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

Tissue and Cell 39 (2007) 353–364

Localization of interchromatin granule cluster and Cajal body components in oocyte nuclear bodies of the hemipterans D.S. Bogolyubov a,∗ , F.M. Batalova a , A. Ogorzałek b a

Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Avenue 4, 194064 St. Petersburg, Russia b Zoological Institute, University of Wrocław, ul. Sienkiewicza 21, 50-335 Wrocław, Poland Received 28 March 2007; received in revised form 5 July 2007; accepted 12 July 2007 Available online 24 September 2007

Abstract 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. © 2007 Elsevier Ltd. All rights reserved. Keywords: Interchromatin granule clusters; Cajal bodies; Nuclear bodies; Oocyte nucleus; Immunogold labeling electron microscopy; Hemiptera

1. Introduction Early electron microscope studies showed that insect oocytes contain numerous and morphologically different nuclear bodies (NBs) (Bier et al., 1967; Gruzova and Parfenov, 1993). Nowadays, their nature, functions and molecular composition remain uncertain although it began possible to draw a parallel between some insect oocyte NBs and interchromatin granule clusters (IGCs) and/or Cajal bodies (CBs) of mammalian somatic cells and amphibian oocytes (see below). In eukaryotic cells, IGCs and CBs are extrachromosomal nuclear organelles, or domains, involved in basic cellular processes (for reviews see Gall, 2000; Lamond and Spector, 2003; Cioce and Lamond, 2005). ∗ Corresponding author at: Laboratory of Cell Morphology, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Avenue 4, 194064 St. Petersburg, Russia. Tel.: +7 812 2976301; fax: +7 812 2970341. E-mail address: [email protected] (D.S. Bogolyubov).

0040-8166/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2007.07.004

IGCs also referred to as “speckles”, “splicing factor compartments (SFCs)”, “SC35-domains”, or B-snurposomes (in amphibian oocytes) represent granular nuclear organelles highly enriched in pre-mRNA splicing factors including small nuclear (sn) RNPs and SR-proteins (Wu et al., 1991; Misteli, 2000; Dundr and Misteli, 2001; Lamond and Spector, 2003). IGCs can be diagnostically revealed with antibodies against non-snRNP splicing factor SC35 (Fu and Maniatis, 1990; Spector et al., 1991). Thus, SC35 protein may be considered as one of the marker IGC components. A widely discussed model of IGC functions assumes this domain to act as reservoirs for splicing factors which are constantly recruited from IGCs to active genes (Mattaj, 1994; Zhang et al., 1994). The recruitment of splicing factors is regulated by the phosphorylation of IGC proteins (Misteli et al., 1997), and the accumulation of splicing factors in transcription sites depends on the C-terminal domain of RNA polymerase II (pol II) (Du and Warren, 1997; Kim et al., 1997).

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Recent works suggested that IGCs also play a direct role in regulation of gene expression, so that IGCs are “hubs” of activity rather than simple stores of inert factors (Hall et al., 2006). Another nuclear domains, CBs, contain a marker protein coilin (Andrade et al., 1991; Raˇska et al., 1991), three RNA polymerases, and other components (proteins and snRNPs) required for transcribing and processing of respective RNA transcripts (for reviews see Matera, 1999; Gall, 2000; Cioce and Lamond, 2005; Stanek and Neugebauer, 2006). In most (but not in all) mammalian somatic cells, coilin colocalizes with the survival of motor neurons (SMN) protein (Matera and Frey, 1998; Young et al., 2000) which play an important role in snRNP assembly (Terns and Terns, 2001; Matera and Shpargel, 2006; Matera et al., 2007). One of the well-known models of CB functions, originally proposed for Xenopus oocytes (Gall et al., 1999), implies the CBs to serve as initial sites for the assembly of macromolecular complexes that function elsewhere in the nucleus (Gall, 2000, 2001; Ogg and Lamond, 2002; Handwerger and Gall, 2006). Many experiments also evidently implicate CBs in remodeling and maturation of splicing and nucleolar snRNAs (Carmo-Fonseca, 2002; Matera, 2003; Cioce and Lamond, 2005; Matera et al., 2007) guided by a class of special snRNAs called small CB-specific (sca) RNAs (Darzacq et al., 2002; J´ady et al., 2003). A member of this class, U85 scaRNA, is a novel clear marker for the CBs (Liu et al., 2006a,b). Another snRNA, U7 snRNA, which play a role in 3 processing of histone pre-mRNAs (Dominski and Marzluff, 1999) should be also mentioned as a major CB component. In HeLa cells (Frey and Matera, 1995) and amphibian oocytes (Wu and Gall, 1993), U7 snRNA is almost exclusively localized in CBs. Recent hypothesis of a modular structure of CBs proposed for the somatic cells (Lemm et al., 2006) is worthy of notice. According to the authors’ opinion, CBs include distinct domains for different nuclear activities, and these modules can be combined and differently exhibited in various cells including oocytes (Matera, 2006). Little is known about the counterparts of IGCs in insect oocytes. Until the present, oocyte NBs comprising snRNPand SC35-containing granules and, thus, interpreted as IGCs have been described in the mealworm Tenebrio molitor (Bogolyubov et al., 2000; Bogolyubov and Parfenov, 2001), the scorpionfly Panorpa communis (Batalova et al., 2005a,b) and the house cricket Acheta domesticus (Stepanova et al., 2007a). Granular NBs morphologically resembling IGCs were also described in oocytes of the dragonfly Cordulia aenea (Halkka, 1981) but it is unknown whether oocyte NBs in this species contain SC35 protein. Insect oocyte CBs were characterized better but still not exhaustively. Oocyte NBs containing coilin, snRNPs, and pol II and, thus, referred to as CBs were identified in T. molitor (Bogolyubov and Parfenov, 2001; Bogolyubov, 2003) and P. communis (Batalova et al., 2000, 2005a,b). Several T. molitor oocyte NBs were also shown to accumulate myc-coilin after

