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Activation of the Ribosomal RNA Genes Late in the Third Cell Cycle of Porcine Embryos¹

^a Departments of Clinical Studies, Reproduction, ^b Anatomy and Physiology, Royal Veterinary and Agricultural University, 1870 Frederiksberg C, Denmark ^c Department of Animal Breeding and Genetics, Danish Institute of Agricultural Sciences, 8830 Tjele, Denmark

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In porcine embryos, nucleoli are first observed during the third postfertilization cell cycle, i.e., at the 4-cell stage. However, direct studies of the initiation of rRNA transcription have not been reported. This transcription was investigated in the present study by simultaneous visualization of the rRNA genes and the rRNA by fluorescent in situ hybridization using a porcine 28S rDNA probe and subsequent visualization of argyrophilic nucleolar proteins by silver staining of extracted and fixed nuclei from in vivo-derived porcine embryos (n = 229). Nucleologenesis was observed by transmission electron microscopy. In general, the 2-cell and 4-cell embryos fixed at 10 and 20 h postcleavage (hpc) showed no signs of rRNA transcription. Four small clusters of fluorescein isothiocyanate (FITC) labeling were visible in interphase nuclei, consistent with hybridization to the rRNA gene clusters only; there was no silver staining at the sites of the rRNA genes and nucleolus precursor bodies. From 30 hpc onwards, most 4-cell embryos had medium size to large clusters of FITC-labeled areas colocalized with silver staining of rRNA gene clusters and fibrillogranular nucleoli. These observations indicate that rRNA transcription had been initiated. These signs of rRNA synthesis could be blocked by actinomycin D, which is a strong inhibitor of RNA polymerase I. The rRNA transcription of porcine embryos is initiated between 20 and 30 hpc, corresponding to the end of the S-phase or the beginning of the G2 phase during the third cell cycle.

developmental biology, early development, embryo, fertilization, gene regulation

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The first few cleavage divisions of mammalian embryos are generally under the influence of the maternal transcripts present in the oocyte. Thus, a period of no or only minor synthesis of RNA is observed after the completion of the oocyte growth phase, and this period extends through the first cleavage divisions [1–3]. Low-grade transcription has been detected during the first 3 cell cycles in bovine embryos [4–7], but studies of porcine embryos have not revealed any transcription until late in the third cell cycle, i.e., at the 4-cell stage [8–12]. At this stage, a typical nucleolus is formed and proteins involved in the transcription and processing of rRNA are allocated to particular compartments of the nucleolar region [8, 12]. Although the presence of pre-rRNA or rRNA transcripts has not been directly demonstrated in porcine embryos, the presence of a nucleolus indicates that the embryonic genome has been activated.

We have previously been able to detect signs of a low-grade transcription of the rRNA genes prior to the formation of a typical fibrillogranular nucleolus by analyzing bovine embryos at the 4-cell stage using fluorescence in situ hybridization (FISH) with an 18S/5.8S/28S rDNA probe [7]. We therefore anticipated that transcripts of the rRNA genes in pig embryos could be detectable prior to the late half of the third cell cycle using FISH with an rDNA probe. The aim of this study was to investigate porcine embryos during their initial cleavage stages for signs of rRNA transcription and processing and to compare this pattern with that of actinomycin D-treated embryos, in which rRNA transcription is blocked.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Collection and Fixation of Embryos

Porcine embryos were collected from 19 sows inseminated at Days 6 and 7 after weaning, i.e., the day where the piglets are taken from the sow around 3 wk after farrowing. The sows were slaughtered at a local abattoir at different times after the last insemination and the oviduct and/or uterine horns were flushed with modified PBS containing 1% cattle serum (Danish Veterinary Laboratory, Frederiksberg C, Denmark). In the first experiment, embryos were collected and fixed at predetermined times after insemination, i.e., at 24, 48, 72, and 120 h postinsemination to obtain 2-cell stage (n = 24), 4-cell stage (n = 66), and 8- to 16-cell stage (n = 22) embryos and blastocysts (n = 16), respectively. In the second experiment, 2-cell embryos (n = 37) were examined every 3 h until cleavage to the 4-cell stage and further cultured for 10, 20, or 30 h postcleavage (hpc) and then processed for fluorescence in situ hybridization (FISH) and silver staining (n = 31) or transmission electron microscopy (TEM) (n = 6). In the third experimental series, the embryos were also observed every 3 h during the second cell cycle, and the 4-cell embryos (n = 36) were then cultured for the first 15 hpc in Beltsville embryo culture medium (BECM-3) [13] followed by 15 h in BECM-3 + actinomycin D (10 µg/ml). Control embryos of the third series (n = 18) were cultured the same way except without the addition of actinomycin D during the 4-cell stage. The embryos from experiment III were processed for FISH and silver staining (n = 48) or TEM (n = 16). In all experiments, cultures were performed in BECM-3 with 5% serum at 39°C in a humidified atmosphere with 5% CO₂, which in our laboratory results in a blastocyst rate per cultured oocyte of 12% [14].

