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BOR - Papers in Press, published online ahead of print November 12, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.018408
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BIOLOGY OF REPRODUCTION 70, 877–886 (2004)
DOI: 10.1095/biolreprod.103.018408
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Immunolocalization of Upstream Binding Factor and Pocket Protein p130 During Final Stages of Bovine Oocyte Growth1

Vladimir Baran3,4, Antonin Pavlok4, Bolette Bjerregaard5, Christine Wrenzycki6, Doris Hermann6, Vlada V. Philimonenko7, Georgios Lapathitis4, Pavel Hozak7, Heiner Niemann6, and Jan Motlik2,4

Institute of Animal Physiology,3 Slovak Academy of Sciences, 040 01Kosice, Slovakia Institute of Animal Physiology and Genetics,4 Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech Republic Department of Anatomy and Physiology,5 The Royal Veterinary and Agricultural University, Copenhagen, Denmark Department of Biotechnology,6 Institute for Animal Science (FAL), Mariensee, D-31535 Neustadt, Germany Institute of Experimental Medicine,7 Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of this study was to describe the dynamic changes in the localization of the key nucleolar protein markers, fibrillarin, B23/nucleophosmin, C23/nucleolin, protein Nopp140, during the final stages of bovine oocyte growth. All these proteins were present in the large reticulated nucleoli of oocytes from the small-size category follicles (<1 mm). The entire nucleolus exhibited strong positivity for UBF (upstream binding factor, RNA polymerase I-specific transcription initiation factor), which displayed a dotted staining pattern. In contrast, protein p130 was diffusely distributed throughout the nucleus and excluded from nucleoli. In oocytes approaching the late period of growth (2–3-mm follicles), UBF localization shifted to the nucleolar periphery. Double staining of UBF-p130 revealed a gradual accumulation of p130 at the periphery shell around the nucleolus. In fully grown oocytes (>3-mm follicles), all studied nucleolar proteins were detected in the small compact nucleoli. The cap structure, attached to the compact nucleolus surface, was positive for UBF and PAF53 (subunit of RNA polymerase I). The UBF-positive cap showed a close structural association with p130. It is concluded that, during the process of oocyte nucleolus compaction, UBF and PAF53, proteins involved in the rDNA transcription, are segregated from fibrillarin and Nopp140, proteins essential for early steps of pre-rRNA processing. The observed changes may reflect the transition from pre-rRNA synthesis to pre-rRNA processing as an analysis of the relative abundance of the developmentally important gene transcripts confirmed. In addition, discovered structural association between UBF and p130 suggests a role for pocket proteins in ribosomal gene silencing in mammalian oocytes.

gamete biology, meiosis, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian oocytes of primordial follicles are arrested in the prophase of meiosis I [1]. During the onset of follicle growth in the primary follicle, the flattened pregranulosa cells become cuboidal and begin to proliferate, and the enclosed oocyte begins to grow. In bovine primordial and primary follicles, the diameter of oocytes ranges between 25 and 30 µm [2, 3] and it increases to 90 µm in small antral follicles [4]. In the growing oocyte, organelles such as mitochondria, Golgi complex, lipid droplets, and vesicles increase in number, relocate to the cell periphery, and gap junctions between an oocyte and granulosa cells are formed [5]. At the end of the growth period, the endoplasmic reticulum decreases, the nucleus migrates to the periphery, and the follicle/oocyte size reflects a developmental competence of the oocyte [6, 7].

The nucleolus of the bovine oocyte is activated in the secondary follicle [8] following progressive increase of activity connected with characteristic changes in the nucleolar fine structure [9, 10]. At the tertiary follicle stage, the oocyte is active in transcription and growth. Bovine oocytes >110 µm in diameter from follicles >3 mm become fully competent to complete meiosis followed by embryonic development. Five types of the bovine oocyte nucleolus are distinguished during the course of tertiary follicle development on the basis of the ultrastructural description. As the oocyte enters the final growth stage, nucleolar transcription activity gradually decreases. Fair et al. [8] have shown that nucleolar morphological changes are associated with migration of nucleolar fibrillar centers. This is characterized morphologically by extrusion of the granular component and the gradual compaction of the nucleolus into a dense fibrillar sphere surrounded by loosely arranged material (halo), referred to as capped lentiform fibrillar centers or cap structure [7]. These nucleolar remnants are present in nuclei of the bovine oocyte at the germinal vesicle stage when ribosome biosynthesis has completely ceased (for a review, see [11]). Taking into consideration the morphological characteristics of the nucleolus during down-regulation of rRNA synthesis, further studies focused on functional morphology aspects of nucleolar activity are necessary.

