Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA

Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA Kseniya V. Shishova a, Elena A. Lavrentyeva a,b, Jerzy W. Dobrucki c, Olga V. Zatsepina a,n a Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Street, 16/10, Moscow 117997, Russian Federation b Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, GSP-1, Leninskiye Gory, MSU, 1-73, Office 433, Moscow 119991, Russian Federation c Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Gronostajowa Street, 730-387 Krakow, Poland

art ic l e i nf o

a b s t r a c t

Article history: Received 20 June 2014 Received in revised form 20 November 2014 Accepted 22 November 2014

It is well known that fully-grown mammalian oocytes, rather than typical nucleoli, contain prominent but structurally homogenous bodies called “nucleolus-like bodies” (NLBs). NLBs accumulate a vast amount of material, but their biochemical composition and functions remain uncertain. To clarify the composition of the NLB material in mouse GV oocytes, we devised an assay to detect internal oocyte proteins with fluorescein-5-isothiocyanate (FITC) and applied the fluorescent RNA-binding dye acridine orange to examine whether NLBs contain RNA. Our results unequivocally show that, similarly to typical nucleoli, proteins and RNA are major constituents of transcriptionally active (or non-surrounded) NLBs as well as of transcriptionally silent (or surrounded) NLBs. We also show, by exposing fixed oocytes to a mild proteinase K treatment, that the NLB mass in oocytes of both types contains nucleolar proteins that are involved in all major steps of ribosome biogenesis, including rDNA transcription (UBF), early rRNA processing (fibrillarin), and late rRNA processing (NPM1/nucleophosmin/B23, nucleolin/C23), but none of the nuclear proteins tested, including SC35, NOBOX, topoisomerase II beta, HP1α, and H3. The ribosomal RPL26 protein was detected within the NLBs of NSN-type oocytes but is virtually absent from NLBs of SN-type oocytes. Taking into account that the major class of nucleolar RNA is ribosomal RNA (rRNA), we applied fluorescence in situ hybridization with oligonucleotide probes targeting 18S and 28S rRNAs. The results show that, in contrast to active nucleoli, NLBs of fully-grown oocytes are impoverished for the rRNAs, which is consistent with the absence of transcribed ribosomal genes in the NLB mass. Overall, the results of this study suggest that NLBs of fully-grown mammalian oocytes serve for storing major nucleolar proteins but not rRNA. & 2014 Published by Elsevier Inc.

Keywords: Nucleolus-like bodies GV oocytes Nucleolar proteins rRNA FITC Proteinase K

Introduction Growth of mammalian oocytes is a long-term process that is accompanied by remarkable functional and structural reorganizations of the nucleolus – the major multifunctional nuclear domain that plays key roles in ribosome biogenesis (Dundr, 2012; Hernandez-Verdun et al., 2010; Shaw and Brown, 2012; Grummt, 2013). In addition to RNAs (mainly, rRNA and snoRNAs), ribosome

Abbreviations: NLB, nucleolus-like body; NSN, non-surrounded nucleolus; SN, surrounded nucleolus; GV, germinal vesicle; MII, metaphase II; rDNA, ribosomal DNA; rRNA, ribosomal RNA; pre-rRNA, precursor rRNA; snoRNA, small nucleolar RNA; ssRNA (ssDNA), single-stranded RNA (DNA); dsRNA (dsDNA), double-stranded RNA (DNA); AO, acridine orange; FITC, fluorescein-5-isothyocyanate; RNase A, ribonuclease A. n Corresponding author. E-mail addresses: [email protected] (K.V. Shishova), [email protected] (E.A. Lavrentyeva), [email protected] (J.W. Dobrucki), [email protected] (O.V. Zatsepina).

biogenesis requires numerous protein factors to ensure rDNA transcription, rRNA processing, and the export of ribosomal particles to the cytoplasm (Cisterna and Biggiogera, 2010). A remarkable feature of the mammalian nucleolus is its functional and morphological divergence that is manifested in various types of cells including oocytes. In this way, nucleoli of growing mammalian oocytes are typical active nucleoli: they contain numerous small fibrillar centers (the harbors of inactive rDNAs and RNA polymerase I), the dense fibrillar component (the site of rRNA synthesis and processing), and a granular compartment that is comprised of maturing ribosomal particles (Fair et al., 2001). Suppression of oocyte growth in antral follicles is accompanied by downregulation of the nucleolar synthetic activity and initiates transformation of the nucleoli into unique structures that are called “nucleolus-like bodies” (NLBs), or “postnucleoli” (Chouinard, 1971). In the fully-grown (or germinal vesicle, GV) oocytes NLBs are seen as prominent, large (up to 10 mm in diameter), spherical bodies whose mass is composed of a tightly and uniformly packed fibrous material. This mass is deprived of any

http://dx.doi.org/10.1016/j.ydbio.2014.11.022 0012-1606/& 2014 Published by Elsevier Inc.

Please cite this article as: Shishova, K.V., et al., Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.11.022i

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morphologically defined counterparts of somatic nucleoli, so that solitary fibrillar centers, the poorly developed dense fibrillar component, and abandoned ribosomal particles are only observed at the NLB surface (Antoine et al., 1988; Biggiogera et al., 1994; Longo et al., 2003). In the vast majority of GV oocytes, only one NLB is present, but other GV oocytes contain two or three NLBs, which are similar in structure and immunochemical properties but may differ in size. Two major types of NLBs have been described in fully-grown oocytes of all mammals studied so far: the NLBs that associate with discrete blocks of heterochromatin (so-called “non-surrounded nucleoli”, NSN), and the NLBs that are surrounded by a layer of heterochromatin (so-called “surrounded nucleoli”, SN) (Debey et al., 1993; Zuccotti et al., 2002; Bellone et al., 2009; Tan et al., 2009). It is accepted that only the NSN-oocytes are able to synthesize rRNA, whereas the SN-type oocytes are transcriptionally silent (Bouniol-Baly et al., 1999; Pesty et al., 2007). However, in contrast to somatic nucleoli, active ribosomal genes have been described only at the NLB surface (Bouniol-Baly et al., 1999; Pesty et al., 2007). SN-oocytes have higher meiotic and developmental competence than NSN-oocytes and correspond to a more advanced stage of oocyte development (Zuccotti et al., 2002, 2011; Inoue et al., 2008). Recent analysis of the global transcriptome profile in mouse NSN- and SN-oocytes showed that they are very similar but not identical: NSN-oocytes are enriched in methylated and acetylated peptides (Monti et al., 2013), whereas SN-oocytes are characterized by higher levels of methylation and acetylation of DNAs (Kageyama et al. 2007). However, expression of nearly 30 genes encoding ribosomal proteins is upregulated in SN-oocytes compared to NSN-oocytes (Monti et al., 2013). NLBs are assembled in fully-grown oocytes of mammals of various species, including mouse, rat, pig, cattle, and human (Chouinard, 1971; Antoine et al., 1988; Kopecny et al., 1996; Hyttel et al., 2001; Parfenov et al., 1989), but their direct homologs have not been described in other animals. These facts point to particular importance and conservative role(s) of NLBs in mammalian oogenesis. In addition, the NLB material is also indispensable for the early steps of embryonal development. Zygotic embryos originating from enucleolated oocytes are incapable of forming nucleolar precursor bodies (NPBs), have severe defects in spatial arrangement of chromatin, contain reduced amounts of the major and minor satellite DNAs, and finally become arrested at the two-cell stage (Ogushi et al., 2008; Ogushi and Saitou, 2010; Inoue et al., 2011; Fulka and Langerova, 2014). However, the reason for the NLB requirement for oogenesis and early embryogenesis remains poorly studied. Numerous cytochemical, autoradiographic, and immunocytochemical studies have been conducted to determine the biochemical composition of the material comprising the NLB mass. Overall, the results of these studies demonstrate that: (1) NLBs do not contain polysaccharides, lipids, or DNAs (Antoine et al., 1988; Kopecny et al., 1995, 1996). (2) NLBs probably contain certain amounts of nuclear RNAs, but the results obtained by different methods are contradictory (Antoine et al., 1989; Kopecny et al., 1996). (3) NLBs most likely contain proteins (Antoine et al., 1988). However, no nucleolar proteins have been revealed within the NLB mass under conventional conditions of immunolabeling of GV oocytes (Zatsepina et al., 2000; Fair et al., 2001; Bjerregaarde et al., 2004; Romanova et al., 2006; Maddox-Hyttel et al., 2007; Pochukalina and Parfenov, 2008; Fulka and Langerova, 2014). Recently, an antigen retrieval achieved by oocyte spread boiling in sodium citrate has shown that NLBs of mouse GV oocytes contain nucleolar proteins (Fulka and Langerova, 2014), but the question whether they are present in the NLBs of both GV-type oocytes remains open. (4) Data on immunoelectron microscopy of NLBs are rather limited or contradictory. For instance, the nuclear splicing factor SC35 has been detected in the NLB mass by some authors (Kopecny et al., 1996) but not by others (Pochukalina and Parfenov, 2008). Overall, survey of the literature data shows that

