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Research Article |
Schemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS,3 Moscow 117997, Russia
Institute of Experimental Medicine RAMN,4 St. Petersburg 197376, Russia
ABSTRACT
The nucleolar precursor bodies (NPBs) are numerous discrete entities present in the nuclei of early mammalian embryos, which structurally support active rRNA genes. However, whether all rRNA genes, including those not transcribed, are spatially associated with NPBs, and moreover what is the general arrangement of ribosomal DNA (rDNA) in early mouse embryos, still remain unanswered questions. In our study, we examined the localization of rDNA in transcriptionally silent (one-cell and early two-cell) and transcriptionally active (late two-cell) mouse embryos by highly sensitive fluorescence in situ hybridization with probes complementary to mouse rDNA repeats. The results obtained showed that irrespective of the rDNA transcriptional status, one or more NPBs per nucleus were not structurally associated with rDNA. These observations support the idea that NPBs are heterogeneous in their ability to recruit rRNA genes and thus to participate in reassembly of the mature nucleolus. As in somatic cells, and despite the absence of the characteristic nucleoli, the general arrangement of rRNA genes in early mouse embryos reflected the intensity of rDNA transcription. Ribosomal RNA genes were unequally distributed with respect to repeat putative copy numbers between nucleolar organizing region (NOR)-bearing chromosomes at the first cleavage division, and more strikingly, between sister chromatid NORs of a single nucleolar organizing chromosome. The latter indicates that sister chromatids might harbor various numbers of rRNA gene copies, and that the genes might be unequally distributed between the two blastomeres during the first cleavage mitosis.
early development, embryo, gene regulation
INTRODUCTION
In higher eukaryotes, ribosomal genes (rDNA) encoding the three major classes of rRNA (18 S, 5.8 S and 28 S) are arranged in clusters of repeating units, the nucleolar organizing regions (NORs), which occupy a defined position in mitotic chromosomes of each species' karyotype [1, 2]. Murine cells are thought to contain 
100–200 copies of rRNA genes, only half of which are transcribed [2, 3]. All transcribed as well as a majority of silent rRNA genes are located within the most prominent nuclear territory, the nucleolus, which is the major factory for ribosome production [4, 5]. In somatic cells, rDNA repeats are generally distributed between two major nucleolar subdomains, namely the fibrillar centers (FCs) and the dense fibrillar component [6, 7]. The morphological features of the FCs, which are major nucleolar storages for nontranscribed rRNA genes, are strictly related to the level of rDNA transcription, so that their total number is increased and average size is diminished when the activity of rRNA genes is upregulated. In contrast, inactivation of rRNA synthesis results in reduction of the FC number and augmentation of their size [7–9]. At the light microscopic level, the arrangement of rDNA in somatic cells has been intensively studied by fluorescence in situ hybridization (FISH) [10–12]. Altogether, these studies demonstrate that the distribution of rDNA reflects the functional architecture of the nucleoli and is characterized by alteration of clustered and extended gene configurations. Clustered rRNA genes predominate in inactive, and extended genes in active nucleoli.
Early development of mammalian embryos is accompanied by a period during which rDNA transcription is switched off. The transcriptionally silent period continues for one or a few of the first cell cycles, whose number depends on the species [13]. In mouse embryos, rRNA synthesis remains downregulated in one-cell and in early two-cell embryos, but resumes in the middle of the second cell cycle (44–45 h post-hCG) [14–18]. Until this time, numerous and optically dense spherical bodies called nucleolar precursor bodies (NPBs) are present instead of typical nucleoli. NPBs appear in zygotic pronuclei shortly after fertilization and persist throughout the first cell cycle. They become dissolved during the first cleavage but reassemble at the beginning of the second cell cycle [13]. Nowadays, it is generally accepted that NPBs serve as a structural support for building the functional nucleoli in early mammalian embryos. In mice, this view is based on numerous observations, which show that in two-cell embryos that commence to transcribe rDNA, activated rRNA genes are located at the NPB periphery [17, 18]. The NPB surface also recruits constituents of the rDNA transcription complex (e.g., the RNA polymerase I genuine subunit RPO1–2 and the transcription factors UBTF and NOLC1), rRNA processing machinery (e.g., fibrillarin, B23/nucleophosmin 1, nucleolin, and pescadillo), Ag-NOR proteins, and snoRNAs [16, 18, 19–22]. However, it remains unknown whether all the rRNA genes, including those not transcribed, are also attached to the NPBs. Moreover, contrary to somatic cells, the topology of rDNA in early mammalian embryos still remains vague. The latter circumstance can be explained in part by the fact that one-cell and early two-cell NPBs lack the morphological features of transcriptionally inert somatic nucleoli, including defined FCs, which harbor inactive rRNA genes in somatic cells [23, 24]. Being transcriptionally inert, rDNA does not interact with Ag-NOR proteins or specific molecular components of the RNA polymerase I transcription machinery, such as RPO1–2 and UBTF, that generally serve as indicators of rRNA gene locations in somatic cells [25–27].