Nuclei were extracted from the embryos (n = 207) and fixed using the method described previously for FISH [15]. Individual embryos were quickly washed in a lysing buffer (0.01 N HCl, 0.1% Tween 20) and transferred in a small droplet to a Superfrost Plus slide (Menzel Gläser, Braunschweig, Germany). The embryos were constantly observed using an inverted phase contrast microscope. After the lysis buffer had been added, the zona pellucida and the blastomere cytoplasm dissolved gradually, and 3:1 methanol:glacial acetic acid was immediately added dropwise to the slide before the nuclei dried out. The specimens were then fixed in 3:1 methanol:glacial acetic acid at 4°C for at least 24 h, air dried, and incubated at 60°C overnight. Slides that were not immediately hybridized were stored at -80°C.

The embryos (n = 22) for ultrastructural analysis by TEM were treated as described by Hyttel et al. [12]. Embryos were fixed in 3% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2). Subsequently, they were washed in buffer, postfixed in 1% OsO₄ in 0.1 M sodium phosphate buffer, stained en bloc in 0.5% uranyl acetate in distilled water, dehydrated, embedded in Epon, and sectioned into semithin sections (2.0 µm). All sections were stained with 1% basic toluidine blue, and selected sections were reembedded for ultrathin sectioning. The ultrathin sections were stained with uranyl acetate and lead citrate and observed in a Philips CM 100 electron microscope.

Fluorescence In Situ Hybridization

The porcine rDNA probe, BHT115, was isolated from a porcine cosmid library using a mouse 6.6-kilobase EcoRI fragment containing 25% of the 18S rDNA, both internal transcribed spacers (ITS1 and ITS2), the 5.8S rDNA, and most of the 28S rDNA [16]. The BHT115 probe produced strong labeling in control hybridizations of metaphase chromosome spreads (not shown) at the p11-p12 of porcine chromosomes 8 and 10, i.e., at the nucleolus organizer regions (NORs) of the porcine karyotype [17]. Purified DNA from cBHT115 was labeled using digoxigenin-11-dUTP by a standard nick-translation reaction. FISH was performed essentially as described by Viuff et al. [7]. The RNase-treated embryos and lymphocyte nuclei were treated with 100 µg/ml RNase A (Sigma, Copenhagen, Denmark) in 2x saline sodium citrate (SSC) for 40 min at 37°C followed by 1 wash of 2 min in 2x SSC at room temperature. All slides (containing RNase-treated as well as the nontreated embryonic nuclei and lymphocyte nuclei) were then fixed in 1% phosphate-buffered formaldehyde for 2 min, washed twice in 2x SSC for 2 min each time, and dehydrated in an ascending ethanol series. Chromosomal DNA was denatured by immersing slides in 70% formamide, 2x SSC (pH 7) for 2 min at 71–72°C and immediately dehydrated in an ice-cold ascending ethanol series. The digoxigenated rDNA probe was added to the hybridization solution (50% deionized formamide, 10% dextran sulfate, 2x SSC, 0.1% salmon sperm DNA, 0.1% porcine genomic DNA) at a final concentration of 20 ng/ml, denatured by incubation at 70°C for 5 min, and quenched on ice. Three-microliter aliquots of the hybridization mix were applied to the area where the embryonic nuclei were concentrated, and the slides were coverslipped and hybridized overnight at 42°C. Slides were then washed 3 times in 0.05x SSC for 3 min each time at 42°C, and hybridization sites were visualized using Anti-Dig-Fluorescein (FITC) (Boehringer Mannheim, Mannheim, Germany). DNA was counterstained with diamidino-phenylindole (1 mg/ml) in DABCO (Sigma) antifade solution. Images of hybridization pattern were recorded for all nuclei using a CCD camera.

