Ribosomal Ribonucleic Acid Is Transcribed at the 4-Cell Stage in In Vitro-Produced Bovine Embryos¹

^c Department of Clinical Studies, Reproduction, ^d Department of Anatomy and Physiology, Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Denmark ^e Embryo Technology Centre, Danish Institute of Agricultural Sciences, Tjele, Denmark

	ABSTRACT

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Ribosomal RNA, rRNA genes, and silver-staining nucleolar proteins were visualized in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst using a sequential fluorescent in situ hybridization (FISH) and a silver-staining procedure. At FISH, the rRNA was differentiated from the signal of the rRNA genes through comparison of RNase- and non-RNase-treated embryos. Both RNase- and non-RNase-treated 2-cell embryos revealed up to 10 small clusters of fluorescein isothiocynate (FITC) labeling in interphase nuclei. The RNase-treated 4-cell embryos displayed the same FITC pattern as the 2-cell embryos. In the non-RNase-treated 4-cell embryos, in contrast, the clusters were larger and included numerous small spots. In 2-cell as well as 4-cell embryos, almost all FITC-labeled clusters colocalized with silver-stained spots. In the RNase-treated 8- to 16-cell embryos, up to 10 clusters of FITC labeling were organized as one or more large spots surrounding a central faint but homogeneously labeled area. The non-RNase-treated 8- to 16-cell embryos displayed similar complexes, but the central areas consisted of small labeled spots. In 8- to 16-cell embryos, all FITC-labeled clusters were again colocalized with silver-stained areas. In the blastocysts, 1–6 big clusters of FITC labeling colocalized with silver staining. In the RNase-treated blastocysts, the FITC labeling was typically located at the edges of the silver-stained areas, whereas in the non-RNase-treated blastocysts, the FITC labeling totally covered the silver-stained areas. In conclusion, there is a close association between the rRNA genes and silver-staining nucleolar proteins in in vitro-produced bovine embryos from the second cell cycle, i.e., the 2-cell stage; the first rRNA is apparently transcribed during the third cell cycle, and during the fourth cell cycle the molecular composition of functional nucleoli is established.

	INTRODUCTION

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The initial period of mammalian preimplantation development is governed by maternal transcripts and polypeptides stored in the oocyte during its development [1]. However, after one to three cleavage divisions, the control of development is gradually taken over by the embryonic genome as maternally derived transcripts and proteins are diluted out or degraded [2, 3]. During this so-called maternal-embryonic transition, the transcription of the 18S, 5.8S, and 28S rRNA genes by RNA polymerase I, and their subsequent processing, lead to the formation of a distinct nuclear structure, the nucleolus. The ultrastructural features of a fully functional nucleolus, i.e., a fibrillo-granular composition, are first detectable during the fourth cell cycle, i.e., the 8-cell stage, in the bovine embryo [4–6]. The activity of the 18S, 5.8S, and 28S rRNA genes during this cell cycle is also supported by the observation that silver staining of the chromosome regions containing these gene clusters, often referred to as the nucleolar organizer region (NOR), can be detected on metaphases at the end of the fourth cell cycle [4]. Silver affinity of the NOR is thought to be caused by proteins that are needed for transcription of the rRNA genes and that remain attached to these genes during mitosis.

Previously, a low level of transcription in bovine embryos during the cell cycles preceding the formation of the nucleolus has been reported [7–10]. It has, however, not been possible to reveal whether this RNA synthesis also includes the rRNA genes. In this context, ultrastructural studies have been of only limited value, since the functional significance of the minor morphological changes of the nucleolus precursor bodies (NPBs) that are observed over the first, second, and third cell cycles of the bovine embryo is unclear [5, 6, 11, 12].

We have used a combination of fluorescent in situ hybridization (FISH) and silver staining to visualize the localization of the 18S, 5.8S, and 28S rRNA genes and their transcripts relative to the silver-staining nucleolar proteins in the early cleavage stages of in vitro-produced bovine embryos.

	MATERIALS AND METHODS

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Embryos and Metaphase Spreads

Slides with spreads of bovine metaphase chromosomes were prepared from phytohaemagglutinin-stimulated lymphocyte cultures of normal bulls using standard cytogenetic techniques [13].