To shed more light on the general arrangement of rRNA genes in relation to their transcriptional activity in preimplantation mammalian embryos, we examined localization of rRNA genes in one- and two-cell mouse embryos by highly sensitive FISH with probes specific to mouse rDNA units. Particular attention was paid to spatial interactions of rDNA with the NPB in transcriptionally inert embryos. The same protocol was also applied to describe the morphology of chromosomal NORs in mouse embryos in the first cleavage metaphase.
MATERIALS AND METHODS
Collection of Embryos
Six- to eight-wk-old CBA/Ca and C57BL/6J mice were used (The Lab Animals Breeding Center). All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. C57BL/6J females were injected with 7 IU of eCG, followed 46–48 h later by 7 IU of hCG, and caged with CBA/Ca males. At 18–28 h after hCG injection, females with vaginal plugs were killed by cervical dislocation. Embryos together with cumulus cells were released from ampullae into warm (37°C) M2 medium containing 300 IU/ml hyaluronidase (type III from sheep testis; Sigma). After removal of cumulus cells, embryos were washed in M2 medium without hyaluronidase, and those with clearly visible second polar bodies were used to prepare spreads. To obtain one-cell embryos blocked at the first cleavage metaphase, embryos were collected 27–28 h after the hCG administration and retained in M16 medium containing 0.034 µg/ml colcemide at 37°C and 5% CO2 for another 2–2.5 h. This treatment arrests one-cell embryos at metaphase, when the paternal and maternal sets of chromosomes remain spatially separated. To obtain joint metaphase plates, embryos were collected at 28–29 h post-hCG and incubated in M16 medium with colcemide for 30 min–1 h. Altogether, about 30 metaphase plates were prepared, among which 20 were with the separated sets of the parental chromosomes and seven contained joint metaphase plates. Two-cell embryos were collected from the mice with vaginal plugs at 32–48 h post-hCG. Only embryos with clearly visible nuclei were used to prepare spreads.
Preparation of Embryo Spreads
Air-dried spreads of embryos including those that were arrested in the first metaphase were prepared according the procedure described previously [16, 28] with the following essential modifications. Embryos were incubated in a mixture of 1.93% citric acid trisodium and 0.56% KCl (3:1 v/v) in double distilled water at 4°C for 5–10 min. Then, they were transferred into cold (0–4°C) standard fixative (a mixture of methanol and glacial acetic acid, 3:1 v/v) in a watch glass and held at 4°C for 5–10 min. Embryos were spread on a thoroughly cleaned slide by addition of a drop of softening solution A (methanol and 75% acetic acid in double distilled water, 1:1 v/v) followed by one drop of softening solution B (methanol and glacial acetic acid, 1:1 v/v). When the softening solution began to dry, the standard fixative was dropped on the slide to complete fixation.