the biochemical composition of NLBs in fully-grown mammalian oocytes remains largely undetermined. Lack of such data makes it difficult to establish the role(s) of NLBs in oogenesis and to explain why the NLB material is required for early development of mammalian embryos. In this study, to elicit the composition and putative functions of mammalian NLBs we optimized conditions for staining mouse paraformaldehyde-fixed oocytes with acridine orange (AO), a metachromatic dye that emits different spectra upon binding DNA or RNA (Bernas et al., 2005). We also devised an approach for staining intracellular proteins with fluorescein-5-isothiocyanate (FITC), a fluorochrome that covalently binds with proteins in vitro (Jullian et al., 2009). The specificity of RNA staining with AO and of protein staining with FITC was verified in mouse somatic fibroblasts and oocytes by their treatment with RNase A or proteinase K before cell exposure to the dyes. The results showed that, irrespective of the functional status, NLBs of fully-grown mouse oocytes contain RNA and proteins similar to nucleoli fully active in rRNA synthesis. By mild digestion of oocytes with proteinase K, we showed that the nucleolar proteins involved in key steps of ribosome biogenesis (UBF, fibrillarin, NPM1, nucleolin, RPL26) are located not only at the NLB surface, but are also immersed into the NLB mass. Conversely, none of the nuclear proteins examined (SC35, NOBOX, topoisomerase II beta, HP1α, or H3) were detected within NLBs. To determine whether accumulation of the nucleolar proteins is accompanied by accumulation of rRNA, we applied fluorescence in situ hybridization (FISH) and oligonucleotide probes targeting 18S and 28S rRNAs. However, the FISH results showed that, in contrast to active nucleoli of growing oocytes and somatic cells (Shishova et al., 2011), the rRNAs are hardly detectable within the NLB mass of GV oocytes. We also failed to reveal transcribed ribosomal genes within NLBs of proteinase K-treated GV oocytes, which was consistent with their absence in NLBs of oocytes examined using BrUTP as a precursor under conventional conditions. Based on our results, we conclude that NLBs of mammalian oocytes serve mainly as storages of nucleolar proteins but not of rRNAs. They may also be impoverished for proteins with nuclear functions.

Materials and methods Cell culture NIH/3T3 mouse fibroblasts were purchased from the Russian Cell Culture Collection (Institute of Cytology of the Russian Academy of Sciences, St. Petersburg, Russia) and were free of mycoplasma. The cells were cultured in DMEM (Pan Eco, Russia) containing 10% fetal bovine serum (HyClone, USA), 2 mM L-glutamine, penicillin, and streptomycin (250 units/ml of each) at 37 1С and 5% CO2. Animals Female C57Bl/6 mice were purchased from the Pushchino Nursery of Laboratory Animals (Pushchino, Russia). The animals were kept under pathogen-free conditions with access to tap water and standard chow ad libitum. All experiments were performed according to the local law and principles of good laboratory animal care. Collection of oocytes Four-to-six-week old females were injected with 7 IU PMSG (pregnant mare's serum gonadotropin) (Sigma-Aldrich, USA) and sacrificed 46–48 h later. Oocytes were collected from ovaries by gentle puncturing of follicles with a needle in М2 medium (Sigma-

Please cite this article as: Shishova, K.V., et al., Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.11.022i

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Aldrich) supplemented with 100 μg/ml dibutyryl-cAMP (dbcAMP, Santa Cruz Biotechnology Inc., USA) to prevent resumption of meiosis. Overall, over 700 oocytes were examined. AO staining NIH/3T3 fibroblasts were briefly rinsed in phosphate buffered saline (PBS, 140 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4, рН 7.2) and fixed with 3% paraformaldehyde (MP Biomedicals, Inc., France) in PBS for 15–20 min. The cells were washed with PBS (3
5 min), permeabilized with 0.2% Triton X-100 for 10 min at 4 1С, washed with PBS for 5 min, and exposed to 8 μg/ml solution of AO (Sigma-Aldrich) in PBS (pH 7.2) or citrate–phosphate buffer (pH 4.6) for 20 min. The oocytes were fixed with 3% paraformaldehyde (PFA) in PBS for 30 min, washed in PBS (3
10 min), and permeabilized with 0.5% Triton X-100 in PBS for 15 min on ice. Then they were placed in 8 μg/ml solution of AO prepared either in PBS (pH 7.2) or citrate–phosphate buffer (pH 4.6) for 40 min. The oocytes were briefly washed in PBS, counterstained with 1 mg/ml Hoechst 33342 in PBS for 15 min, and mounted in Vectashield. To assess the specificity of AO-staining RNA, before exposure to the dye the oocytes were incubated with 1 mg/ml ssRNA-specific RNase A (Sigma-Aldrich) in PBS for 1 h at room temperature. In the control, the oocytes were kept in PBS alone for the same length of time. FITC staining FITC (fluorescein-5-isothyocyanate isomer I, Biotium Inc., USA) was dissolved in DMSO and used at working concentration of 1 mg/ml in PBS. NIH/3T3 fibroblasts were fixed and permeabilized as described above, placed in the FITC solution for 60 min, and mounted in Vectashield. All procedures were performed at room temperature unless stated otherwise. The oocytes fixed and permeabilized as described above were stained with 1 mg/ml FITC in PBS for 120 min, briefly washed in PBS, counterstained with 1 mg/ml Hoechst 33342 in PBS for 15 min, and mounted in Vectashield. To test the specificity of FITC binding with proteins, before exposure to the dye, the oocytes were incubated with 1 mg/ml proteinase K (Sigma-Aldrich) in PBS for 20 min at room temperature. Oocytes of the control group were kept in PBS for the same time period. Proteinase K treatment and immunolabeling Oocytes were fixed with 3% PFA in PBS for 30 min, treated with 0.5% Triton X-100 for 15 min, washed in PBS (3
10 min), and transferred to a solution of 1 mg/ml proteinase K (Sigma-Aldrich) in PBS for 15–20, 40–45 min, or 80–90 min at room temperature. At each time-point, more than 10 oocytes were collected, washed in PBS (3
10 min), and incubated with specific antibodies to nucleolar or nuclear proteins (Table 1) for 1 h at room temperature. After washing in PBS (3
10 min), the oocytes were transferred to relevant secondary antibodies for 45 min at room temperature. As the antibodies, goat anti-human IgG conjugated with FITC (SigmaAldrich, cat. F 3512) (to detect UBF), Alexa Fluors 488 goat antirabbit IgG (HþL) (Molecular probes Inc., USA, cat. A-11034) (to detect fibrillarin, nucleolin, RPL26, NOBOX, topoisomerase II beta, HP1α, H3), or Alexa Fluors 488 goat anti-mouse IgG (HþL) (Molecular probes Inc., cat. A11029) (to detect NPM1) or Alexa Fluors 568 goat anti-mouse IgG (Hþ L) (Molecular probes Inc., cat. A11004) (to detect SC35) were used. The antibodies were dissolved in PBS following recommendations of the suppliers. The oocytes were washed in PBS (3
10 min), stained with 1 mg/ml Hoechst 33342 for 15 min, and mounted in Vectashield. The oocytes of the control group were incubated in PBS without proteinase K and processed for mounting under the same conditions.