Silver Staining

Silver staining was performed according to the method of Lindner [18]. The slides were incubated in 1% dithiothreitol for 12 min at room temperature followed by careful rinsing with distilled water. Slides were then covered with 100 µl of AgNO₃ solution (a freshly prepared 3:1 mixture of 50% AgNO₃ [Merck, Rahway, NJ]:2% gelatine [Sigma] and 1% formic acid [Merck]), coverslipped, and incubated for 1 h at 37°C. After a rinse in distilled water, slides were mounted in DABCO antifade solution (pH 8). Cells for which the FITC labeling pattern had been previously recorded were relocated using bright field microscopy, and an image was recorded.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Three experiments were performed to determine the onset of rRNA transcription in porcine embryos. Experiment I was aimed at determining the cell cycle where the first pre-rRNAs were detectable at the site of the rRNA genes. Experiment II was set up to determine at which time during that cell cycle (the third cell cycle) the rRNA transcription was observed. Experiment III was included specifically to observe whether the signs of rRNA transcription were blocked by an RNA polymerase I inhibitor.

Experiment I: First Sign of rRNA Synthesis

For the 2-cell embryos (n = 24), both RNase-treated and nontreated embryos had 4 small foci of FITC labeling, consistent with hybridization only to the rRNA gene clusters at chromosome 8 and 10 homologs (Fig. 1A). No specific silver staining was observed (Fig. 1B), in agreement with the absence of detectable rRNA. For the 4-cell stages (n = 66), all except 1 of the RNase-treated embryos (n = 13) resembled the 2-cell embryos with respect to hybridization pattern and silver staining, indicating that rRNA transcription had not started. The exceptional embryo showed FITC clusters surrounded by areas with more dispersed FITC labeling colocalized with foci of silver staining, indicating decondensation and a beginning of transcription of the rRNA genes (data not shown). Of the 53 nontreated 4-cell embryos, 48 showed the same pattern as observed in the 2-cell embryos, whereas the remaining 5 embryos displayed small, medium, or large round areas of FITC labeling in some of the nuclei colocalized with larger spots of silver staining (similar to the pattern in Fig. 1, C and D), indicating rRNA transcription initiation. In the 5- to 8-cell embryos, all embryos of the RNase-treated group (n = 9) had up to 4 large areas with dispersed FITC labeling. The center of the area typically had no or faint labeling, but the periphery of each area generally included 1 or a few peripheral clusters of labeling (Fig. 1E), which colocalized with foci of NOR proteins and subsequent silver staining (Fig. 1F). The nontreated 5- to 8-cell embryos (n = 13) displayed similar complexes, but the central areas of the clusters had stronger FITC labeling, indicating the preservation of rRNAs in these embryos (Fig. 1G). The hybridization sites of the rDNA probe colocalized with foci of silver staining (Fig. 1H). The blastocysts (n = 16) in the RNase-treated and nontreated groups resembled the 5- to 8-cell embryos.

View larger version (80K): [in this window] [in a new window]   FIG. 1. Analysis of rRNA gene expression and presence of silver-stained proteins in interphase nuclei from porcine embryos at different stages of development. A) FISH to an interphase nucleus of a non-RNase-treated porcine 2-cell embryo showing the localizations of the rRNA gene clusters as 4 small clusters of FITC labeling. B) Silver staining of the nucleus from A demonstrating no silver labeling. C) FISH to an interphase nucleus of a non-RNase-treated late (30 hpc) porcine 4-cell embryo showing 2 large and 2 medium size clusters of FITC labeling. D) Silver staining of the nucleus from C demonstrating 4 spots of silver deposits colocalized with the FITC signals. E) FISH to an interphase nucleus of an RNase-treated porcine 8-cell embryo showing 4 dispersed clusters of FITC labeling. F) Silver staining of the nucleus from E demonstrating 4 large colocalized spots of silver deposits. G) FISH to an interphase nucleus of a non-RNase-treated porcine 8-cell embryo showing 3 large clusters of FITC labeling. H) Silver staining of the nucleus from G demonstrating 3 large colocalized spots of silver deposits.