The nucleolus is the most prominent nuclear organelle. Although recently it has been implicated in a variety of processes, such as cell cycle control [12], nuclear protein export [13], cell aging [14], cell growth [15], and probably translation of some ribonucleoproteins [16], the main function of the nucleolus is ribosomal biogenesis. Transcription of ribosomal genes, rRNA processing, and formation of preribosome particles are spatially organized within the nucleolar structure. Morphologically, the activated fibrillogranular nucleolus of the secondary follicle oocyte possesses three basic subcomponents—fibrillar centers (FCs), a dense fibrillar component (DFC), and a granular component (GC). The relative sizes and configuration of individual compartments can vary greatly depending on the cell type and metabolic state of the cell [17, 18]. The active nucleolus represents a functional alliance of a rDNA transcription complex and a pre-rRNA processing complex containing specific proteins, participating in the various steps of ribosomal biogenesis. The primary assembly of the rDNA transcription complex is dependent on two basic factors (SL-1 and upstream binding factor [UBF]) binding to the rDNA promotor [19]. The process is controlled by specific cyclin-dependent kinases [20] followed immediately by association with RNA polymerase I [21]. UBF converts the rDNA chromatin into a transcriptionally competent form [22] after its rephosphorylation by the cdk kinases [23]. The nascent pre-rDNA molecules are immediately associated with proteins involved in a pre-rDNA processing complex containing fibrillarin, C23/nucleolin, B23/nucleophosmin, Nopp140, and others taking part in preribosome assembly machinery. The compartmentalization of specific nucleolar proteins within a nucleolar body reflects the actual status of ribosomal metabolism in the course of the cell cycle. All nucleolar subdomains are organized around rDNA gene repeats (nucleolar organizing regions—NORs) which are anchored in the FC. The FCs are intended to be storage sites for RNA polymerase I [24–26]. Transcription of rDNA was shown to be located in the FC/DFC border (for a review, see [27, 28]). Early steps of rRNA processing also take place in DFC, while in GC, the later stages of maturing ribosomal particles are accumulated [29, 30]. Well-defined nucleolar protein markers (fibrillarin, C23/nucleolin, B23/nucleophosmin), transcription factors (UBF, TIF-1B/SL1), and splicing components of small nucleolar ribonucleoprotein particles (snoRNP) complexes have been shown to be involved in chromatin remodeling at the promotor region level, followed by association of RNA polymerase I to form a stable preinitiation complex [19]. Fibrillarin, C23/nucleolin, and B23/nucleophosmin are also required for rRNA processing. Taken together, the nucleolar proteome seems to be very dynamic in response to the claims of the cell metabolic state [31].

The retinoblastoma family of pocket proteins includes the tumor suppressor pRb and related p107 and p130 proteins [32]. The pocket proteins implement their function through interaction with transcription factors, such as E2F1-4 (pRb), E2F4, and E2F4 (p107 and p130) [33]. Moreover, it was discovered that pRb-mediated repression of rDNA transcription includes interaction with the RNA polymerase I transcription factor UBF [34]. Additionally, pRb has been shown to regulate also most genes transcribed by RNA polymerase III [35].

The aim of this study was to describe the dynamic changes in the localization of key nucleolar protein markers of the transcription complex (UBF and RNA polymerase I), the pre-rDNA processing complex (fibrillarin, Nopp140, B23/nucleophosmin, and C23/nucleolin), and a pocket protein (p130) during the final stages of bovine oocyte growth. The level of expression of genes coding for UBF, RNA polymerase I, Poly A, and Hsp70.1 was estimated by the relative abundance of respective mRNAs.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Follicles/Oocytes Collection and Fixation