microinjection of myc-tagged coilin mRNA into the ooplasm (Bogolyubov, 2003). Another example of insect oocyte CBs is a large NB in the oocytes of the violet ground beetle Carabus violaceus (Jaglarz, 2001). This nuclear organelle was shown to contain pigpen, a member of EWS family of oncoproteins highly enriched in CBs (Alliegro and Alliegro, 1996), snRNPs and coilin (Jaglarz, 2001). The peculiar NB of A. domesticus oocytes originally referred to as the Binnenk¨orper or endobody (J¨orgensen, 1913; Bier et al., 1967) also evidently represents a CB (Gall et al., 1995). In this species, the CB is the only one oocyte NB targeting the fluorescein-tagged U7 snRNA injected into the ooplasm (Stepanova et al., 2007a). A. domesticus oocyte CB is also enriched in coilin, fibrillarin, snRNPs (Gall et al., 1995; Tsvetkov et al., 1997) but does not contain pol II (Stepanova et al., 2007a) which is a component of the CBs in mammalian somatic cells (Schul et al., 1998) and amphibian oocytes (Gall et al., 1999; Morgan et al., 2000). At the same time, some basic components directly involved in pol II transcription (the basal transcription factor TFIID and transcription coactivators CBP/p300) were revealed in A. domesticus oocyte CBs (Stepanova et al., 2007b). In Drosophila oocytes, a prominent NB also known as the Binnenk¨orper (Bier et al., 1967; Mahowald and Tiefert, 1970) was identified as a CB because it contains splicing snRNAs, SMN protein, and, the most diagnostically, U85 scaRNA (Liu et al., 2006a,b). However, U7 snRNA in Drosophila cells is localized in a separate NB called the histone locus body (HLB) (Liu et al., 2006a,b). It should be also noted that oocyte NBs in some insects still cannot be identified as IGCs, CBs, or any else. For example, NBs that do not contain ICG or CB markers were described in the oocytes of Drosophila (Liu et al., 2006a), ´ atek and Jaglarz, 2004) and lice (Zelazowska ˙ weevils (Swi˛ and Jaglarz, 2004). All these examples of extremely high heterogeneity of oocyte NBs in insects underline the importance of further comparative studies on this topic. In the present work we provide first ultrastructural and immunocytochemical observations on oocyte NBs in three species of hemipteran bugs, Notostira elongata, Capsodes gothicus and Velia caprai. Oocyte nuclear structures in hemipterans are extremely poor known. Earlier investigations carried out on hemipteran oocytes concerned other aspects of oogenesis and did not consider extrachromosomal NBs. Our work aimed revealing the IGC and CB counterparts in the hemipteran oocytes.

