BIOLOGY OF REPRODUCTION 59, 626–631 (1998)
Ribosomal Ribonucleic Acid Is Transcribed at the 4-Cell Stage in In Vitro-Produced
Bovine Embryos1
Dorthe Viuff,2,3 Poul Hyttel,4 Birthe Avery,3 Gabor Vajta,5 Torben Greve,3 Henrik Callesen,5
and Preben D. Thomsen4
Department of Clinical Studies, Reproduction,3 Department of Anatomy and Physiology,4 Royal Veterinary and
Agricultural University, DK-1870 Frederiksberg C, Denmark
Embryo Technology Centre,5 Danish Institute of Agricultural Sciences, Tjele, Denmark
ABSTRACT
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.
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-RNasetreated 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 4cell 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 2cell 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 16cell 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 silverstained 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.
MATERIALS AND METHODS
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 48C for at least 24 h. The slides were
then air dried and hardened at 608C overnight before the
FISH procedure was initiated. Slides that were not immediately hybridized were stored at 2208C.
INTRODUCTION
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 maternalembryonic 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
Accepted April 28, 1998.
Received January 21, 1998.
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;
e-mail: [email protected]
626
627
rRNA GENE ACTIVATION IN BOVINE EMBRYOS
FISH
The porcine ribosomal (r) DNA probe, BHT115, was
isolated from a porcine cosmid library using a mouse 6.6kilobase 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-11dUTP 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 mg/ml RNase A (Sigma) in doublestrength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate) for 30 min at 378C 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% phosphatebuffered 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 65–688C 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 mg salmon sperm
DNA) at a final concentration of 50 ng/ml, denatured by
incubation at 708C for 5 min, and quenched on ice. Aliquots
(15–30 ml) of this solution were placed on each slide, coverslipped, sealed, and incubated overnight at 378C. After
hybridization, slides were washed twice in 45% formamide,
double-strength SSC for 3 min and three times in doublestrength SSC for 3 min, all at 428C. After washing, slides
were preincubated at 378C 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-phenylindole (DAPI, 1 mg/ml) in Dabco (Sigma) antifade solution
(pH 8). Chromosomes were R-banded using 20–40 mg 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.
TABLE 1. Total number of embryos and nuclei examined at different developmental stages.
Parameter
Total embryos
Total nuclei
Average nuclei/embryo
2 Cells
4 Cells
8–16
Cells
Blastocysts
27
37
1.4
22
38
1.7
13
57
4.4
10
. 50
.5
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 ml 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 378C. 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
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 5 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 5 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).
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 5 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 4cell embryos, up to 10 medium (n 5 11) or large (n 5 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 2cell embryos. Almost all FITC clusters were colocalized
with silver-stained spots (Fig. 1, F and H).
628
VIUFF ET AL.
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).
rRNA GENE ACTIVATION IN BOVINE EMBRYOS
629
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VIUFF ET AL.
Eight- to Sixteen-Cell Embryos
In the RNase-treated 8- to 16-cell embryos (n 5 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 5 9) displayed similar complexes,
but the central areas of the clusters were not homogeneous,
instead consisting of small spots (Fig. 1K). The 8- to 16cell 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. 1, 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 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 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
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 socalled 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 GENE ACTIVATION IN BOVINE EMBRYOS
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.
12.
13.
ACKNOWLEDGMENTS
14.
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.
15.
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