Experiment II: Onset of rRNA Synthesis During the Third Cell Cycle

Four-cell embryos were fixed at 10, 20, or 30 hpc and processed for FISH and TEM analysis without RNase treatment. At 10 and 20 hpc, 22 of the 24 embryos showed the same FITC and silver staining pattern as seen in the 2-cell embryos in experiment I. The remaining 2 embryos had foci of silver staining proteins, some of which were colocalized with the FITC labeling. At 30 hpc, 6 of 7 embryos displayed small, medium, or large round clusters of FITC labeling (Fig. 1, C and D). The sites of FITC labeling were colocalized with foci of silver staining in 4 of these 6 embryos. Ultrastructurally, all nuclei in the two 4-cell embryos fixed at 10 hpc and the two 4-cell embryos fixed at 20 hpc had electron-dense nucleolus precursor bodies (NPBs). Slightly condensed chromatin was dispersed throughout the nuclei. One of the two 4-cell embryos fixed at 30 hpc looked like the 4-cell embryos fixed at 10 and 20 hpc. In the other embryo, the nuclei displayed different stages of nucleolus formation, ranging from NPBs to fibrillogranular nucleoli with semilunar formations of fibrillar centers, dense fibrillar components, and granular components on the surface of the NPBs that were more or less encapsulated. Different stages of nucleolus development were observed within the same nucleus.

Experiment III: Block of rRNA Transcription

The rRNA transcription in 4-cell embryos (n = 37) at 30 hpc was inhibited by actinomycin D. Four-cell embryos at 15 hpc were incubated for 15 h in medium containing 10 µg/ml actinomycin D. As in experiment II, we did not include any RNase treatment of nuclei during preparation for FISH. After FISH with the rRNA probe, we observed only 4 small foci of FITC labeling and no accumulation of silver staining at the sites of rRNA genes. The control group, which was not treated with actinomycin D, included eleven 4-cell embryos isolated at 30 hpc, and all except 1 of these embryos displayed medium to large areas of FITC labeling that were colocalized with foci of silver staining. The remaining embryo of the control group had only small foci of FITC labeling and no silver staining, which indicated that this embryo had not yet started rRNA transcription. Ultrastructurally, the actinomycin D-treated embryos (n = 9) displayed NPBs (Fig. 2, a and b) but no fibrillogranular nucleoli. In contrast, 6 of 7 of the control group had fibrillogranular nucleoli (Fig. 3, a and b). The last embryo of the control group had only NPBs and was similar in this respect to the 4-cell embryos at 10 and 20 hpc, in which no rRNA transcription had been initiated.

View larger version (96K): [in this window] [in a new window]   FIG. 2. Transmission electron micrographs of actinomycin D-inhibited 4-cell porcine embryos. a) Nucleus with an NPB. x3740. b) Detail showing the NPB composed of densely packed fibrilles. x23 000

View larger version (97K): [in this window] [in a new window]   FIG. 3. Transmission electron micrographs of non-actinomycin D-treated 4-cell porcine embryos. a) Nucleus with an NPB and a fibrillogranular nucleolus (N). x4130. b) Detail showing the fibrillogranular nucleolus established on the surface of an NPB. Note the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC). x17 800

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
These experiments produced evidence that embryonic rRNA transcription is first detectable during the third cell cycle in the porcine embryo, more specifically at 30 hpc, whereas no transcription was observed at 20 hpc. Thus, rRNA transcription is initiated in the 4-cell embryo between 20 and 30 hpc. Our data include both FISH-based detection of rRNA, the more indirect ultrastructural analysis, and detection of argyrophilic proteins at the NORs. These results provide a significant addition to previous ultrastructural data and immunolabeling studies reported by Hyttel et al. [12] by the direct detection of the expression of the rRNA genes. These data also support those of earlier studies suggesting that the maternal genome controls development in the oocyte and the 2-cell and early 4-cell embryos and that the embryonic genome controls development beyond the 4-cell stage [8–11].