Bovine embryos were produced exactly as described earlier [14, 15] except that the maturation medium contained 1 mg/ml polyvinylalcohol (Sigma, Copenhagen, Denmark) instead of serum.

Embryos at the 2-cell stage were collected at 27–33 h postinsemination (hpi); 4-cell embryos were collected at 45–50 hpi; 8- to 16-cell embryos were collected at 70–74 hpi; and blastocyst stages were collected at 170 hpi. Slides with fixed blastomeres were prepared as described by King et al. [16]. The embryos were placed in 1% w:v sodium citrate for 5–15 min at room temperature, spread on clean glass slides with drops of a 1:1 mix of glacial acetic acid and methanol, and dried by gently blowing on the spread. The specimens were subsequently fixed in 1:3 glacial acetic acid : methanol at 4°C for at least 24 h. The slides were then air dried and hardened at 60°C overnight before the FISH procedure was initiated. Slides that were not immediately hybridized were stored at -20°C.

FISH

The porcine ribosomal (r) DNA 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 [17]. DNA sequencing of subclones from BHT115 confirmed that BHT115 contained sequences highly similar to 18S and 28S rDNA from the human and mouse. DNA from cBHT115 was labeled using biotin-14-dATP or labeled using digoxigenin-11-dUTP by a standard nick-translation reaction [18]. FISH was performed essentially as described by Thomsen et al. [19]. Briefly, the RNase-treated embryos and lymphocytes were treated with 100 µg/ml RNase A (Sigma) in double-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate) for 30 min at 37°C and then washed once, for 2 min, in double-strength SSC at room temperature. All slides (RNase-treated as well as the non-RNase-treated slides) were then fixed in 1% phosphate-buffered paraformaldehyde for 2 min, washed twice in double-strength SSC for 2 min, and dehydrated in an ascending ethanol series. Chromosomal DNA was denatured by immersing slides in 70% formamide, double-strength SSC (pH 7) for 2 min at 65–68°C and thereafter immediately dehydrated in an ice-cold ascending ethanol series. The biotinylated or digoxigenated rDNA probe was added to the hybridization solution (50% deionized formamide, 10% dextran sulfate, double-strength SSC, 10 µg salmon sperm DNA) at a final concentration of 50 ng/ml, denatured by incubation at 70°C for 5 min, and quenched on ice. Aliquots (15–30 µl) of this solution were placed on each slide, coverslipped, sealed, and incubated overnight at 37°C. After hybridization, slides were washed twice in 45% formamide, double-strength SSC for 3 min and three times in double-strength SSC for 3 min, all at 42°C. After washing, slides were preincubated at 37°C for 10 min in 4-strength SSC, 0.1% Tween 20 containing 5% skim milk powder in order to reduce nonspecific antibody binding. Hybridization sites of biotinylated probes were visualized using fluorescein (FITC)-avidin (Vector Laboratories, Albertslund, Denmark) after one round of amplification using biotinylated goat anti-avidin antibodies (Vector Laboratories). Hybridization sites of digoxigenated probes were visualized using the fluorescent antibody enhancer set (Boehringer Mannheim, Kvistgård, Denmark). Nuclei were counterstained with either propidium iodide (PI, 400 ng/ml) or diamidino-phenyl-indole (DAPI, 1 µg/ml) in Dabco (Sigma) antifade solution (pH 8). Chromosomes were R-banded using 20–40 µg propidium iodide per milliliter alkaline (pH 11) mounting medium as suggested by Lemiaux et al. [20]. The slides were examined using epifluorescence microscopy, and images of FITC and DAPI or PI fluorescence were recorded separately using a Quantix CCD camera (Photometrix, Tucson, AZ) and subsequently merged using the multigene extension for IPLab Spectrum (Signal Analytics, Vienna, VA).

FISH with biotinylated cBHT115 DNA as probe on RNase-treated metaphase spreads from bovine lymphocytes revealed FITC labeling at the telomere region of 9–10 chromosomes, corresponding well to the previously reported localization of the rRNA gene clusters on bovine chromosomes. The large interphase lymphocyte nuclei showed clusters of FITC labeling arranged in a circular or semicircular pattern. Faint tracks of more homogeneous FITC labeling were often radiating into the central part of such areas.