2. Material and methods The specimens of three bugs, N. elongata, C. gothicus (Hemiptera: Miridae) and V. caprai (Hemiptera: Veliidae) were collected in June near Wrocław (Lower Silesia). Insect ovaries were isolated in OR2 medium (Wallace et al., 1973)

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or in Ringers’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, Inc.) in 0.1 M phosphate buffer, pH 7.4 for 1.5 h following by the postfixation in 1% OsO4 in the same buffer for 1.5 h. After dehydration in ethanol series the specimens were embedded in Epon 812 (Serva, Heidelberg, Germany). Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined in a Zeiss EM 900 electron microscope at 80 kV. For ultrastructural immunocytochemistry, single ovarioles were prefixed for 2 h in 4% paraformaldehyde and 0.5% glutaraldehyde in PBS and then fixed overnight in 2% paraformaldehyde at 4 ◦ C. After rinsing in PBS containing 0.05 M NH4 Cl and subsequent dehydration in ethanol series, ovarioles were embedded in LR White resin (Sigma). Ultrathin sections were incubated for 10 min in a blocking buffer containing 0.5% fish gelatin (Sigma) and 0.02% Tween-20 in PBS, pH 7.4. After blocking, the sections were incubated in first antibody solution overnight at 4 ◦ C in a moist chamber. Primary antibodies for immunostaining included the following mAbs: anti-SC35 (5 ␮g/ml) against the non-snRNP splicing factor SC35 (Fu and Maniatis, 1990), Y12 (1:1 or undiluted culture supernatant) against the Sm-epitope of snRNPs (Lerner et al., 1981), and 8WG16 (1:200) against the nonphosphorylated C-terminal domain of RNA polymerase II (Thompson et al., 1989). Coilin was detected with rabbit polyclonal serum R288 (1:200) against the carboxy-terminal fragment (14 kDa) of human p80-coilin (Andrade et al., 1991). After rinsing in PBS, the sections were incubated with secondary goat anti-mouse or goat anti-rabbit antibodies conjugated with colloidal gold particles of 10 nm (BBInternational, USA). As a control, additional sections were incubated only with secondary antibodies. The sections were contrasted with uranyl acetate and analyzed in a JEM-7A electron microscope. In some cases, the contrast of images was digitally enhanced using Adobe Photoshop.

3. Results The female gonads of the Hemiptera consist of meroistic telotrophic ovarioles (Figs. 1 and 3). Oocyte nuclei of the bugs contain numerous nuclear bodies (NBs) which vary in size and shape (Figs. 2 and 4). 3.1. N. elongata Among different N. elongata oocyte NBs, a single large NB is observed (Fig. 2, arrow). Early previtellogenic oocytes located in the “neck” region of the germarium already contain this NB which is preserved in the nucleus during the subsequent stages of oocyte growth. The largest oocyte NB has complex fine structure and consists of several morphological parts: a central irregularly shaped fibrillar zone with numerous vacuoles and electron dense “bumps” located at the

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periphery (Figs. 5–8). The “bumps” consist of intermingled fibrils and unclearly seen granules. Immunogold labeling microscopy revealed that anti-coilin serum R288 and monoclonal antibody (mAb) 8WG16 against the non-phosphorylated RNA polymerase II cross-react with all parts of the largest NB (Figs. 6 and 7). However, the central part of this NB was seen devoid of labels using anti-SC35 mAb. On the contrary, the peripheral “bumps” were strongly labeled with this antibody (Fig. 8). Apart from the largest NB, N. elongata oocytes contain smaller NBs of different morphology. Several NBs consist of a central fibrillar body with peripherally located structures of two types: (i) electron dense amorphous aggregates of a fibrillar material (Figs. 9–12, arrowheads), and (ii) fibrillar spherical structures partially immersed into the central body (Figs. 10 and 12, arrows). No antibody used in this study labeled the latter structures, and their nature remains uncertain. The central spherical part of the described NBs was selectively labeled with anti-coilin serum (Fig. 9) and mAb Y12 (Fig. 10). The latter antibody was originally found to detect the “Sm-epitope” of splicing snRNPs (Lerner et al., 1981) but now is known to react with symmetrical dymethylarginines typical for several Sm-proteins (Brahms et al., 2000) and coilin (Hebert et al., 2002). Thus, the labeling of the NBs with mAb Y12 may be due to the presence either Sm-proteins, or coilin, or them both. Also, given NBs were labeled with mAb 8WG16 but the labeling of the peripheral amorphous material was not so considerable as compared to the central body (Fig. 11). Contradictory results were obtained with anti-SC35 mAb. Unlike the largest NB demonstrating the labeling only the peripheral “bumps” with this antibody, a strong labeling of the central (coilin-containing) part of smaller NBs rather than the labeling of the peripheral structures was observed (cf. Figs. 8 and 12). Finally, N. elongata oocytes contain a few small NBs which are simple granular bodies (Figs. 13 and 14). These NBs were weakly but still selectively labeled with antiRNA polymerase II mAb 8WG16 (Fig. 13) and additionally were found to contain perceptible amounts of SC35 protein (Fig. 14). 3.2. C. gothicus As in the previous species, C. gothicus oocytes contain numerous and morphologically complicated NBs (Figs. 15–20). A large group of C. gothicus oocyte NBs represents fibrillar bodies that comprise irregularly shaped zones differed in electron and packing density of the fibrils (Figs. 15–19). Such NBs are strongly positive when anti-coilin or anti-SC35 antibodies were used for immunogold labeling (Figs. 15–17 and 19, on the right). Peripheral parts of these NBs display characteristic electron dense fibrillar “caps” protruded by electron transparent interstices (Figs. 17–19). These caps were labeled with anti-SC35 mAb