Bovine ovaries were collected at a local slaughterhouse and within 1 h transferred to the laboratory in physiological saline at 25–30°C, rinsed briefly in ethyl alcohol, and washed twice in PBS. The follicles were dissected, classified into three size categories: small (<1 mm), middle (2–3 mm), and large (>3 mm) and separately placed in the basic culture medium [36]. Approximately 250 cumulus oocyte complexes were isolated per each category, their compact cumuli were removed mechanically, and 50 cumulus-free oocytes per category were used for reverse transcription-polymerase chain reaction (RT-PCR). The diameter of follicles were measured with an ocular micrometer at 200x magnification immediately after isolation. In other oocytes, the zona pellucida was very gently dissolved by controlled incubation in the medium supplemented with 0.2% pronase at 39°C. After washing in PBS, zona-free oocytes were fixed in 3.7% paraformaldehyde in PBS at 4°C for 50–60 min.

Immunofluorescence

Fixed oocytes were washed in PBS and free aldehydic groups were blocked with 50 mM ammonium chloride in PBS. After washing in PBS containing 0.25% bovine serum albumin (PBS/BSA), the oocytes were permeabilized with 0.5% TritonX-100 for 15 min at room temperature and preincubated with inactivated 2% normal goat serum in PBS/BSA containing 0.05% saponine (PBS/BSA-sap) for 10–15 h at 4°C. The PBS/BSA solution supplemented with saponine was used in each step up to the final embedding in Mowiol. The oocytes were incubated with primary antibodies diluted in PBS/BSA-sap overnight at 4°C and, after exhaustive washing with affinity-purified secondary antibodies, were coupled with fluorescent dye (FITC or TRITC) for 60 min at room temperature. For double-staining experiments, both primary antibodies were employed simultaneously overnight at 4°C followed by detection of antibody-binding sites with respective immuno-conjugates applied separately. Controls of immunostaining specificity were carried out by omitting primary antibodies or using another species-specific secondary antibody conjugate. After the final washing in PBS, the oocytes were mounted in Mowiol and examined with a confocal laser scanning microscope Leica TCS SP (Leica Microsystems AG, Wetzlar, Germany). Image files were edited with Corel Draw computer software (Corel Corporation, Ottawa, ON, Canada).

Antibodies

A UBF was detected by human autoimmune serum B15 containing antibodies against the RNA pol I transcription factor UBF and rabbit polyclonal serum against the PAF53, subunit of RNA polymerase I. The fibrillarin-nucleolar protein marker of DFC was immunostained with mouse monoclonal antibody AFB01 (Cytoskeleton Inc., Denver, CO) or human autoimmune serum S4. For immunodetection of B23/nucleophosmin, the mouse serum containing a monoclonal antibody against the protein marker of GC was used. The C23/nucleolin-protein marker mainly of DFC was detected by mouse monoclonal antibody 4E2 (Med and Biol Labs Co., Nagoya, Japan). Protein Nopp140—a component of ribonucleoprotein particles associated with pre-rDNA posttranscription processing—was immunostained by rabbit serum RF12 containing polyclonal antibodies against the protein. Protein p130—a member of the pocket protein family—was detected by mouse monoclonal antibody DSC211.6 or rabbit polyclonal antibody sc-317 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For clarification, the list of applied antibodies is given in Table 1. About 20 oocytes from the three follicular categories were used per each single and double immunostaining on the light-microscopy level.


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  		TABLE 1. Antibodies used for immunostaining

RNA Isolation and RT-PCR

After washing three times in PBS containing 0.1% polyvinyl alcohol, cumulus-free oocytes were stored individually at -80°C in a minimum volume (5 µl or less) of medium while awaiting experimental use. Poly(A)+RNA was isolated from single oocytes, as was described recently [30], and used immediately for reverse transcription, carried out in a whole volume of 20 µl using 2.5 µM random hexamers (Perkin-Elmer, Vaterstetten, Germany). Prior to RNA isolation, 1 pg of rabbit globin RNA (BRL, Gaithersburg, MD) was added as an internal standard. The reaction mixture consisted of 1x RT buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3; Perkin-Elmer), 5 mM MgCl2, 1 mM of each dNTP (Amersham, Brunswick, Germany), 20 IU RNase inhibitor (Perkin-Elmer), and 50 IU MuLV reverse transcriptase (Perkin-Elmer). The mixture was overlaid with mineral oil to prevent evaporation.