Ribosomal DNA Hybridization Probes
The original pMr974, pMr100, and pMr3'Eco plasmids containing fragments derived from various regions of mouse rDNA units were kindly provided by Dr. I. Grummt (German Cancer Research Center, Heidelberg, Germany). The rDNA fragments were subcloned into pBluescript II KS vector (Stratagene), purified with Wizard DNA Purification System (Promega), and labeled with digoxigenin-UTP using the Nick Translation Kit (Roche) following recommendations of the manufacturers. Probe 1, an 11.35-kb EcoRI-EcoRI fragment, comprised parts of the nontranscribed spacer (NTS), the 5' external transcribed spacer (ETS), and the majority of the 18S sequence (residuals –5715 to +5635 relative to the transcription start site). Probe 2, a 6.6-kb EcoRI-EcoRI fragment, comprised a part of the 18S sequence, the internal transcribed spacers (ITS1 and ITS2), the 5.8S sequence, and the majority of the 28S sequence (residuals +5635 to12235). Probe 3, a 4.4-kb EcoRI-EcoRI fragment, was complementary to a minor part of the 28S sequence, the 3' ETS, and a short NTS sequence (residuals +12,235 to +16,535) (Fig. 1). Unless specially indicated, a mixture of these three probes was used for FISH.
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Fluorescence In Situ Hybridization
Embryo spreads were pretreated with 100 µg/ml RNase A (Roche) in 2x SSC (0.3 M NaCl and 0.03 M sodium acetate, pH 7.3) at 37°C for 1 h, washed in 2x SSC, and exposed to 0.01% pepsine (Sigma) in 0.01 HCl (pH 2) for 4–8 min at room temperature. After washing in PBS (137 mM NaCl, 2.7 mM KCl, 6.7 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.3), they were fixed with 3.7% paraformaldehyde in PBS for 10 min at room temperature and dried. Ten microliters hybridization mix that contained 10–25-µg rDNA probe(s), 500 ng/ml carrier salmon sperm DNA (Sigma), 500 ng/ml tRNA (Sigma), 10% dextran sulfate, and 50% deionized formamide (Sigma) in 2x SSC were placed on a spread and covered by a 18 x 18 mm coverslip. Denaturation of the target rDNA sequences and rDNA probe(s) was carried out simultaneously at 85°C for 10 min. The slides were subsequently transferred to moist chambers at 37°C for 12–14 h. Specimens were washed in 50% formamide in 4x SSC at 42°C, then in 2x SSC at room temperature, and incubated with mouse antidigoxigenin antibodies conjugated with rhodamine (Roche; diluted 1:20 in PBS containing 0.05% Tween 20 and 5% nonfat dry milk) followed by incubation with Texas red-conjugated rabbit anti-mouse immunoglobulins (Dianova; diluted 1:100 in PBS) for another 30 min. Spreads were stained with DAPI (Sigma) for 10 min, mounted in Mowiol (Calbiochem), and studied with an epifluorescence microscope Axiovert 200 (Carl Zeiss) using objectives 100x/NA 1.3 Fluar and 40x/NA 0.75 Plan-Neofluar. Images were acquired with a 13 bit black/white CoolSnapcf CCD camera (Roper Scientific), and analyzed with Adobe Photoshop software, version 6.
RESULTS
Localization of rRNA Genes in One-Cell Embryo Metaphase Chromosomes
In Mus musculus, NORs are known to be located in several chromosomes of the karyotype and occupy a position adjacent to the centromeric heterochromatin [2, 29–31]. In C57BL/6J inbred mice, which were used in this study, four NOR-bearing chromosomes have been identified by Ag-NOR staining technique [31]. Therefore, we first used embryo chromosome spreads to test the specificity and sensitivity of the FISH reaction in our experimental conditions. Chromosome spreads of F1 C57BL/6JxCBA hybrids were hybridized with either the mixture of three rDNA probes, which together recognize approximately half of the mouse rDNA repeat, or only with probe 2, which was complimentary to a transcribed part of the rDNA unit (Fig. 1). Only metaphase plates, which contained 40 chromosomes, e.g., the number characteristic for the mouse karyotype [29, 30], were used for FISH. Typical patterns of metaphase plates hybridized with the mixture of three rDNA probes (Fig. 2, a and b) or only with probe 2 (Fig. 2c) are shown in Figure 2. One can see that in all metaphase plates fluorescent signals were revealed as double or singular spots positioned in the proximal chromosome regions near the blocks of centromeric heterochromatin, which were more intensely stained with DAPI than the chromosome arms (Fig. 2, a–c). In total, seven NORs were present in diploid metaphase plates (Fig. 2a) and the combined haploid chromosome complements originating from parental pronuclei (Fig. 2, b and c). In some spreads, associations between NORs of two nucleolar organizing (NO) chromosomes lying in close proximity were observed (Fig. 2c). Because C57BL/6J mice contain four NO chromosomes per haploid set [31], and in our work females of this strain were used to obtain F1 hybrid embryos (see Materials and Methods), we concluded that the chromosome complements with four NORs were of the maternal origin, whereas those with three NORs originated from the paternal (CBA/Ca mice) pronuclei. Taken all together, these observations provided evidence that our conditions for the FISH reaction were adequate to map rDNA in mouse one- and two-cell mouse embryos.