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Figs. 1–4. Isolated ovarioles and semithin sections of oocyte nuclei of Notostira elongata (Figs. 1 and 2) and Capsodes gothicus (Figs. 3 and 4). Arrow in Fig. 2 indicates the largest Cajal body of N. elongata oocytes. Scale bars = 20 ␮m in Figs. 1 and 3 and 10 ␮m in Figs. 2 and 4.

(Figs. 17 and 18) but the most prominent labeling with this antibody was observed in spherical homogeneous fibrillar inclusions also found within these NBs (Fig. 17, asterisk). Several other NBs appear in sections as fibrillar “rings” of low electron density with a fibrillar spherical body inside. The latter was prominently labeled with anti-coilin serum (Fig. 20). Some NBs have irregular shape and consist of tightly packed homogeneous fibrillar material (Fig. 19, on the left).

Curiously, anti-SC35 mAb was found to decorate only some local areas within these NBs, and no morphological differences between the labeled zones and the rest of an NB was revealed (Fig. 19). 3.3. V. caprai In this species, an oocyte contains one large and numerous small NBs. All NBs have a fibrillar organization. The largest

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Figs. 5–8. (Fig. 5) N. elongata oocyte nuclear bodies as viewed by routine electron microscopy. Scale bars = 1 ␮m. (Figs. 6–8) The largest Cajal body of N. elongata oocytes after immunogold labeling using anti-coilin serum R288 (Fig. 6), mAb 8WG16 against the non-phosphorylated RNA polymerase II (Fig. 7) and anti-SC35 mAb (Fig. 8). Note, that SC35 protein is localized only in the peripheral electron dense “bumps” (asterisks). Scale bars = 0.5 ␮m.

NB has a complex morphology. It generally consists of electron dense vacuolated central part surrounded by a material of lower electron density and peripheral irregular areas comprising a filamentous material (Fig. 21). The central part of the NB is labeled with anti-coilin serum (Fig. 22) while anti-

SC35 mAb decorates only the peripheral filamentous zones (Fig. 23). Among smaller NBs, there are fine fibrillar bodies which also give a clear positive reaction with the serum R288 against coilin (Fig. 24).

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Figs. 9–12. Smaller nuclear bodies of N. elongata oocytes labeled with antibodies against coilin (Fig. 9), Sm-epitope of snRNPs (Fig. 10), non-phosphorylated RNA polymerase II (Fig. 11) and SC35 protein (Fig. 12). Arrowheads indicate the accumulations of an amorphous fibrillar material at the periphery of nuclear bodies; arrows point spherical fibrillar structures of unknown nature. Scale bars = 0.5 ␮m.