The RT reaction was carried out at 25°C for 10 min, 42°C for 1 h, followed by denaturation at 99°C for 5 min, then flash cooling on ice. PCR was achieved with cDNA equivalents (as described in Table 2) from oocytes of different follicle sizes as well as 50 fg of globin RNA in a final volume of 50 µl of 1x PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl; Gibco BRL, Eggenstein, Germany), 1.5 mM MgCl2, 200 µM of each dNTP, 1 µM of each sequence-specific primer (globin: 0.5 µM) using a PTC-200 thermocycler (MJ Research, Watertown, MA). To ensure specific amplification, a hot start PCR was used by adding 1 IU Taq DNA polymerase (Gibco) at 72°C. PCR primers were designed from the coding regions of each gene sequence using the OLIGO program (Molecular Biology Insights Inc., Cascade, CO). The sequences of the primers used, the annealing temperatures, the fragment sizes and the sequence references are summarized in Table 2.


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  		TABLE 2. Primers used for PCR

The PCR program involved an initial step of 97°C for 2 min and 72°C for 2 min (hot start) followed by different cycle numbers of 15 sec each at 95°C for DNA denaturation, 15 sec at different temperatures for annealing of primers, and 15 sec at 72°C for primer extension. The last cycle was followed by a 5-min continuation at 72°C and cooling to 4°C. As negative controls, tubes were prepared in which RNA or reverse transcriptase was omitted during the RT reaction (data not shown).

The RT-PCR products were subjected to electrophoresis on a 2% agarose gel in 1x Tris-borate-EDTA buffer (90 mM Tris, 90 mM borate, 2 mM EDTA, pH 8.3) containing 0.2 µg/ml ethidium bromide. Additional ethidium bromide in the same concentration was added to the running buffer. The image of each gel was recorded using a charged coupled device camera (Quantix; Photometrics, München, Germany) and the IPLab Spectrum program (Scanalytics, Fairfax, VA). The intensity of each band was assessed by densitometry using an image analysis program (IPLab Gel Analysis; Scanalytics). The relative amount of the mRNA of interest was calculated by dividing the intensity of the band for each developmental stage by the intensity of the globin band for the corresponding stage. Experiments were repeated with at least eight oocytes for each mRNA.

For each pair of gene-specific primers, semilog plots of the fragment intensity as a function of cycle number were used to determine the range of cycle number over which linear amplification occurred, with the number of PCR cycles being kept within this range [37]. Because the absolute efficiency of amplification for each set of primers during each cycle is not known, such an assay can only be used to compare the relative abundance of one mRNA among different samples [38].

The number of replicates was calculated to get an acceptable ability to repeat the assay (0.90). The average repeatable precision of the assay varied from 0.60 to 0.70. Therefore, a minimum of 10 replicates was carried out.

Statistical Analysis

Relative abundance was analyzed using the SigmaStat 2.0 (Jandel Scientific, San Rafael, CA) software package. After testing for normality (Kolmogorov-Smirnov test with Lilliefor correction) and testing for equal variance (Levene Median test), an ANOVA test followed by multiple pairwise comparisons using the Tukey test was employed. Differences of P <= 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In oocytes of all size groups, the nuclei were located eccentrically or peripherally. Only one fifth of the oocytes from the small category assumed a central location of the germinal vesicle. A positive correlation between the oocyte diameter (<100 µm = small, 110–120 µm = middle, >120 µm = large) and the follicle size category (<1 mm = small, 2–3 mm = middle, > 3 mm = large) was observed in a pool of about 200 bovine tertiary follicles per each size category. The small and middle categories were attributed to growing oocytes and the large category to fully grown oocytes. The standard fluorescence pattern observed in the majority of evaluated oocytes in each category is presented.

Growing Bovine Oocytes

The nucleoli in oocytes of the small-size category were intensively immunostained for fibrillarin (Fig. 1a), B23/nucleophosmin (Fig. 1d), C23/nucleolin (Fig. 1g), and Nopp140 (not shown). The protein Nopp140 displayed the same staining pattern as for fibrillarin. The irregularly shaped nucleoli were large and exhibited a strong fluorescence signal in the whole nucleolus but the signal was not spread homogeneously.