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Assessment of rRNA synthesis Microinjections were performed as described earlier (Zatsepina et al., 2000). GV oocytes were incubated in M2 medium containing 100 mg/ml dbcAMP and 10 mg/ml alpha-amanitin (Sigma-Aldrich) for 30 min at 37 1C and microinjected into the cytoplasm with 1 70.5 pl of a solution containing 100 mM BrUTP (5-bromouridine 50 -triphosphate sodium salt, Sigma-Aldrich), 50 mg/ml alpha-amanitin, 140 mM KCl, and 2 mM Pipes (pH 7.4). The oocytes were recovered in M2 medium containing dbcAMP and alpha-amanitin for 30 min at 37 1C, rinsed in PBS, fixed with 3% PFA in PBS for 30 min, and treated with 0.5% Triton X-100 for 10 min. The oocytes were treated with 1 mg/ml proteinase K in PBS for 40–45 or 80– 90 min at room temperature. Control oocytes were kept in PBS alone for the same length of time. The oocytes of both groups were stained in a mixture of a mouse monoclonal antibody to BrdU (Roche, USA) and rabbit polyclonal antibodies to fibrillarin for 1 h at room temperature. After washing in PBS (3
10 min), the oocytes were incubated in a mixture of Alexa Fluors 568 goat anti-mouse IgG (H þL) and Alexa Fluors 488 goat anti-rabbit IgG (HþL) antibodies dissolved in PBS, stained with 1 mg/ml Hoechst 33342 for 15 min, and mounted in Vectashield. Fluorescence in situ hybridization Fluorescence in situ hybridization (FISH) was performed with antisense oligonucleotide probes recognizing the mouse 47S prerRNA. The probe “18S rRNA” (50 cca tta ttc cta gct gcg gta tcc agg cgg) hybridizes with the 18S rRNA sequence (residuals þ4847/ þ4876 relative to the transcription start site), and the probe “28S rRNA” (50 gag gga acc agc tac tag atg gtt cga tta) hybridizes with the 28S rRNA sequence (residuals þ9571 to þ 9600 relative to the transcription start site). The sense probes were used as control. The probes were conjugated with Cy3 at the 50 -terminal end and synthesized by DNA-synthesis Ltd (Russia). The concentration of each probe in a stock solution was about 2 μg/μl. NIH/3T3 fibroblasts were fixed with 4% PFA in PBS for 20 min at room temperature, washed with PBS (3
5 min), treated with 0.5% Triton X-100 (10 min at 4 1С), and briefly washed with PBS followed by two washing (for 10 min each) in the saline-sodium citrate buffer (2
SSC, 0.3 M NaCl, 0.03 M Na3С6Н5О7, рН 7.0). The hybridization mixture contained 50% deionized formamide (Sigma-Aldrich), 10% dextran sulfate (Loba Chemie, Fischamend, Austria), 5% 20
SSC (3 M NaCl, 0.3 M Na3С6Н5О7, pH 7.0), and 8 ng/ml oligonucleotide probes. Hybridization was performed in a humid chamber for 18 h at 42 1C. The cells were sequentially washed with 50% formamide (Panreac, Spain) in 2
SC (3
10 min) at 42 1C, 2
SSC at 42 1C (10 min), and 2
SSC (10 min) at room temperature. Oocytes were fixed with 4% PFA in PBS for 30 min, washed with PBS (3
10 min), treated with 0.5% Triton X-100 in PBS (10 min at 4 1С), washed with PBS (10 min), and finally washed in 2
SSC (2
5 min). All the hybridization steps, unless stated otherwise, were performed as described above. Before mounting in Vectashield, the oocytes were counterstained with 1 μg/ml Hoechst 33342 for 15 min and washed in PBS for 5 min. Confocal imaging Confocal 8-bit digital images were obtained using a DuoScanMeta LSM510 laser-scanning microscope (Carl Zeiss, Germany) equipped with a Plan-Apochromat 63
/1.40 numerical aperture immersion lens. The image acquisition parameters were as follows: AO green fluorescence (DNA staining) with excitation at 488 nm and emission at 505–550 nm; AO red fluorescence (RNA staining), excitation at 561 nm, emission at 575 nm; and FITC (protein staining), excitation at 488 nm, emission at 505–550 nm.

Please cite this article as: Shishova, K.V., et al., Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.11.022i

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Table 1 Nucleolar and nuclear proteins and antibodies used for their immunolocalization in fully-grown mouse oocytes in the control and following proteinase K treatment. The nucleolar proteins are shaded. Protein

Function

Reference

Antibody

Working dilution

Reference or resource

UBF

RNA polymerase I transcription factor

Ueshima et al. (2014)

1:200

Fibrillarin

Ueshima et al. (2014)

Zatsepina et al. (1993) Abcam, ab5821

RPL26

Early rRNA processing factor, the major nucleolar methyltransferase Multifunctional protein, participates in late steps of ribosome biogenesis Multifunctional protein, participates in late steps of ribosome biogenesis Large ribosome subunit protein

Preti et al. (2013)

Anti-UBF human autoimmune serum P419 Anti-fibrillarin rabbit polyclonal antibodies Anti-nucleophosmin/B23 mouse monoclonal antibody Anti-nucleolin/C23 rabbit polyclonal antibody Anti-RPL26 rabbit polyclonal antibody

SC35

Splicing factor

Lin et al. (2008)

Anti-SC35 mouse monoclonal antibody 1:200

NOBOX

Transcription factor essential for oocyte growth

Belli et al. (2013)

Anti-NOBOX rabbit polyclonal antibody 1:200

Topoisomerase II beta HP1α

DNA transcription and replication

Chen et al. (2013)

Gene transcription

H3

Core nucleohistone

Hiragami-Hamada et al. (2011) Hiragami-Hamada et al. (2011)

Anti-Topoisomerase II beta rabbit polyclonal Anti-HP1α rabbit polyclonal antibody

NPM1 Nucleolin

Lindström (2011) Gaume et al. (2011)

Two different approaches were used to analyze fluorescence intensities. (1) Fluorescence intensity profiles were analyzed using Zeiss LSM software. Straight lines were drawn on images of comparable NLBs that were recorded using the same laser power output and image acquisition parameters. The pixel intensities (in 0–255 grayscale) were quantified along the lines. (2) Fluorescence intensity per pixel in a region of interest (ROI) was examined using ImageJ software (http://imagej.nih.gov/ij/download.html), and images of 3–5 representative oocytes were taken from every comparable group. More than 20 measurements per ROI were performed, and the average pixel intensities and corresponding standard deviations (S.D.) were calculated using Microsoft Office Excel 2007 software.