No rRNA transcription was detected in the embryos at the 2-cell stage, in the embryos fixed at either 10 or 20 hpc to the 4-cell stage, and in most of 4-cell embryos (60 out of 66) collected without knowing where in the cell cycle they were examined. The 6 exceptional embryos (5 nontreated and 1 RNase-treated) from the latter group displayed rRNA transcription by showing initiation of 2 rRNA gene clusters (Fig. 1, C and D). This pattern of activation may be interpreted as an activation of the rRNA locus first on chromosome 10 homologs and then on the chromosome 8 homologs in those cells beginning rRNA transcription because the rRNA gene cluster on chromosome 10 is more actively transcribed [19]. In the 4-cell embryos fixed at 30 hpc, 6 of 7 showed the same pattern as seen in the 5 nontreated 4-cell embryos described above, thus demonstrating active rRNA transcription. The last embryo from this group showed no rRNA transcription initiation. In addition, TEM revealed only NPBs and no fibrillogranular nucleoli in two of nine 4-cell embryos fixed at 30 hpc. Those embryos may have been close to the onset of the rRNA transcription or they may have stopped developing. Furthermore, some of the 4-cell embryos showed silver stained foci without rRNA labeling. This pattern may reflect the beginning of organization of the nucleolus-specific proteins towards the rRNA genes; silver-stained foci beyond the 4-cell stage were always associated with the rRNA genes and their transcripts. This finding is in agreement with the presence of a fibrillogranular nucleolus in the late 4-cell embryos (30 hpc) and with recently published data from Hyttel et al. [12]. However, Hyttel et al. [12] did not observe any labeling with anti-nucleolin or with anti-nucleophosmin antibodies, a fact that seems to disagree with a positive silver staining reaction in the present study, because nucleolin and nucleophosmin are strongly stained by silver [20]. However, the discrepancy may be related to the limitations in the sensitivity of the immunocytochemical method, because the late 4-cell embryos also lack immunolabeling of UBF and topoisomerase I. These proteins are involved in assembling a stable RNA polymerase I transcription initiation complex by binding to the promoter of the rRNA genes and by uncoiling the DNA, respectively [21, 22].

The signs of rRNA transcription were successfully blocked in 4-cell embryos at 30 hpc by adding actinomycin D to a final concentration of 10 µg/ml medium and cultivating embryos for a further 15 hpc and until fixation at 30 hpc. Actinomycin D is frequently used to inhibit transcription, and a final concentration of 250 µg/ml will inhibit all 3 RNA polymerases [23]. However, very low concentrations (0.04 µg/ml) will selectively inhibit RNA polymerase I and lead to formation of mininucleoli in HeLa cells [24]. In the present study, the actinomycin D-induced block of rRNA transcription at the end of the third cell cycle was inferred from 1) the lack of detectable rRNA at the site of transcription, 2) the lack of specific silver staining, and 3) the presence of NBPs as revealed by TEM. Although this evidence is indirect, it further supports the hypothesis that rRNA transcription in porcine embryos is initiated at the 4-cell stage between 20 and 30 hpc, i.e., at the end of the S-phase or the beginning of the G2 phase during the third cell cycle [11, 25].

The transcription of the rRNA genes is first detectable late in the third cell cycle in both pigs, as shown in this study, and cattle [7]. However, only in the pig is embryonic rRNA transcription initiation accompanied by the simultaneous formation of a fibrillogranular nucleolus. Although the difference is well documented, it may be more artifactual than real because the studies in cattle have been conducted on embryos produced by in vitro fertilization because of the ease of obtaining specimens by this technique. Porcine embryos are generally developed in vivo, because in vitro techniques are still in their infancy. Thus, ultrastructural studies and FISH-based analyses of rRNA transcription initiation in cattle embryos developed in vivo must be conducted before a meaningful comparison of nucleologenesis can be made between porcine and bovine embryos.

	ACKNOWLEDGMENTS

 
The mouse 18S/5.8S/28S probe was generously provided by Dr. Sune Frederiksen (Department of Biochemistry, University of Copenhagen, Copenhagen, Denmark), and the pig cosmid library for screening was kindly provided by Dr. Bjørn Høyheim (Veterinary University, Oslo, Norway). We are grateful to Mrs. Inger Heinze, Mrs. Zaida R. Rasmussen, Mrs. Anne F. Petersen, and Mrs. Jytte Nielsen for excellent technical assistance.

	FOOTNOTES

 
First decision: 13 August 2001.

¹ This work was supported by the Danish Agricultural and Veterinary Research Council, the Danish Biotechnology Program, and Novo Nordisk A/S.

² Correspondence and current address: Dorthe Viuff, Department of Molecular Pharmacology, Novo Nordisk A/S, Novo Nordisk Park, 2760 Malov, Denmark. FAX: 45 4443 4587; dviu{at}novonordisk.com

Accepted: October 12, 2001.

Received: July 12, 2001.

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