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  		FIG. 1. Single immunocytochemical localization of the nucleolar protein in the bovine oocytes. Light microscopy single staining of fibrillarin (a–c), B23/nucleophosmin (d–f), C23/nucleolin (g–i) in different size categories of bovine oocyte—small (a, d, g), middle (b, e, h), and large (c, f, i). The fully reticulated nucleolus compacted to small nucleolar remnants with cap substructure (open arrows) attached to the compact nucleolar body (full arrows). The nuclear area (germinal vesicle) is free of immunosignal (double arrows). Bar = 20 µm

The oocytes of the middle-size category possessed more compacted nucleoli (smaller in diameter) in comparison with the small-size category of oocytes. Fibrillarin (Fig. 1b), Nopp140 (not shown), B23/nucleophosmin (Fig. 1e), and C23/nucleolin (Fig. 1h) were located in the nucleoli. In addition to the nucleolar staining, B23/nucleophosmin and C23/nucleolin were also detected in the nucleoplasm, displaying a light diffuse staining pattern.

The immunostaining for UBF (protein of the rDNA transcription complex) displayed a dotted staining pattern in the whole large nucleoli of oocytes from the small follicles (Figs. 2, a' and d', and 3e). In the middle-size oocyte category, UBF was located mainly on the periphery of the nucleolus (Figs. 2, b' and e', and 3f, open arrow). Double labeling of UBF and fibrillarin established more diffuse localization of fibrillarin (Fig. 2, a and b) in contrast with the dotted labeling pattern of UBF (Fig. 2, a' and b') in nucleoli of small (Fig. 2, a and a') and middle oocytes (Fig. 2, b and b'). This pattern fully reflected the role of these proteins in transcription and early posttranscriptional events in the reticulated nucleolus. While during gradual compaction of the nucleolus, UBF was shifted toward the nucleolar periphery (Figs. 2e' and 3f), fibrillarin remained in the compact nucleolar body. Double immunostaining of UBF (Fig. 2, d' and e') and PAF53 (Fig. 2, d and e) revealed their colocalization in fully active nucleolus of the small- (Fig. 2, d and d') as well as the middle- (Fig. 2, e and e') size category of growing follicles.



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  		FIG. 2. Double immunocytochemical localization of the nucleolar protein in the bovine oocytes. Light microscopy double staining of fibrillarin (a–c) with UBF (a', b', c') in bovine oocytes of small- (a, a'), middle- (b, b'), and large- (c, c') size category as well as double staining of PAF53 (d–f) with UBF (d', e', f') in bovine oocytes of small- (d, d'), middle- (e, e'), and large- (f, f') size categories. UBF and PAF53 are gradually marginalized in the periphery of the compacted nucleolus and finally localized in cap structure (open arrow) during compaction of the nucleolus. Bar = 20 µm. Additional magnification x3 (f,f' merge inset)



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  		FIG. 3. Immunocytochemical localization of the nucleolar proteins in the bovine fully grown oocytes and localization of p130 in growing and fully grown oocytes. Light-microscopy double staining of Nopp140 (a, b) with C23/nucleolin (a') or UBF (b') in bovine oocytes of large-size category. Double staining of fibrillarin (c, d) with p130 (c', d') in bovine oocytes of small- (c, c') and large- (d, d') size category. Double immunostaining of UBF (e–h) with p130 (e', f', g', h') in bovine oocytes of small- (e, e'), middle- (f, f', g, g'), and large- (h, h') size categories. Protein p130 is distributed through the whole nucleoplasm except for the nucleolar area in the small-size oocyte category. During the termination of oocyte growth (middle-size category), p130 gradually concentrates in the periphery shell of the nucleolus (full arrows). UBF is marginalized in the nucleolus and finally located in the cap structure (open arrows). The association of p130 and UBF is observed during the final stages of oocyte growth (merge of g and g'). Nopp140 is localized mainly in the cortex of nucleolar compact bodies. Double staining of Nopp140 with C23/nucleolin shows colocalization of these two proteins in the nucleolar body cortex. Double staining of Nopp140 with UBF suggests the presence of UBF in the cap substructure. Bar = 20 µm

Protein p130, a member of the pocket proteins family that regulates UBF activity, was observed to be diffusely distributed throughout the nucleus except for the nucleolar area in growing oocytes of the small category (Fig. 3, c' and e'). In all oocytes in the middle category, UBF was located at the nucleolar periphery (Fig. 3, f and g); however, immunostaining of p130 showed two staining patterns. In one half of oocytes, p130 was still diffusely distributed in nucleoplasm (Fig. 3f'), while in the second half of oocytes, the p130 signal was concentrated in few patches (Fig. 3g'). Double staining of UBF and p130 revealed a close structural association between these proteins (see merge of Fig. 3, g and g').