4. Discussion Our present work was a first attempt to reveal, using immunogold labeling electron microscopy, IGC and CB equivalents in hemipteran oocytes. We regarded coilin as a marker antigen of CBs (Andrade et al., 1991; Raˇska et al.,

1991) and SC35 protein as that of IGCs (Fu and Maniatis, 1990; Spector et al., 1991). We found that both these proteins, as well as RNA polymerase II (pol II), are revealed in oocyte NBs of the studied species. But our findings presented here, together with previous immunomorphological studies on oocyte NBs in other insects, evidently suggest that

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Figs. 13 and 14. Typical interchromatin granule clusters of N. elongata oocytes after immunogold labeling using antibodies against the non-phosphorylated RNA polymerase II (Fig. 13) and SC35 protein (Fig. 14). Scale bars = 0.1 ␮m.

the nucleus of insect oocytes is structurally more complex as compared to the nuclei of mammalian somatic cells and even amphibian oocytes. In particular, morphological and biochemical criteria seem not to be sufficient to determine IGCs and/or CBs in insect oocytes. 4.1. Insect oocyte Cajal bodies Among the studied species, only N. elongata oocytes contain an NB which may be evidently regarded as a CB. It is a large NB containing both coilin and pol II. Morphological features of other oocyte NBs in this and other studied species are not so obvious to say whether they represent CBs. In the hemipterans studied in this work, oocyte CBs (coilin-containing NBs) are highly complicated structures consisting of a fibrillar material of several types. In other insects, oocyte CBs are also heteromorphous organelles and they may be fibrillar, fibrogranular, or even granular structures (Bogolyubov and Parfenov, 2001; Jaglarz, 2001; Bogolyubov, 2003; Batalova et al., 2005a,b; Liu et al., 2006a; Stepanova et al., 2007a). In T. molitor, several oocyte NBs were shown to contain coilin whereas others with the same morphology do not (Bogolyubov, 2003). This is also true for the pol II (Bogolyubov and Parfenov, 2004). It should be also noted that even in N. elongata and C. gothicus which belong to the same family, oocyte CBs are morphologically different. 4.2. Insect oocyte interchromatin granule clusters In many insects, anti-SC35 antibody that commonly used to designate IGCs (Fu and Maniatis, 1990; Spector et al., 1991), labels not only granular oocyte NBs but also marks several types of a fibrillar material. Similar material was found in oocyte NBs of all three species of the bugs. The

labeling of fibrillar oocyte NBs with anti-SC35 antibody was previously reported for the scorpionfly P. communis (Batalova et al., 2005a,b). For this reason, the term “SC35domain” (Hall et al., 2006) appears more attractive for insect oocytes, since it does not concern an ultrastructural aspect. Among the studied bugs, more distinct IGC equivalents that would simultaneously have a granular appearance and contain SC35 protein were found only in N. elongata oocytes. Some of these NBs were seen located freely in the nucleoplasm. These are small roundish structures containing SC35-positive granules. Other IGC counterparts in N. elongata oocytes are the peculiar “bumps” located at the periphery of coilin-containing NBs and enriched in SC35 protein. These bodies look similar to the structures described in the accessory nuclei of wasp oocytes (Bili´nski and Kloc, 2002; Jaglarz et al., 2005). The accessory nuclei of the wasp Vespula germanica contain heteromorphous inclusions consisting of a fibrillar coilin- and SMN-containing body with granular electron dense hemispheres attached to its periphery (Bili´nski and Kloc, 2002; Jaglarz et al., 2005). However, the authors did not report whether they contain SC35 protein. Also, the “bumps” of N. elongata oocyte NBs look similar to amphibian oocyte IGCs termed B-snurposomes that are associated with or even embedded into the matrix of the CBs (Wu et al., 1991; Gall et al., 1995, 1999; reviewed in Gall, 2000; Gall et al., 2004). However, these nuclear organelles seem not to be absolutely identical. We found that non-phosphorylated pol II is localized in all parts of complex N. elongata oocyte NBs whereas amphibian oocyte IGCs/Bsnurposomes do not contain pol II (Doyle et al., 2002), and mAb 8WG16 against the non-phosphorylated pol II stains only the CB matrix in amphibian oocytes (Gall et al., 1999; Morgan et al., 2000; Doyle et al., 2002). From the other hand, IGCs/speckles of mammalian somatic cells (Xie et al., 2006)

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Figs. 21–24. (Fig. 21) A large complex nuclear body in an oocyte of Velia caprai. Scale bars = 1 ␮m. (Figs. 22 and 23) Similar nuclear body of V. caprai oocytes after immunogold labeling with antibodies against coilin (Fig. 22) and SC35 protein (Fig. 23). (Fig. 24) A small coilin-containing oocyte nuclear body in V. caprai. Scale bars = 0.5 ␮m.