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Like chromosomal NORs in somatic cells of various mammalian species [10, 26, 32], NORs observed in a single chromosomal plate of one-cell mouse embryos also varied in size. In addition to this chromosome-to-chromosome polymorphism, in all metaphase plates examined, unexpected differences in size or fluorescence intensity, or both, were revealed in sister chromatid NORs belonging to a single NO chromosome (Fig. 2, a'''–c'''). In the literature, the differences in banding pattern between adjacent regions of sister chromatids have been called lateral asymmetry [33]. Therefore, we used this term to designate asymmetry in sister chromatid NORs in embryo metaphase chromosomes as well. Lateral asymmetry was observed in chromosome NORs of various sizes, being most evident in relatively small ones (Fig. 2, a'''– c''').
Localization of rDNA in Transcriptionally Silent Embryos
As mentioned above, in mouse embryos resumption of rDNA transcription takes place at 44–45 h post-hCG, or in the middle of the second cell cycle [18]. Therefore, we used one-cell and early two-cell embryos to study the general arrangement of rDNA units when they remained transcriptionally inert. The hybridization mixture contained either three rDNA probes (Fig. 3, b, d, and e) or only probe 2 (Fig. 3, a and c). The results obtained were essentially similar. Figure 3 a–d represents typical pronuclei of one-cell embryos after 21 h (Fig. 3a; 8 pronuclei were examined), 24 h (Fig. 3b; 10 pronuclei were examined), 26 h (Fig. 3c; 11 pronuclei were examined), and 29 h (Fig. 3d; 10 pronuclei were examined) post-hCG. In Figure 3, a and b, the paternal pronuclei; in Figure 3, c and d, the maternal pronuclei; and in Figure 3e the second polar body are shown. In all spreads, fluorescent signals were revealed as bright spots of various sizes, among which larger ones predominated. In the second polar bodies, which were committed to degenerate, only large fluorescent granules were observed (Fig. 3e). In pronuclei, the rDNA signals were arranged in small and rather compact groups. The total number of these groups generally was equal to two or three; rarely, four groups were revealed in the female pronuclei (Fig. 3c). When zygotic nuclei were processed for FISH and afterwards counterstained with DAPI, NPBs became visible because of a higher local concentration of the chromatin at their surface (Fig. 3, a'–d'). Previously, a chromatin layer outlining NPBs was described in living mouse embryos after incubation with a chromatin dye, Hoechst 33342 [34], and by electron microscopy immunocytochemistry [24]. Overlapping of the two images—FISH signals and DAPI-stained pronuclei—clearly demonstrated that in nearly every pronucleus examined, from one to several NPBs (generally, two or three) were not structurally associated with rDNA (Fig. 3, a'', b'', and d''). In approximately 15% of pronuclei we observed rDNA signals located within the nucleoplasm without obvious association with NPBs (Fig. 3, c and d). In some embryos, rDNA-free NPBs and NPB-free rDNA were seen in the same pronucleus (Fig. 3d).
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In early two-cell embryos, fixed at 32 h post-hCG (a total of 20 nuclei were examined), NPBs were also seen after staining with DAPI. Following the FISH procedure, rDNAs were revealed as distinct clusters of signals, which number was augmented as compared with that seen in one-cell embryos (compare Fig. 4, a and a', with Fig. 3, a–d). However, as in zygotic embryos, the vast majority of FISH signals was coincident with or located in close proximity to the NPBs. For example, in a typical early two-cell embryo shown in Figure 4, a and a', the upper nucleus contains seven groups and the lower nucleus six groups of rDNA signals, among which only one group per nucleus does not associate with the NPBs. As in one-cell embryos, a certain number of NPBs did not interact with rDNA. In general, the more NPBs could be revealed within nuclei, the more NPBs lacking rDNA signals were observed in early two-cell embryo spreads (Fig. 4, a and a').