Fully Grown Bovine Oocytes

In oocytes isolated from follicles of the large-size category, the nucleolar proteins fibrillarin (Fig. 1c), B23/nucleophosmin (Fig. 1f), and C23/nucleolin (Fig. 1i) were detected in compact nucleoli, which were visible as small foci in nuclei (germinal vesicles). In addition, the weak signal for B23/nucleophosmin and C23/nucleolin were present throughout the whole nucleoplasm, displaying a similar staining pattern as in the middle-size category of oocytes. Protein Nopp140 was also detected in the compact nucleoli (Fig. 3a) but the immunosignal was accumulated mainly in the peripheral rim of the compact nucleoli. The absence of positivity in the central core was clearly evident on a single optical section in confocal microscopy (Fig. 3b). In addition, double staining of Nopp140 (Fig. 3a) and C23/nucleolin (Fig. 3a') showed colocalization of these two proteins in the nucleolar cortex.

In close apposition with the compact nucleolar body, the loosely arranged cap structure was seen (see inset of Fig. 1c, open arrows). This associated structure was only poorly stained with an antibody against fibrillarin or Nopp140 showing a picture of a shadowed halo. Moreover, the double staining of fibrillarin (Fig. 2c) and UBF (Fig. 2c') demonstrated that the fibrillarin-positive compact core is surrounded by the UBF-positive cap structure, in which UBF and PAF53 colocalized (Fig. 2, f and f'). This observation was also confirmed by double staining of Nopp140 and UBF because UBF was present in the cap structure and Nopp140 in the cortex of compact nucleoli (Fig. 3, b and b').

The double staining of fibrillarin (Fig. 3d) and p130 (Fig. 3d') as well as UBF (Fig. 3h) and p130 (Fig. 3h') illustrated the gradual accumulation of p130 around the compact nucleolus during the final period of oocyte growth.

These data proved that, during the process of oocyte nucleolus compaction, UBF and PAF53 proteins, involved in the rDNA transcription, are segregated from fibrillarin and Nopp140 proteins that are essential for nascent rRNAs processing. In addition, the close mutual structural association between UBF and p130 revealed a possible role of pocket proteins in ribosomal gene silencing in mammalian oocytes.

Determination of the Relative Abundance of Developmentally Important Gene Transcripts in Single Bovine Oocytes

A representative gel photograph of a semiquantitative RT-PCR analysis of RNA pol I, UBF, Hsp, Poly A, and the corresponding globin bands in oocytes from follicles of different size is shown in Figure 4. The expression pattern for Hsp and UBF transcripts was similar in oocytes isolated from all follicular categories (Fig. 5). However, RNA polymerase I expression was significantly higher in oocytes derived from small follicles, whereas the amount of Poly A transcripts significantly increased in oocytes from the middle and large follicular category.



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  		FIG. 4. Semiquantitative RT-PCR analysis of RNA polymerase I, UBF, Hsp, and Poly A transcripts in oocytes from small-, middle-, and large-size follicles and corresponding globin fragments



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  		FIG. 5. Effects of follicle size in the expression pattern of RNA polymerase I, UBF, Hsp, and Poly A transcripts in oocytes from small- (open bars), medium- (gray bars), and large- (black bars) size follicles


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Functional Architecture of the Nucleolus in Bovine Growing Oocytes