Results AO and FITC staining of NIH/3T3 fibroblasts Acridine orange is widely used in flow cytometry (Darzynkiewicz et al., 2004) and epifluorescence microscopy of both fixed and living somatic cells (Bernas et al., 2005). However, some authors recommend staining cells with AO at a low pH (pH 3.5–4.6) to avoid nonspecific interaction of the dye with proteins, whereas others use AO at physiological pH (7.2–7.4) to facilitate AO binding with DNA (Zelenin, 1999). In addition, somatic cells are generally fixed with denaturing fixatives, whereas oocytes are traditionally fixed with aldehyde fixatives. Therefore, to elaborate conditions for AO staining of paraformaldehyde-fixed oocytes, we first performed pilot experiments using mouse NIH/3T3 fibroblasts. The most convincing results were obtained when fibroblasts were fixed with 3% paraformaldehyde, permeabilized with Triton X-100, and exposed to 8 mg/ml AO for 20 min either at pH 4.6 or 7.2. As shown in Fig. 1, at both pH values AO dimers (binding with RNA and emitting red fluorescence) brightly decorate nucleoli and the cytoplasm (Fig. 1a and b). The AO monomers, which preferentially bind with DNA and emit green fluorescence, stain the diffuse chromatin and chromocenters (Fig. 1a’ and b’). A weak green fluorescence observed over nucleoli and the cytoplasm most likely originates from interaction of AO monomers with RNA duplexes (dsRNAs) formed by rRNAs (Fig. 1a’ and b’). Similar to AO, FITC is also used in flow cytometry of somatic cells (Crissman et al., 1990), but we failed to find literature on application of

Anti-H3 C-terminal rabbit polyclonal antibody

1:200 1:200 1:100 1:200

1:200 1:200 1:100

Sigma-Aldrich, B0556 Abcam, ab70493 Abcam, ab59567 Abcam, ab11826 Abcam, ab41521 Abcam, ab15565/500 Sigma-Aldrich, H2164 Active Motif, 39163

FITC for analysis of intracellular proteins by microscopy. Therefore, we determined conditions appropriate for microscopic localization of FITCstained proteins experimentally. The most convenient results were observed by staining formaldehyde-fixed fibroblasts with 1 mg/ml of FITC in PBS for 60 min (Fig. 1c). Under these conditions, nucleoli as intracellular areas with the highest local concentration of proteins (Boisvert et al., 2010) were stained most intensely. Prominent but less bright fluorescence was also observed in the nucleoplasm and cytoplasm (Fig. 1c).

AO and FITC staining of oocytes Representative oocytes stained with AO at pH 7.2 and counterstained with Hoechst 33342 are shown in Fig. 2 (n ¼35). The AO green fluorescence reveals the oocyte type: in the NSN-oocytes AO brightly stains chromocenters (Fig. 2b), and in the SN-oocytes, it reveals a chromatin layer around NLBs (Fig. 2e). The AO green fluorescence coincides well with the Hoechst 33342 fluorescence (Fig. 2c), which supports the specificity of chromatin staining with the AO monomers. Similar patterns of oocyte staining were also observed at pH 4.6 (data not shown). Fig. 2a and d illustrates red fluorescence emitted by the AO dimers bound with RNAs in the same oocytes. In both types of oocytes, NLBs are conspicuously stained for RNAs. In some oocytes, discrete aggregates of RNA inside NLBs can be seen (Fig. 2d). These inclusions may correspond to local accumulations of ribosomes that have been described in NLBs of fully-grown mouse oocytes by electron microscopy (Parfenov et al., 1989; Pochukalina and Parfenov, 2008). Red fluorescence was also seen in the cytoplasm of all oocytes examined (Fig. 2a and d). The specificity of RNA staining with AO dimers was further confirmed by treating GV oocytes with RNase A (1 mg/ml, 1 h) before their exposure to the dye. Fig. 3 (a–f, and g) shows that RNase A significantly (more than 60%) reduces the red fluorescence of NLBs, which however exceeds the background level or the fluorescence level outside the oocyte (Fig. 3g). In contrast, the NLB green fluorescence was not affected by RNase A (Fig. 3h), thereby supporting the assumption that it originates from dsRNAs present in the NLB mass (Fig. 3c and f). It is noteworthy that neither red (corresponding to ssRNAs) nor green (corresponding to dsRNAs) fluorescence of NLBs was affected by proteinase K (data not shown).

Please cite this article as: Shishova, K.V., et al., Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.11.022i

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Fig. 1. Mouse NIH/3T3 fibroblasts stained with 8 μg/ml acridine orange (AO) at pH 7.2 (a, a’) or 4.6 (b, b’), 1 μg/ml fluorescein-5-isothiocyanate (FITC, c), or hybridized in situ with a Cy3-conjugated oligonucleotide probe targeting 18 S rRNA (d). The cells were fixed with 3% paraformaldehyde, treated with Triton X-100, and processed for staining with the fluorochromes or for FISH. At both pH values, AO red fluorescence emitted mainly by the RNA-bound AO dimers is seen in nucleoli and the cytoplasm (a, b). In the same cells, the AO monomers emitting green fluorescence stain chromatin and, in addition, decorate nucleoli and the cytoplasm (a’, b’). The brightest FITC-signals are observed in nucleoli, and a weaker fluorescence is seen over the nucleoplasm and the cytoplasm (c). 18 S rRNA is detectable in nucleoli and the cytoplasm. Arrows – nucleoli; arrowheads – chromocenters. Bars, 10 μm.

Fig. 2. Mouse GV oocytes of NSN-type (a–c) and SN-type (d–) stained with AO (pH 7.2) (a, b, d and e), counterstained with Hoechst 33342 (c), and visualized under phase contrast (f). The oocytes were fixed with 3% paraformaldehyde for 30 min, treated with 0.5% Triton X-100 for 15 min, and stained with 8 μg/ml AO for 40 min. The oocytes were counterstained with 1 μg/ml Hoechst 33342 for 15 min before mounting to Vectashield. The AO red fluorescence corresponds mainly to RNA (a, d), and the AO green fluorescence corresponds to DNA (b, e). In oocytes of both types, the most intense red fluorescence is seen over NLBs. NLB – nucleolus-like body; nu – nucleoplasm; cyt – cytoplasm; arrows – chromatin. Bars, 10 μm.

Thus, the results of oocyte staining with AO reveal that RNA is a chemical constituent of NLBs in fully-grown mouse oocytes of both types. We did not observe any significant differences between the patterns of NLB staining with AO at neutral (7.2) and acidic (4.6) pH values.

Typical patterns of GV oocytes stained with FITC and counterstained with Hoechst 33342 are shown in Fig. 4 (n ¼22). In the NSN-oocyte (Fig. 4a and b) as well as in the SN-oocyte (Fig. 4c and d), FITC stains the “nucleoli” most brightly. If two NLBs are present

Please cite this article as: Shishova, K.V., et al., Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.11.022i

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Fig. 3. Analysis of AO binding ssRNA (red fluorescence) and dsRNA (green fluorescence) in NLBs of GV oocytes (SN-type oocytes are shown as examples). The oocytes were fixed as described in the legend to Fig. 2. Oocytes of the control group were incubated in PBS (a–c, c’), and oocytes of the experimental group were incubated in PBS containing 1 μg/ml RNase A (d–f, f’) for 1 h. Oocytes of both groups were stained with AO (8 μg/ml, 40 min, pH 7.2), mounted in Vectashield, and their 8-bit digital images were acquired using the same image acquisition parameters. The pixel fluorescence intensities in the red channel (for ssRNA) and the green channel (for dsRNA) were measured along the lines drawn on the images (c’, f’) or inside a ROI (g, h) and displayed as 0–255 grayscale values. (c’, f’); vertical axes, pixel fluorescence intensities; horizontal axes, distance (in mm) from the starting point along the lines shown in (c, f). (g, h) Bar graphs illustrating average pixel intensities and corresponding standard deviations in a ROI that were quantified in the control (n¼ 3) and RNase A-treated (n ¼5) oocytes. In each group, Z 50 measurements were performed in every ROI. The average fluorescence intensity/pixel of the NLB mass in the control and treated oocytes differs statistically significantly (t-test, p o 0.001) in the red channel, but it does not differ in the green channel. NLB – nucleolus-like body; nu – the nucleoplasm; ROI – region of interest; background – ROI outside the oocytes. Bars, 10 μm.