Silver Staining

Silver staining was performed according to Lindner [21]. The slides were incubated in 1% dithiothreitol for 12 min at room temperature and then carefully rinsed with distilled water. Slides were then covered with 100 µl of AgNO₃ solution (a freshly prepared 3:1 mixture of 50% AgNO₃ : 2% gelatine : 1% formic acid [Merck, Rahway, NJ; Sigma; and Merck, respectively]), coverslipped, and incubated for 1 h at 37°C. Slides were mounted in Dabco antifade (pH 8) solution after a rinse in distilled water. Cells for which the FITC-labeling pattern had been previously recorded were relocated using brightfield microscopy, and an image was recorded.

	RESULTS

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Only embryos in which at least one interphase nucleus had been successfully exposed to both FISH and silver staining were included in the results. In some embryos only one such nucleus could be identified, whereas in others several or all nuclei were used (Table 1). An FITC signal was considered positive when it was 1) markedly stronger than the background and/or 2) colocalized with a positive silver staining.

Two-Cell Embryos

In the RNase-treated 2-cell embryos (n = 11), FITC labeling was localized to up to 10 small clusters dispersed throughout the interphase nuclei (Fig. 1A). Each cluster typically consisted of one or two larger spots, which in some cases were surrounded by some smaller spots. In the non-RNase-treated 2-cell embryos (n = 16), the same pattern of FITC labeling was noticed (data not shown). The silver staining of the 2-cell embryos was localized to many small spots and up to 12 larger spots. Almost all FITC clusters were colocalized with silver-stained spots (Fig. 1B).

View larger version (250K): [in this window] [in a new window]   FIG. 1. Sequential recording of FISH and silver staining showing the localization of the rRNA gene clusters and rRNA and of silver-staining proteins on interphase cells from early bovine embryos (A, B, E–P) and on metaphase spreads (C, D). A) FISH to an interphase nucleus of an RNase-treated bovine 2-cell embryo showing the localization of the rRNA gene clusters as 7 small spots of FITC labeling. B) Silver staining of the nucleus from A demonstrating 7 small colocalized spots of silver deposits (arrowheads). C) FISH with the porcine rRNA probe to a metaphase spread from an RNase-treated bovine 4-cell embryo. The hybridization sites at the telomere region of 9 bovine chromosomes are visualized using FITC (yellow), and the chromosomes are counterstained with DAPI (blue). D) Silver staining of the metaphase from C to demonstrate that there were no silver deposits at hybridization sites of the rDNA probe. E) FISH to an interphase nucleus from an RNase-treated bovine 4-cell embryo showing 10 small spots of FITC labeling. F) Silver staining of the nucleus from E demonstrating several small colocalized spots of silver deposits (arrowheads). G) FISH to an interphase nucleus from a non-RNase-treated bovine 4-cell embryo showing 7 larger clusters of FITC labeling. The nucleus is counterstained with PI (red). H) Silver staining of the nucleus from G demonstrating 7 colocalized spots of silver deposits (arrowheads). I) FISH to an interphase nucleus from an RNase-treated bovine 8- to 16-cell embryo showing 7 large clusters of FITC labeling. J) Silver staining of the nucleus from I demonstrating 7 large colocalized spots of silver deposits (arrowheads). K) FISH to an interphase nucleus from a non-RNase-treated bovine 8- to 16-cell embryo showing 4 large clusters of FITC labeling. L) Silver staining of the nucleus from K demonstrating 4 large colocalized spots of silver deposits (arrowheads). M) FISH to an interphase nucleus from an RNase-treated bovine blastocyst showing 6 large clusters of FITC labeling. N) Silver staining of the nucleus from M demonstrating 6 large colocalized spots of silver deposits (arrowheads). O) FISH to an interphase nucleus from a non-RNase-treated bovine blastocyst showing 2 large clusters of FITC labeling. P) Silver staining of the nucleus from O demonstrating large colocalized spots of silver deposits (arrowheads).