Nucleolar activity is initiated at the time of the secondary follicle stage and progressively increases during the period of oocyte growth correlated with characteristic changes in the nucleolar fine structure [8, 9]. The ultrastructural and autoradiographic studies [9, 10] documented that the growing bovine oocytes isolated from small antral follicles possess the fibrillogranular nucleoli actively involved in rRNA synthesis. The nucleoli are gradually compacted into inactive electron-dense fibrillar spheres [11] when the oocyte enters the final growth stage. Thus, the ultrastructural description of the dense fibrillar spheres resembles a compact nucleoli observed at the light-microscopy level. In this study, the immunostaining experiments revealed reticulated nucleoli in growing oocytes in the small- and middle-size categories of bovine follicles. These fully active nucleoli displayed large positive fluorescent patches diffusely stained with antibodies against fibrillarin, C23/nucleolin, B23/nucleophosmin, and Nopp140. At the light-microscopy level, the reticulated character of the nucleoli would be derived from the unhomogeneous appearance of the staining pattern. The UBF immunostaining pattern in the fully active nucleoli thus shows multiple sites of rDNA transcription activity scattered throughout the whole nucleolus during the growing stage of the oocyte. These sites represent FCs as documented by colocalization of UBF with PAF53, a subunit of RNA polymerase I.

Relocation of Oocyte Nucleolar Proteins During Nucleolus Compaction

The immunodetection of the nucleolar proteins confirm the results of Fair et al. [10] that the bovine oocyte nucleolus gradually compacts during the course of its tertiary follicle development. During the downregulation of rRNA synthesis in somatic cells (at the end of interphase or after the induced transcription arrest), the nucleolus disintegrates. Despite the absence of substrate for pre-rDNA processing, the rDNA transcription machinery remains assembled in mitotic NORs in opposition to the processing machinery relocated at the chromosomal periphery recruited in prenucleolar bodies [39]. We assume that functional aspects of rDNA transcription and processing machinery of pre-rDNA molecules are similar or nearly identical in both somatic cells and growing oocytes. However, the morphological features of nucleolar disintegration in somatic cells and oocyte/cleavage embryos are significantly different.

In the final stages of the oocyte growth period, the nucleolus compacts into transcriptionally inactive small, dense fibrillar spheres surrounded by cap structures that are composed of less dense material. The dense fibrillar sphere (nucleolar remnant body) of the fully grown mouse [40], pig [41], and cattle [9, 10] oocyte is composed of tightly packed fibrous material and lacks fibrillar centers. As shown in this study on fibrillarin, C23/nucleoli and B23/nucleophosmin immunostaining pattern, the creation of the compact nucleolar body is a gradual process during which C23/nucleolin and B23/nucleophosmin are extruded to the nucleoplasm except for small parts of the proteins remaining in the compact nucleolus body up to the germinal vesicle breakdown [42]. We demonstrate that fibrillarin occupies the round compact (residual) sphere surrounded by a cap structure containing UBF and PAF53. Thus, the double-staining experiments revealed segregation of these nucleolar protein markers. In addition, during the final stages of oocyte growth, protein Nopp140 accumulates in the cortical region of the compact sphere. In this cortical region, Nopp140 partially colocalizes with UBF and C23/nucleolin. The low immunosignals for Nopp140, C23/nucleolin, and fibrillarin observed in the cap structure are interpreted as residual amounts of the proteins during the gradual nucleolar segregation and compaction into the final nucleolar bodies (remnants) having a diameter of 1–2 µm.

Downregulation of rRNA transcription in a mouse oocyte was not associated with the detachment of the RNA polymerase I transcription complex from the nucleolar surface [43]. Also in serum-deprived or actinomycin D-treated cells, rDNA templates were redistributed in a few large perinucleolar clusters [44, 45], which retained the proteins of the transcription machinery [46]. The physiological block of transcription, which accompanies acquiring of meiotic competence in an oocyte, is also characterized by clustering the rDNA encoding genes and the appearance of large UBF/RNA polymerase I foci [43]. In current experiments, the compact nucleolus formation was first accompanied by the dispersion of nucleolin and nucleophosmin into the nucleoplasm; however, remnants of these proteins were detectable between the dense fibrillar sphere and its cap structure. In accordance with the mouse model, UBF was located exclusively in the nucleolar cap. But in contrast with the mouse nucleolus, where no visible amounts of fibrillarin and B23/nucleophosmin were present in the nucleolus-like body [43], fibrillarin and Nopp140 were still present in the cattle compact nucleoli, clearly segregated from UBF.