in a nucleus, both are stained, although a smaller NLB is often stained more weakly than a larger one (Fig. 4c). To assay the specificity of FITC binding with proteins, oocytes were incubated with 1 mg/ml proteinase K in PBS for 20 min before exposure to the dye; control oocytes were kept in PBS during the same time period (n¼ 11). Fig. 5a and d shows that proteinase K almost completely abolishes FITC fluorescence in the cytoplasm and the nucleoplasm and significantly (more than two-fold) reduces the fluorescence of NLBs as evidenced by quantification of the pixel intensities along the lines drawn on the images (Fig. 5c and f). The average pixel intensity in NLBs was 104.7 75.1 in the control oocytes and 48.23 74.33 in the oocytes treated with proteinase K (Fig. 5g). In the oocytes of both groups, the average NLB fluorescence intensities were significantly higher than the background fluorescence (po 0.001). Strikingly, the attenuation of NLB fluorescence was not accompanied by changes in the NLB optical density or size (Fig. 5b and e). We failed to abolish NLB fluorescence completely: a longer (more than 90 min) incubation of oocytes with proteinase K significantly increased their adhesiveness to substrates (capillaries and plastic dishes), which made impossible any manipulations with the oocytes. The proteinase K-resistant fluorescence may be due to strong protein intermolecular cross-linkages caused by fixation.

FISH analysis of 18S and 28S rRNAs It is well known that in nucleoli active in ribosome synthesis the majority of RNA is represented by ribosomal RNA (Hernandez-Verdun et al., 2010; Cisterna and Biggiogera, 2010; Fig. 1d). Therefore, we decided first to test the assumption that the RNA comprising the NLB mass is rRNA. To accomplish this, we used FISH and Cy3-conjugated oligonucleotide probes targeting 18S and 28S rRNAs. NIH/3T3 fibroblasts hybridized with the probe to 18S rRNA (Fig. 1d) and a pre-antral oocyte hybridized with the probe to 28S rRNA (Fig. 6a and b) are shown as references. In both cases, FISH-signals are clearly seen within nucleoli. In the nucleolus of the pre-antral oocyte (Fig. 6a), 28S rRNA forms characteristic nucleolonema-like threads that have been described by electron microscopy (Chouinard 1971; Mirre et al., 1980). FISH-signals are also observed in the cytoplasm of the oocyte and in adjacent follicular cells (Fig. 6a). Similar results were obtained with the probe to 18S rRNA (data not shown). In contrast, under the same hybridization conditions 18S rRNA and 28S rRNA were not seen inside NLBs in the vast majority of NSN-oocytes (n¼57) and in all SN-oocytes (n¼65) examined. Only in 10% of NSN-oocytes NLBs hybridized with the probes (Fig. 6c, c’, k and k’). In approximately 40% of the oocytes, the FISH-signals were seen at the NLB periphery, but they were absent from the NLB interior (Fig. 6e and m). In half of the

Please cite this article as: Shishova, K.V., et al., Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.11.022i

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Fig. 4. NSN-type (a, b) and the SN-type (c, d) GV oocytes stained with FITC (a, c) and Hoechst 33342 (b, d). The oocytes were fixed as described in the legend to Fig. 2, stained with 1 μg/ml FITC for 2 h, and counterstained with 1 μg/ml Hoechst 33342 for 15 min before mounting in Vectashield. Nucleolus-like bodies (NLBs) are stained most brightly; a weaker fluorescence is detected in the cytoplasm (cyt) of both oocytes and in the nucleoplasm (nu) of the NSN-oocyte. The arrows indicate chromocenters (b) and the chromosomal rim at the NLB surface (d). Bars, 10 μm.

NSN-type oocytes, the NLBs did not hybridize with the probes to 18S or 28S rRNA (Fig. 6g and o). We also failed to detect rRNA within NLBs of all examined SN-oocytes (Fig. 6i and q), although several modifications of the standard FISH conditions (longer hybridization time, higher concentration of the probes in hybridization mix) and of stringent washing conditions were attempted. We also failed to increase the efficacy of the rRNA detection by applying the protocol recommended for FISH-detection of mRNA in NLBs of mouse GV oocyte that includes the oocyte treatment with proteinase K before hybridization (Flemr and Svoboda, 2011). In the control, when the sense probes to 18S or 28S rRNA were used, no FISH-signals were observed (data not shown). Therefore, summarizing these results, we assumed that during oocyte maturation their “nucleoli” gradually loose rRNA. Immunolabeling of oocytes with antibodies to key nucleolar and nuclear proteins Staining of fully-grown oocytes with FITC suggests that the NLB mass contains proteins (Fig. 4a and c). However, no nucleolar protein has been detected within the NLB mass by conventional immunocytochemistry (Zatsepina et al., 2000; Bjerregaarde et al. 2004; Romanova et al., 2006; Maddox-Hyttel et al. 2007). Similar observations were made in the current work using antibodies to UBF (Fig. 7a), fibrillarin (Fig. 7g), NPM1 (Fig. 8a), nucleolin (Fig. 9a), or the large ribosomal subunit RPL26 protein (Fig. 8g). It is know that in somatic nucleoli the binding of specific antibodies with such nucleolar proteins as fibrillarin and NPM1 can be prevented by their excess (Sheval et al., 2005; Svistunova et al., 2012), and that treatment of cells fixed with aldehyde

fixatives with proteases can facilitate antigen retrieval (D'Amico et al., 2009; Svistunova et al., 2012). Taking into account these data, in the current study we applied a protease digestion assay to examine putative presence of the nucleolar proteins in the NLB mass. The oocytes were fixed with paraformaldehyde and permeabilized with Triton X-100 under standard conditions (Zatsepina et al., 2000) and then exposed to 1 mg/ml proteinase K for 20 min–1.5 h. The most convincing results were obtained when the oocytes were preincubated with proteinase K for 40– 45 min at room temperature (22 1C). After a shorter treatment (15–20 min), oocyte labeling remained similar to that of untreated controls, while a longer incubation (80–90 min) led to the complete disappearance of immunosignals (data not shown). The antibodies used in the study are listed in Table 1, and the results obtained are shown in Figs. 7–9. In Fig. 7b and h, one can see that a mild treatment of oocytes with proteinase K (40–45 min) readily reveals UBF and fibrillarin within the NLB mass of NSN-oocytes, where locations of both proteins are reminiscent of those in somatic nucleoli: UBF is observed in numerous small dots (Fig. 8b), and fibrillarin forms discrete patches (Fig. 8h). However, in SN-oocytes the proteins become almost uniformly distributed over the NLBs (Fig. 7c and i). Proteinase K treatment also reveals NPM1 (Fig. 8b and c) and nucleolin (Fig. 9g and j) in the NLB mass, where their locations were also similar to those in somatic nucleoli (Zatsepina et al., 1997; Tajrishi et al., 2011). If two NLBs were present in an oocyte nucleus, both were labeled (Fig. 8b). Proteinase K completely removed NPM1 (Fig. 8b and c) and nucleolin (Fig. 9g and j) from the nucleoplasm which, in addition to the NLB surface, was the location of the protein in the control (Figs. 8a and 9a, d).