Four-Cell Embryos

One 4-cell embryo displayed two metaphase spreads where the FITC labeling was localized to the telomere region of 9 chromosomes (Fig. 1C). It was not possible to detect any silver staining of these areas (Fig. 1D). In the RNase-treated 4-cell embryos (n = 7), the FITC labeling was localized to up to 10 small clusters dispersed throughout the interphase nuclei, thus resembling the situation in the 2-cell embryos (Fig. 1E). In the non-RNase-treated 4-cell embryos, up to 10 medium (n = 11) or large (n = 4) clusters of FITC labeling were observed (Fig. 1G). Each cluster typically consisted of one or more peripherally located large spots and a central portion comprising numerous small spots. The silver staining of the 4-cell embryos revealed a combination of small and larger spots, of which the latter were, in general, more prominent than in the 2-cell embryos. Almost all FITC clusters were colocalized with silver-stained spots (Fig. 1, F and H).

Eight- to Sixteen-Cell Embryos

In the RNase-treated 8- to 16-cell embryos (n = 4), the FITC labeling was localized to up to 10 clusters dispersed throughout the interphase nuclei. Each cluster included one or more large spots typically placed in the periphery of a central area, presenting a faint but more or less homogeneous labeling (Fig. 1I). In contrast, the non-RNase-treated 8- to 16-cell embryos (n = 9) displayed similar complexes, but the central areas of the clusters were not homogeneous, instead consisting of small spots (Fig. 1K). The 8- to 16-cell embryos presented large silver-stained foci including heavily stained black areas and sparsely stained brown areas. All FITC clusters were colocalized with silver-stained foci (Fig. 1X, J and L). The black areas in the silver-stained foci were typically colocalized with the strongest FITC signals on RNase-treated nuclei (Fig. 1, I and J).

Blastocysts

In the RNase-treated blastocysts (n = 5), the FITC labeling was localized to up to 6 big clusters (Fig. 1M). Each cluster was typically delineated by large spots, while the central area presented a faint but more or less homogenous labeling. In contrast, the non-RNase-treated blastocysts (n = 5) presented a strong labeling of the central area in addition to the peripheral labeling (Fig. 1O). The silver staining of the blastocyst revealed large stained foci, which were colocalized with the FITC-labeled areas (Fig. 1>, N and P).

	DISCUSSION

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In this report we extend our previous studies showing a low-grade transcriptional activity in 2- and 4-cell bovine embryos [7, 8]. Using an rDNA probe for FISH on fixed, air-dried bovine embryo cells, we found evidence of rRNA transcription at least during the third cell cycle, i.e., at the 4-cell stage. This is the first direct evidence that rRNA is transcribed before the nucleolus is formed during the fourth cell cycle in bovine embryos. We do note, however that Bilodeau-Goeseels and Schultz [22] observed a small but repeatable increase in the rRNA hybridization signal on Northern blots of RNA from 2- to 4-cell embryos as compared with 1-cell embryos.

During the fourth cell cycle, both the FITC labeling and the silver staining assumed characteristics that were comparable to those observed in the blastocysts, where the embryonic genome has attained a full somatic level of activation, and in lymphocytes used as actively transcribing control cells. We are confident that we get a valid estimation of rRNA content and localization in these cells by comparing non-RNase-treated nuclei to RNase-treated nuclei, because our earlier autoradiographic studies showed that [³H]uridine labeling of blastocysts could be completely removed by RNase treatment [8]. The FISH labeling and the silver staining indicate that the molecular organization of functional nucleoli is gained during the fourth cell cycle. This observation is in accordance with several sets of previously published data. Firstly, the ultrastructural organization of the fibrillo-granular nucleolus is obtained during this cell cycle [4–6]. Secondly, it has been demonstrated that the NPBs develop silver-staining characteristics during this cell cycle [23], most likely because the nucleolar proteins C23 and B23 become localized to the NPBs [24]. Up to the fifth cell cycle, we observed up to 10 FITC-labeled clusters, whereas the number decreased to a maximum of 6 in the later stages (blastocysts). This feature is probably due to association of two or more NORs in the formation of each nucleolus [25].