An altered relative abundance of transcripts was detected within the oocytes of different follicle size categories when looking at RNA polymerase I and Poly A mRNAs. However, UBF transcripts were not affected in their relative amounts. The decrease of RNA polymerase I and the increase of Poly A transcripts in oocytes from growing and fully grown follicles might be due to the transition from rRNA synthesis to pre-rDNA processing.

Involvement of Pocket Proteins in Downregulation of Nucleolar Transcription

Cavanaugh et al. [34] suggested that pRb-mediated repression of rDNA transcription includes interaction with the RNA polymerase I transcription factor UBF. The pRb, but not p107, acts as a transcription repressor by interfering with the assembly of the transcription initiation complex [23, 47, 48]. Also, the p130 protein, which possesses growth-restraining activity [48, 49], was found in a complex with UBF [50]. In accordance with this observation, the cellular content of p130 inversely correlated with the rate of rDNA transcription [50]. In a growing cattle oocyte, p130 was localized exclusively in nucleoplasm and it moved to a perinucleolar position during the compact nucleolus formation. Double immunostaining of UBF with p130 showed a close structural association between these proteins only in the nonactive nucleolar entity. This close association is probably in close correlation with downregulation of rRNA synthesis in a fully grown oocyte. Studies on somatic cells suggest that pocket proteins (involved in the regulation of the cell proliferation) block rRNA synthesis by direct interaction with the UBF [20]. Thus, localization of p130 in close proximity to compact nucleoli observed in a fully grown oocyte might suggest its function in the event of downregulation of rDNA transcription. In a fully grown oocyte, the p130 fluorescent signal was in close apposition with UBF localized in the nucleolar cap structure. These observations indicate that p130 does associate with the nucleolus during the final period of oocyte growth and interacts with UBF during decreasing of rDNA transcription in the fully grown mammalian oocytes.

Conclusion

We conclude that downregulation of the rRNA transcription in cattle oocytes is accompanied by the separation of proteins of the rDNA transcription (UBF, RNA polymerase I) and pre-rDNA processing (fibrillarin, Nopp140) complex (see Fig. 6). Upon the silencing of rRNA synthesis in fully grown oocytes, UBF and RNA polymerase I were relocalized to the cap structure, remaining as a lentiform [11] substructure located on the surface of the small compact nucleoli. Association of UBF with p130 in the final stage of oocyte growth suggests a possible role for pocket proteins in downregulation of rRNA synthesis.



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  		FIG. 6. Scheme of oocyte nucleologenesis in the bovine tertiary follicles. Fibrillar centers (FC) are located inside the reticulated nucleolus and marginalized toward the end of oocyte growth when dense nucleolar bodies are formed. During this compaction, UBF and RNA polymerase I are relocated into the cap substructure associated with protein p130. Fibrillarin is concentrated in the core of the compact nucleolus and Nopp140 together with C23/nucleophosmin and B23/nucleophosmin located mainly in its cortex. In addition, a great amount of C23/nucleolin and B23/nucleophosmin (granular component) is extruded in nucleoplasm (not shown in the scheme). DFC = dense fibrillar component


   ACKNOWLEDGMENTS
 
For primary antibodies, the authors thank the following: Prof. Chan PK (Baylor College of Medicine, Houston, TX), Dr. Hansen K (Inst Cancer Biol, Copenhagen, Denmark), Dr. Hernandez-Verdun D (J Monod Inst, Jussieu, France), Dr. Meier UT (Yeshiva University, New York, NY), Prof. Ochs RL (Scripps Research Institute, La Jolla, CA), and Dr. Grummt I (German Cancer Res, Heidelberg, Germany).


   FOOTNOTES
 
1 Supported by EU Grant QLK3-CT1999-00104, by the Ministry of Education, Youth and Sport of the Czech Republic, projects LN00A065 and ME573, and Slovak Academy of Sciences grant VEGA 2/3065/23. Back

2 Correspondence: Jan Motlik, Department of Reproduction, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech Republic. FAX: 420 315 697186; motlik{at}iapg.cas.cz Back

Received: 5 May 2003.

First decision: 27 May 2003.

Accepted: 3 November 2003.


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 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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