Please cite this article as: Shishova, K.V., et al., Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.11.022i

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Fig. 5. Analysis of the specificity of protein staining with FITC in GV oocytes. The oocytes were fixed and treated with detergent as described in the legend to Fig. 2. Oocytes of the control group were incubated in PBS (a–c), and oocytes of the experimental group were incubated in PBS containing 1 μg/ml proteinase K (d–f) for 20min before staining with 1 μg/ml FITC for 2 h and mounting in Vectashield. (b, e) phase-contrast images. The pixel fluorescence intensities (c, f) were measured along the lines drawn on the 8-bit digital oocyte images or in regions of interest (ROI) (g) and displayed as 0–255 grayscale values. Images of the control and proteinase K-treated oocytes were acquired using the same image acquisition parameters. (c, f) Vertical axes, pixel fluorescence intensities; horizontal axes, distance (in mm) from the starting point along the lines shown in (a, d). (g) Columns and small vertical bars, average pixel intensities and standard deviations, correspondingly, in the control (n¼ 3) and the proteinase K-treated (n ¼4) oocytes. In each group, Z 50 measurements were performed over a ROI. The pixel intensities in the control and the proteinase K-treated NLBs differ statistically significantly (t-test, po 0.001). NLB – nucleolus-like body; nu – nucleoplasm; cyt –cytoplasm; background – ROI outside the oocytes. Bar, 10 μm.

Unlike NPM1 and nucleolin, the RPL26 protein was clearly observed only within NLBs of the NSN-type oocytes (Fig. 8h) but was barely visible in the NLBs of SN-oocytes (Fig. 8i). To examine whether NLBs can also contain nuclear proteins, we immunolabeled proteinase K-treated oocytes with antibodies to the nuclear splicing factor SC35, NOBOX (essential for folliculogenesis and regulation of oocyte-specific genes; Belli et al., 2013), topoisomerase II beta (involved in RNA transcription and DNA replication; Chen et al., 2013), heterochromatin protein HP1α (involved in regulation of gene transcription; Canzio et al., 2014), and the core nucleohistone H3 (Table 1). All these proteins except for SC35 and NOBOX have been reported in the proteome of mouse somatic nucleoli (Kar et al., 2011). However, we have not been able to detect any of these proteins in the NLB mass of GV oocytes (n¼ 72). Fig. 9 shows typical oocytes simultaneously immunolabeled for SC35 (b, e, h and k) and nucleolin (a, d, g and j) in the control (a–f) and following treatment with proteinase K (g–l). In proteinase K-treated oocytes, only nucleolin is clearly detected within NLBs (Fig. 9g and j).

proteinase K pretreatment (n¼15). As expected, in the control BrUrRNA (Fig. 10b) and fibrillarin (Fig. 10c) were seen only at the NLB periphery, where the signals were virtually colocalized (Fig. 10d). After oocyte exposure to proteinase K for 40–45 min, distinct fibrillarin foci become seen inside NLBs (Fig. 10g), but the BrUTP signals remain located solely at the NLB surface (Fig. 10f). A longer incubation of oocytes with proteinase K (80–90 min) caused a partial disappearance of the fibrillarin patches, so that only small fibrillarin foci were seen in the NLB mass (Fig. 10k). However, even under these conditions no incorporation of BrUTP into the NLB mass was observed (Fig. 10j). NLBs of the SN-type oocytes incorporated BrUTP neither in the control (data not shown, Bouniol-Baly et al., 1999; Zatsepina et al., 2000) nor after proteinase K treatment (Fig. 10m), while fibrillarin was easily detected inside NLBs in the latter case (Fig. 10n). These observations suggest that, regardless of the fibrillarin and UBF topology, in NSN-oocytes transcribed ribosomal genes are most likely present only at the NLB surface.

Discussion BrUTP labeling of oocytes The active nucleoli-like distribution of UBF (Fig. 7b) and fibrillarin (Fig. 7h) in the NLBs of the NSN-oocytes contradicts the literature data on the absence of transcribed ribosomal genes in the NLB mass (Bouniol-Baly et al., 1999; Zatsepina et al., 2000; Longo et al., 2003). To examine whether, similar to the nucleolar proteins, the transcribed rDNA can also be unmasked by a mild proteinase K treatment of GV oocytes, we applied a BrUTP microinjection assay (Zatsepina et al., 2000). The oocytes were fixed, exposed to 1 mg/ml proteinase K, simultaneously immunolabeled with antibodies to BrdU and fibrillarin, and counterstained with Hoechst 33342 to determine the oocyte type (n¼19). Control oocytes were processed for immunolabeling without

Results of this study allow us to make several major conclusions: (1) NLBs of fully-grown mouse oocytes contain RNAs as judged by their positive staining with the RNA-binding dye acridine orange. (2) NLBs contain proteins as demonstrated by their bright staining with the protein-binding dye FITC. (3) NLBs contain the nucleolar proteins that are involved in the main steps of ribosome biogenesis, including rDNA transcription (UBF), early rRNA processing (fibrillarin), late rRNA processing (NPM1 and nucleolin), and ribosome assembly (RPL26). (4) NLBs are impoverished for 18S and 28S rRNAs and apparently for proteins with nuclear functions. (5) The biochemical composition of NLBs in fully-grown mouse oocytes of the NSN- and SN-types differs.

Please cite this article as: Shishova, K.V., et al., Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.11.022i

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Fig. 6. Fluorescence in situ hybridization of a pre-antral follicle (a, b), NSN-oocytes (c-h, c’, k–p, k’), and SN-oocytes (i, j, q, r) with Cy3-conjugated oligonucleotide probes targeting 28S rRNA (a, k, k’, m, o, q) or 18S rRNA (c, c’, e, g, i). (b, d, f, h, j, l, n, p, r), Hoechst 33342 staining. FISH signals are seen over the nucleolus (nuo), the cytoplasm (cyt), and in follicular cells (FC) surrounding the oocyte (a). Positive FISH-signals corresponding to 18S rRNA (c, c’) and 28S rRNA (k, k’) were observed in the NLB mass of 10% of the NSN-type oocytes. In 40% of the NSN oocytes, FISH signals were seen at the periphery of NLBs but absent inside NLBs (e, m). No FISH signals were observed in 50% of the NSN-type oocytes (g, o) and in all the SN-type oocytes (i, q) examined. NLB – nucleolus-like body; nuo – nucleolus; cyt – cytoplasm; FC – follicular cells; arrows – local accumulation of rRNA in the NLB mass. Bars, 10 μm.

Please cite this article as: Shishova, K.V., et al., Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.11.022i

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Fig. 7. Immunolabeling of NSN-type oocytes (b, e, h, k) and SN-type oocytes (a, c, d, f, g, i, j, l) with antibodies against UBF (a–c) or fibrillarin (g–i) in control (a, g) and after incubation with 1 μg/ml proteinase K for 40–45 min (b, c, h, i,). Hoechst 33342 staining of the same oocytes is shown in (d–f, j–l). In the untreated control oocytes, both proteins are observed only at the NLB surface (a, g), while in the proteinase K-exposed oocytes the proteins are reveals in the NLB mass (b, c, h, i). In the proteinase K treated NSN-oocytes, they form discrete foci (b, h). Bar, 10 μm.