Figs. 15–20. Oocyte nuclear bodies in Capsodes gothicus after immunogold labeling using antibodies against coilin (Figs. 15 and 20) and SC35 protein (Figs. 16–19). Asterisk in Fig. 17 indicates a fibrillar area densely labeled with anti-SC35 antibody; other parts of this body including the peripheral “caps” (arrowhead), similar to that shown in Fig. 18 at higher magnification, are also labeled with this antibody. In Fig. 19, on the left, a local SC35-containing area of a fibrillar nuclear body is seen; the rest of this body is devoid of labels. Another complex nuclear body on the right of Fig. 19 is intensely labeled with anti-SC35 antibody. Scale bars = 0.5 ␮m in Figs. 15–17 and 19 and 0.1 ␮m in Figs. 18 and 20.

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and oocytes (Parfenov et al., 2000, 2003) contain all forms of the pol II. SC35-containing loose areas located at the periphery of large coilin-containing oocyte NBs were also observed in V. caprai. 4.3. Oocyte NBs may share features of CBs and IGCs We found that CB and IGC markers (coilin and SC35, respectively) can be revealed in the same oocyte NBs of the hemipterans. It is well-known that in mammalian somatic cells, CBs lack SC35 protein which is localized in IGCs/speckles (Raˇska et al., 1991; Spector et al., 1991; Gall, 2000). This protein was readily demonstrable in purified fractions of IGCs from mouse hepatocytes (Mintz et al., 1999); correspondingly, it was not revealed in large-scale isolated CBs from HeLa cells (Lam et al., 2002). In oocytes, the situation is somewhat different. Even in amphibians, while SC35 protein is really concentrated in IGCs/B-snurposomes (Wu et al., 1991; Gall, 2000), a weak anti-SC35 staining of the CB matrix is still registered by laser scanning confocal microscopy (Gall et al., 1999). Among insects, similar pictures are observed in the structurally complex oocyte CB of the house cricket A. domesticus (Stepanova et al., 2007a). In earlier works (Gall et al., 1995; Tsvetkov et al., 1997), anti-SC35 staining of A. domesticus oocyte CB was seen limited by the “internal” IGC which is located inside the CB (Filek et al., 2002). Later, a cross-reaction of the CB matrix with anti-SC35 antibody was revealed by confocal microscopy. This finding was also confirmed by double immunogold labeling of the CB with antibodies against SC35 and coilin (Stepanova et al., 2007a). It was also shown that diplotene oocyte nuclei of P. communis accumulate a peculiar granular material containing SC35 protein, coilin and pol II (Batalova et al., 2005b). Apart from the insects, colocalization of SC35 protein and coilin in the same NBs was recently observed by laser scanning confocal microscopy in spider oocytes (Bogolyubov and Bogolyubova, 2007). 4.4. Conclusion remarks Immunolocalization experiments alone cannot shed light on NB functions in insect oocytes. Comparative studies in this respect still state more questions than find answers. Why insect oocyte IGCs and CBs are so heterogeneous? Why the morphology and molecular composition of oocyte IGCs and/or CBs are significantly different in various species regardless of their taxonomic/phylogenetic position and the type of oogenesis? Why there is a poor correlation between the morphological appearance of an NB and its molecular contents? Finally, why specific IGC and CB components may be united within the same NB and what is the functional significance of such consolidation? An oocyte is an unusual cell as compared to the somatic one, and the features of the oocyte may affect to its nuclear

structure. Correspondingly, the functions of oocyte nuclear organelles may be somewhat different then those of the somatic cells (Gall et al., 2004). Oocyte NBs may represent special large stores for macromolecules that are not used in the oocytes but needed for early embryogenesis (Bogolyubov et al., 2000; Jaglarz, 2001; Bili´nski and Kloc, 2002; Jaglarz et al., 2005). Also, oocyte NBs may represent just the “garbage bins” (Matera, 2003) for inactive factors disengaged from RNA transcription/processing cycles. This seems more likely for the insects that are characterized by active oocyte nuclei at early stages of oogenesis and the cessation of transcription activity to the end of oocyte growth (Gruzova and Parfenov, 1993; Bogolyubov, 2007).

Acknowledgements We are grateful to the following persons for providing primary antibodies for this work: E.K.L. Chan for R288 serum, K.G. Murti for mAb 8WG16, and J.G. Gall for mAbs Y12 and anti-SC35. We also thank Yu.I. Gukina for technical assistance. This work was supported by Russian Foundation for Basic Research (grant 06-04-48904), by the research grant 1018/DS/IZ/2006, and by the State support for leading scientific school of Russian Federation (grant no. NS-1125.2006.4).

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