The procedure used in this study to prepare embryos for FISH includes air-drying of methanol : acetic acid-fixed embryonic cells. The spatial relationships of the nuclear structures may therefore be distorted as compared, for example, to those in sections for transmission electron microscopy. Nevertheless, it is very unlikely that structures that were separated in the living cell are systematically brought together by the air-drying technique. We therefore consider structures that are colocalized in several embryos to be representative of the spatial relationship in the cell in toto rather than artifacts. Further, it is tempting to compare the large clusters of FITC labeling in the non-RNase-treated 4-cell embryos, i.e., the presumptive rRNA gene and rRNA complexes, with the clusters of electron-dense granules that are observed at the ultrastructural level during the initial three cell cycles of the developing bovine embryo. The chromatin-like cap of the latter clusters observed in the electron microscope could correspond to the rRNA gene clusters and associated heterochromatin. In this light, the affinity between the NPBs and these granule clusters as previously described [11, 12] seems logical. At the ultrastructural level, however, the granule clusters are observed already from the first cell cycle, whereas the rRNA hybridization signal in the present experiment did not appear until the third cell cycle. At the ultrastructural analysis, proteinaceous material is contrasted, whereas rRNA sequences are detected at FISH. We therefore hypothesize that the granule clusters during the first and second cell cycle are exclusively proteinaceous whereas during the third cycle they are enriched with rRNA. It may be significant in this respect that we detected colocalization of silver-staining material and the rRNA gene clusters from the second cell cycle onward. This is remarkable since the NORs on metaphase chromosomes do not display silver staining until the fourth cell cycle. Thus, the spatial organization of the molecules that make up the nucleolus is clearly different during the first, second, and third cell cycle of the bovine embryo as compared with somatic cells; and the NOR of such cells may, during interphase, possess an affinity to pre-ribosomal particles that is independent of rRNA transcription. An experiment at the ultrastructural level in which rRNA genes or silver-staining proteins and granule clusters could be localized sequentially on the same specimen would clarify whether this hypothesis is correct or whether it could be discarded.

Our data indicate that embryonic rRNA production precedes the formation of a true nucleolus. This may not be surprising, because experiments in yeast [26] have shown that an intact nucleolar structure is not absolutely required for ribosome biosynthesis. A mutant yeast strain deficient in RNA polymerase I, which was genetically engineered to produce rRNA by RNA polymerase II, showed ribosome biosynthesis but lacked nucleoli. Instead, rRNA gene transcription took place in solitary foci reminiscent of the so-called pre-nucleolar bodies observed at the telophase in somatic cells, where rRNA transcription starts again after mitosis. It may be that nucleolus formation is dependent on a number of proteins, including RNA polymerase I, and that it simply needs time to form in the bovine embryo. Time is likely to be a critical factor because the cleavage divisions preceding the activation of the embryonic genome are merely reduced to an S-phase, a short G₂-phase, and mitosis [27].

In conclusion, there is a close association between the rRNA genes and silver-staining nucleolar proteins in in vitro-produced bovine embryos from the second cell cycle onward, i.e., the 2-cell stage; the first rRNA is apparently transcribed during the third cell cycle, and during the fourth cell cycle the molecular composition of functional nucleoli is established.

	ACKNOWLEDGMENTS

 
The mouse 18S/5.8S/28S probe was generously provided by Dr. Sune Frederiksen, Dept. of Biochemistry, Copenhagen; and pig cosmid library for screening was kindly provided by Dr. Bjørn Høyheim, Veterinary University, Norway. We are grateful to Ms. Anne Katrine Winterø for assistance with automated DNA sequencing and to Mrs. Inger Heinze and Ms. Eva Tøt Nielsen for technical assistance.

	FOOTNOTES

 
¹ Supported by the Danish Agricultural and Veterinary Research Council.

² Correspondence: Dorthe Viuff, Department of Clinical Studies, Reproduction, Royal Veterinary and Agricultural University, Bülowsvej 13, DK-1870 Frederiksberg, Denmark. FAX: 45 35 28 29 72; dv{at}kvl.dk

Accepted: April 28, 1998.

Received: January 21, 1998.

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