NLBs of fully-grown mouse oocytes contain RNA but are impoverished for rRNA Different assays were employed to visualize RNA within NLBs of fully-grown mammalian oocytes, but the results remain contradictory: some authors observed RNA within the NLB mass by autoradiography (Kopecny et al., 1995, 1996), whereas others were unable to detect it by cytochemical assays (Antoine et al., 1988). Austin and Bishop (1959)

were the first who applied AO to examine RNA- and DNA-compounds in living mammalian oocytes. However, in their study mainly green fluorescence corresponding to DNA was described in living rat eggs, while the AO-RNA red signals were fuzzy and unconvincing. Our observations, in general, support these early data: in living mouse GV oocytes the AO dimers were actively pumped in by cytoplasmic lysosomes, and a very weak, if any, AO red fluorescence was detected within NLBs (data not shown). In paraformaldehyde-fixed oocytes,

Please cite this article as: Shishova, K.V., et al., Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.11.022i

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Fig. 8. Immunolabeling of NSN-type oocytes (a, b, d, e, h, k) and SN-type oocytes (c, f, g, i, j, l) for NPM1 (nucleophosmin/B23) (a–c) or RLP26 (g–i) in control (a, g) and after incubation with 1 μg/ml proteinase K for 40–45 min (b, c, h, i,). The oocytes were counterstained with Hoechst 33342 (d–f, j–l) prior to mounting in Vectashield. In all control oocytes the proteins are not observed inside NLBs (a, g), while treatment with proteinase K reveals them in the NLB mass of NSN-oocytes (b, h). Within NLBs of SN-type oocytes, only NPM1 (c) but not RPL26 (i) is clearly seen. Bar, 10 μm.

which are routinely used in immunochemical and immunocytochemical studies, AO green fluorescence emitting by DNA-bound AO monomers perfectly colocalizes with the chromatin stained with the specific DNA-binding dye Hoechst 33342 (Fig. 2b and c). In the same oocytes, the AO red dimers, which have a high affinity to RNA, most brightly stained the NLB material (Fig. 2a and d). The sensitivity of the AO red fluorescence to RNase A (Fig. 3g) confirms the presence of RNA within the NLB mass. However, our attempts using the FISH technique failed to reveal 18S and 28S rRNAs in the NLB mass of the majority of NSN-oocytes

and in all SN-oocytes examined, despite the fact that under the same conditions the rRNAs were easily detected in active nucleoli of pre-antral oocytes (Fig. 6a) and in cultured mouse fibroblasts (Fig. 1d; Shishova et al., 2011). Taking into account that NLBs evolve from active nucleoli, which contain a large amount of 18S and 28S rRNAs, these observations were unexpected. We can propose two major explanations for these findings. The first is based on the knowledge that growing mammalian oocytes accumulate a huge amount of RNA (e.g., a fully-grown mouse oocyte contains about 0.5 ng RNA, that is 25–50 times greater than the amount of RNA per

Please cite this article as: Shishova, K.V., et al., Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.11.022i

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Fig. 9. Double immunolabeling of NSN-type oocytes (a–c, g–i) and SN-type oocytes (d–f, j–l) with antibodies against nucleolin (a, d, g, j) and SC35 (b, e, h, k) in control (a–f) and after incubation with 1 μg/ml proteinase K for 40–45 min (g–l). The oocytes were counterstained with Hoechst 33342 (c, f, i, l) prior to mounting in Vectashield. Nucleolin is revealed inside NLBs only in the proteinase-treated oocytes (g, j). SC35 is located within discrete patches in the control (b, e), is hardly visible within the NLB of the NSN-oocyte (h), and is absent in the NLB mass of the SN-oocyte (k). Bar, 10 μm.

somatic cell (Bachvarova, 1985; Olszanska and Borgul, 1993)). A fraction of this RNA, including rRNA, can be stored within NLBs. Being immersed into the tightly packed NLB material, the rRNA becomes inaccessible to hybridization probes. To “loosen” the NLB material, we treated GV oocytes with proteinase K according to the protocol described previously for FISH detection of mRNA (Flemr and Svoboda, 2011). However, this and other modifications of the standard FISH protocol we tested did not improve the efficacy of rRNA-FISH in GV oocytes. The impoverishment of NLBs for 18S and 28S rRNA was also supported by the lack of BrUTP incorporation into the NLBs under conventional conditions (Fig. 10b and d; Bouniol-Baly et al., 1999; Zatsepina et al., 2000) and after the oocyte treatment

with proteinase K (Fig. 10f–h, j and m). Therefore, we assume that NLBs of the majority of fully-grown mouse oocytes are indeed impoverished for rRNA, and the RNA detected within NLBs with AO may belong to other RNA classes, including nuclear RNAs. Indeed, the Sm antigen of nucleoplasmic small nuclear RNPs (snRNPs) and the methyl-3 guanosine (m3G) cap of snRNAs have been located within mouse NLBs by immunoelectron microscopy (Kopecny et al., 1996). Flemr and Svoboda (2011) showed that in mouse oocytes treated with proteinase K prior to FISH, the NLBs hybridized with a probe targeting dormant MOS kinase mRNA. The absence of rRNA within NLBs of SN-oocytes is indirectly supported by our observations on their depletion in ribosomal protein RPL26 (Fig. 8i) and is in line with

Please cite this article as: Shishova, K.V., et al., Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.11.022i

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Fig. 10. Immunodetection of BrU-labeled RNA (b, f, j, m) and fibrillarin (c, g, k, n) in NSN-type oocytes (a–k, d, h) and in SN-type oocytes (l–n) in control (a–d) and after treatment with 1 μg/ml proteinase K for 40–45 min (e–h, l–n) or 80–90 min (i–k). Hoechst 33342 staining is shown in (a, e, i, l). BrUTP incorporation is observed only at the surface of the NSN-type NLBs in both the control (b) and the proteinase K-treated oocytes (f, j). BrUTP sites are well colocalized with fibrillarin in the control (d) and are poorly colocalized with fibrillarin in the proteinase K-treated oocytes (h). Prolonged treatment with proteinase K leads to disappearance of the majority of fibrillarin foci (k), but no BrU-RNA is revealed within NLBs (j). No incorporation of BrUTP is observed in the SN-oocytes (m), although they contain fibrillarin-positive NLB (n). NLB – nucleoluslike body; arrowheads and red in (h) – BrUTP incorporation sites; arrows and green in (d) – fibrillarin foci; asterisks – sites of BrUTP and fibrillarin colocalization. Bars, 10 μm.

the idea that in mammalian oocytes maternally-derived ribosomes are mainly stored in cytoplasmic lattices (Yurttas et al., 2008; Monti et al., 2013). Thereby, the results summarized above indicate that the NLBs of fully-grown mammalian oocytes contain RNA, but this RNA is most likely not ribosomal RNA. NLBs contain nucleolar proteins that are involved in the main steps of ribosome biogenesis In contrast to rRNA, NLBs of GV oocytes accumulate a vast amount of protein, as judged by their intense staining with the protein-binding

dye FITC (Figs. 4 and 5) and by positive immunolabeling with antinucleolar antibodies achieved after mild treatment with proteinase K (Figs. 7–9). Our observations are in line with the results of a recent study by Fulka and Langerova (2014) where another approach for the retrieval NLB antigens was applied. Both studies show that the NLB mass contain the nucleolar proteins involved in the key stages of ribosome biogenesis: UBF (a specific nucleolar cofactor of RNA polymerase I), fibrillarin (an early rRNA processing factor and a snoRNP member), NPM1 (a late rRNA processing factor that is involved in the assembly of ribosomal particles), nucleolin (it plays roles at different stages of rRNA processing), and the ribosomal protein

Please cite this article as: Shishova, K.V., et al., Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.11.022i

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Table 2 Biochemical composition of NLBs in fully-grown mouse oocytes of the NSN- and SN-types as evidenced by results of the current work and literature data. NSNtype

SNtype

Reference

UBF

þ

þ

Fibrillarin

þ

þ

NPM1 (B23/ nucleophosmin) Nucleolin (C23)

þ

þ

þ

þ

RPL26 SC35 NOBOX Topoisomerase II beta HP1α H3 Nucleoplasmin 2 (NPM2)

þ þ /–     þ*

     

c.w., Fulka and Langerova, 2014* c.w., Fulka and Langerova, 2014* c.w., Fulka and Langerova, 2014* c.w., Fulka and Langerova, 2014* c.w. c.w. c.w. c.w. c.w. c.w. Inoue and Aoki (2010)

þ/ þ/ þ* þ*

 

Proteins

RNAs 18S rRNA 28S rRNA snRNA mRNA (MOS kinase mRNA)

c.w. c.w. Kopecny et al. (1996) Flemr and Svoboda, 2011

þ prominent (strong) signals irrespective of their localization; þ /  , faint signals;  , not detected. n

The oocyte type has not been defined; c.w., current work.

