., zone I), we examined the labeling of MCBs with the antifibrillarin antibody. The results are shown in Fig. 4. The anti-fibrillarin antibody predominantly labeled zones I and IV and to a lesser extent zone V. These results suggest that MCBs contain fibrillarin, a protein common to both nucleoli and CBs. The nucleolus contains a major shuttling protein, B23, which may have chaperone-like functions during ribosome assembly and nuclear protein import [22]. This protein, however, is absent from CBs [10]. Since

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the results thus far indicated that MCBs contain both nucleolar and CB proteins, we examined the labeling of B23 in the MCBs. The highest level of labeling with the anti-B23 antibody was found in zone V (see Fig. 5 insert), with decreasing levels of labeling in zones I, IV, II, and III (Fig. 5). From these results, it is clear that B23 is a component of MCBs. In summary, the MCBs contain coilin, fibrillarin, snRNPs, and B23. Table 1 summarizes the labeling of each of the zones in MCBs with the antibodies used. The data are based on an examination of sections of 10 different MCBs. Significance of MCBs. A variety of nuclear bodies containing coilin or coilin-related proteins has been described in the literature (reviewed in ref. 27). These in-

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or localized within the nucleolus surrounded by nucleoplasmic material. The MCBs described here contain both nucleolar and CB antigens but they seem different Zones of from products of a simple association of nucleoli and MCB Coilin snRNP Fibrillarin B-23 CBs. The zone V of MCBs resembles a CB associated with the periphery of a nucleolar body as described in I 0 / /// // the past literature [3]. Zone IV is an extensive zone II 0 0 0 / III 0 0 0 / composed of coiled threads in intimate association with IV ///// ///// /// // fibrillarin-containing zone (I) as well as other zones V // // // /// containing fibrillar material (zone II) and a fine fibrillar meshwork of low electron density (zone III). This zone differs from the typical CB in that the coiled threads clude CBs of somatic nuclei, ‘‘prenucleolar bodies’’ in are packed much tighter and their electron density is nuclei assembled in vitro with Xenopus egg extract, much lower. Thus, the material of zone IV appears sphere organelles in amphibian oocyte nuclei, and bin- more like precursor material of CBs or material originenko¨rper of insect germinal vesicles [27]. All these nu- nated by the dissolution of CBs. Another striking feaclear bodies are spherical, range in size from õ1 to ú10 ture of both zones IV and V is that they label for B23, mm, and contain snRNPs. CBs, prenucleolar bodies, and a nucleolar shuttling protein. This is highly unusual binnenko¨rper contain the nucleolar marker fibrillarin because typical coiled bodies do not label with anti-B23 while sphere organelles do not. The prenucleolar bodies antibodies [10]. Thus, MCBs appear unique in terms also contain other nucleolar markers such as nucleolin, of their composition. The fact that they occur at a stage B23, and a 180-kDa nucleolar protein. Recently, it was of development of oocyte at which the synthetic activipostulated that all these different nuclear bodies may ties are at their lowest level suggests that MCBs may carry out similar functions in diverse cell types [27]. The represent common storage structures for CB and nucleMCBs described in this paper share certain similarities olar components which are needed for early stages of with the above, but differ from them in some respects. embryogenesis. Another interesting observation to Morphologically, the MCBs are distinct from all other come out of the present study is the resistance of MCBs nuclear bodies in that they have an ovoid or elliptical to the extraction conditions that remove most of the shape with clearly demarcated zones. Some of these chromatin and soluble proteins. This suggests that zones resemble nucleolar regions (zones I and III), while MCBs, like the residual nucleoli [13] and CBs [4, 23], others (zones IV and V) have CB characteristics. Zone are part of the karyoskeleton and that the spatial relaIV of MCBs, however, appears to be similar to the matrix tionship of these components is maintained by the of the sphere organelle in both ultrastructure and com- structural framework of the nucleus. position (i.e., presence of coilin and snRNPs). In terms of the overall composition, the MCBs resemble the preThe authors thank Dr. William Crist for support and encouragenucleolar bodies. Both contain fibrillarin, B23, coilin, ment and Dr. A. G. Tsvetkov for stimulating discussion. The authors and snRNPs [27, 28], but the prenucleolar bodies are also thank Dr. Scott Kaufman for providing the anti B23 antibody, Dr. E. K. L. Chan for the anti-coilin antibody, Dr. Joan Steitz for the spherical, smaller (0.15 to 0.4 mm), and electron dense, anti-Sm antibody, and Dr. Y Ren for the anti-fibrillarin antibody. with no demarcated zones [29]. This work was supported by a Cancer Center Support (CORE) grant It is possible that MCBs represent a fusion product and CA-68237 (K.G.M.) from the National Institutes of Health, by of nucleoli with CBs or CB homologues described above. the American Lebanese Syrian Associated Charities (ALSAC) of St. In some previous studies, intimate association of CBs Jude Children’s Research Hospital, and by the International Scientific fund. with nucleoli was observed. In neuronal cells, CBs were seen associating with or detaching from the nucleoli REFERENCES [10]. Staining of these cells with anti-p80 coilin antibody confirmed this intimate association [10]. In cy1. Spector, D. L. (1993) Annu. Rev. Cell. Biol. 9, 265–315. cling cells, the CBs were not observed to associate with 2. Ramon, Y., and Cajal, S. R. (1903) Trab. Lab. Invest. Biol. 2, the nucleoli but treatment of cells with agents that 129–221. inhibit rRNA synthesis (actinomycin D) or rRNA pro3. Lamond, A., and Carmo-Fonseca, M. 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Received October 27, 1995 Revised version received July 17, 1996

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