RPL26 (Table 2). However, we failed to observe within NLBs nuclear proteins that regulate transcription of Polymerase II-genes (NOBOX, topoisomerase II beta, HP1α), mRNA splicing (SC35), or play a structural role (histone H3) (Tables 1 and 2). To the best of our knowledge, only nucleoplasmin 2, a specific oocyte nuclear protein, has been located within NLBs of mouse oocytes by expression of EGFPfusions (Inoue and Aoki, 2010). Overall, these data suggest the idea that NLBs primarily accumulate the nucleolar proteins. Immunoblot analysis showed that some of them (UBF, RPA116, and fibrillarin) become degraded by the MII stage or during the first embryonic cell cycle (Zatsepina et al., 2000, 2003; Fulka and Langerova, 2014). However, detectable amounts of the maternally derived fibrillarin, NPM1, and nucleolin have been described in pronuclei and NPBs of zygotic embryos (Zatsepina et al., 2003; Romanova et al., 2006; Fulka and Langerova, 2014). These and other maternally-derived nucleolar proteins apparently play multiple roles in early embryonal development: they can be utilized for ribosome production until full reactivation of the embryonal genome that generally occurs at the morula/blastocyst stage (Wang et al., 2004; Hamatani et al., 2004; Zeng et al., 2004; Hamatani et al., 2008). The nucleolar proteins are involved in maintenance of the integrity and expression of satellite DNA and in completion of the zygotic stage (Fulka and Langerova, 2014). The presence of common proteins between nucleoli of growing oocytes and NLBs of GV oocytes could explain why the growing oocyte nucleoli support the development of enucleolated GV oocytes (Kyogoku et al., 2014). Interestingly, localization of some nucleolar proteins in the NLB mass of NSN- and SN-oocytes differs. For example, in NSN-type oocytes the UBF (Fig. 7b) and fibrillarin (Fig. 7h) form local aggregates that are characteristic of transcriptionally active nucleoli. However, neither literature data (Bouniol-Baly et al., 1999; Zatsepina et al., 2000; Pesty et al., 2007) nor our current observations indicate the presence of transcribed ribosomal genes in the NLB mass (Fig. 10b, d, f, h, j and m). Our results also strengthen the idea that active NLBs of NSN-type oocytes are more heterogeneous in biochemical composition than the silent NLBs of the SN-oocytes. Indeed, only a portion of NLBs in the NSN-oocytes (Fig. 6g and o), but all NLBs

67 in the SN-oocytes (Fig. 6i and q), did not hybridize with probes to 18S 68 and 28S rRNAs. A loss of rRNA from the NLB mass may be an 69 additional feature of developing mouse oocytes along with changes 70 in their transcriptome profile (Kageyama et al. 2007; Monti et al., 71 2013). 72 Taking into account results of the current study and the literature 73 data, we conclude that a function of NLBs in fully-grown mamma74 lian oocytes is sequestering nucleolar proteins temporally excluded 75 from the ribosome assembly or the dormant nucleolar proteins. 76 NLBs in preovulatory oocytes are impoverished for rRNA, but they 77 may contain mRNA and snRNA. A mild treatment of GV oocytes 78 with proteinase K that removes some NLB material retrieves 79 antigenic protein determinants that are normally hidden. A partial 80 biochemical composition of NLBs in fully-grown mouse oocytes of the NSN- and SN-types available today is summarized in Table 2. Q3 81 82 83 Uncited references Q4 84 85 (Schultz, 2002; Wang et al., 2010). 86 87 88 Acknowledgments 89 90 This study was funded by the Russian Scientific Foundation 91 (Grant 14-34-00033). We gratefully acknowledge the support of 92 bilateral scientific exchanges between the Russian and Polish 93 Academies of Sciences. We sincerely thank Miss M. Kordon 94 (Jagiellonian University, Krakow, Poland) for her help with pre95 liminary experiments using acridine orange and Dr. Yuri Khodor96 ovich (Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry 97 Russian Academy of Sciences, Moscow) for help with BrUTP 98 microinjections. 99 100 101 References 102 103 Antoine, N., Lepoint, A., Baeckeland, E., Goessens, G., 1988. Ultrastructural cytochemistry of the nucleolus in rat oocytes at the end of the folliculogenesis. 104 Histochemistry 89, 221–226. 105 Antoine, N., Thiry, M., Goessens, G., 1989. Ultrastructural and cytochemical studies 106 on extranucleolar bodies in rat oocytes at preovulatory follicle stage. Biol. Cell 65, 61–66. 107 Austin, C.R., Bishop, M.W.H., 1959. Differential fluorescence in living rat eggs 108 treated with acridine orange. Exp. Cell Res. 17, 35–43. 109 Bachvarova, R., 1985. Gene expression during oogenesis and oocyte development in mammals. Dev. Biol. 1, 453–524. 110 Belli, M., Cimadomo, D., Merico, V., Redi, C.A., Garagna, S., Zuccotti, M., 2013. The 111 NOBOX protein becomes undetectable in developmentally competent antral 112 and ovulated oocytes. Int. J. Dev. Biol. 57, 35–39. Bellone, M., Zuccotti, M., Redi, C.A., Garagna, S., 2009. The position of the germinal 113 vesicle and the chromatin organization together provide a marker of the 114 developmental competence of mouse antral oocytes. Reproduction 138, 115 639–643. Bernas, T., Asem, E.K., Robinson, J.P., Cook, P.R., Dobrucki, J.W., 2005. Photochem. 116 Photobiol. 81, 960–969. 117 Biggiogera, M., Martin, T.E., Gordon, J., Amalric, F., Fakan, S., 1994. Physiologically 118 inactive nucleoli contain nucleoplasmic ribonucleoproteins: immunoelectron 119 microscopy of mouse spermatids and early embryos. Exp. Cell Res. 213, 55–63. Bjerregaarde, B., Wrenzycki, C., Philimonenko, V.V., Hozak, P., Laurincik, J., Niemann, 120 H., Motlik, J., Maddox-Hyttel, P., 2004. Regulation of ribosomal RNA synthesis 121 during the final phases of porcine oocyte growth. Biol. Reprod. 70, 925–935. 122 Boisvert, F., Lam, Y.W., Lamont, D., Lamond, A.I., 2010. A quantitative proteomics analysis of subcellular proteome localization and changes induced by DNA 123 damage. Mol. Cell. Proteomics 9, 457–470. 124 Bouniol-Baly, C., Hamraoui, L., Guibert, J., Beaujean, N., Szöllösi, M.S., Debey, P., 125 1999. Differential transcriptional activity associated to chromatin configuration in fully-grown GV mouse oocytes. Biol. Reprod. 60, 580–587. 126 Canzio, D., Larson, A., Narlikar, G.J., 2014. Mechanisms of functional promiscuity by 127 HP1 proteins. Trends Cell Biol. 24, 377–386. 128 Chen, S.H., Chan, N.L., Hsieh, T.S., 2013. New mechanistic and functional insights into DNA topoisomerases. Annu. Rev. Biochem. 82, 139–170. 129 Chouinard, L.A., 1971. A light- and electron-microscopic study of the nucleolus 130 during growth of the oocyte in the prepubertal mouse. J. Cell Sci. 9, 637–663. 131 Cisterna, B., Biggiogera, M., 2010. Ribosome biogenesis: from structure to dynamics. Int. Rev. Cell Mol. Biol. 284, 67–111. 132

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