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It is noteworthy that the total number of rDNA clusters did not exceed the diploid number of the chromosomal NORs, equal to seven in F1 CBL57/6J x CBA/Ca hybrid embryos (Fig. 2a). This quantitative correlation suggests that the majority of these clusters correspond to individual chromosomal NORs.
Localization of rDNA in Transcriptionally Competent Embryos
Results of rDNA in situ hybridization in embryos fixed at 46–48 h post-hCG (a total of 26 late two-cell embryo nuclei were examined) are shown in Figure 4, b and c, and Figure 5. In these embryos, which are known to actively transcribe rDNA [18], the rDNA topology was significantly changed as compared to that in transcriptionally inert (i.e., one-cell and early two-cell) embryos. In late two-cell embryos, hybridization signals became much more decondensed and formed either extended arrays (Fig. 4, c–c'') or loosely packed clusters (Fig. 4, b–b''). At the end of the second cell cycle (48 h post-hCG), it became particularly evident that the clusters were formed by variable numbers of small rDNA comprising foci (Fig. 5). In the largest clusters up to a few decades of foci could be recognized (Fig. 5, b and d). All rDNA clusters were structurally associated with the NPBs revealed either with DAPI (Fig. 4, b and b') or under phase contrast (Fig. 5, a and c). Nevertheless, as in transcriptionally inert embryos, in transcriptionally competent embryos a portion of NPBs was lacking contacts with rDNA. For example, in Figure 4, b and b', three rDNA-free NPBs among seven and in Figure 5, a–c, four rDNA-free NPBs among 10 can be recognized. The total number of the rDNA clusters per nucleus did not exceed the number of the NO chromosomes in F1 hybrid embryos (seven; Figs. 5, a–c, and 2, a–c).
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DISCUSSION
The main purpose of this study is to shed more light on the general arrangement of rRNA genes in early mammalian embryos in relation to ongoing rDNA transcription. To this end, we applied a highly sensitive FISH with probes to mouse rDNA to map rDNA in transcriptionally silent (one-cell and early two-cell) and competent (late two-cell) mouse embryos. The results of our work allowed us to make the following major conclusions.
General Arrangement of rRNA in Early Mouse Embryos
The general arrangement of rDNA in early mouse embryos strictly relates to the rRNA gene transcription status. When synthesis of rRNA is suppressed (one-cell embryos, early two-cell embryos, and polar bodies), rRNA genes are organized in relatively small clusters comprising from one to a few rather large fluorescent foci. The most compact organization of rRNA genes was observed in the second polar bodies, which are known to remain transcriptionally silent and eventually degenerate [35] (Fig. 3e). In contrast, in late two-cell embryos, which actively transcribed rRNA, the rDNA clusters became much more relaxed, and the number of their comprising foci was profoundly augmented (Figs. 4c and 5). In such embryos, the loose clusters were composed of numerous small punctuate fluorescent foci, which arrangement and regular size were quite compatible with the assumption that one focus corresponds to an rDNA unit or compact aggregates of a few units [27, 36] (Figs. 4c'' and 5d). These observations are in good agreement with results on localization of rDNA in transcriptionally active versus inactive nucleoli of somatic cells, wherein activation of rDNA transcription is also accompanied by structural relaxation and augmentation of the rDNA focus numbers [10, 12, 25, 26]. They also fit with the finding described in porcine embryos, wherein rDNA forms compact clusters in transcriptionally inert (one-cell) embryos and dispersed clusters in transcriptionally active (eight-cell) embryos [37]. Therefore, one can conclude that unraveling of rDNA in response to activation of rRNA synthesis is a general feature of somatic cells and early mammalian embryos, even through the embryos lack the characteristic nucleoli. Apparently, unfolding of rDNA clusters facilitates interactions of RNA polymerase I and its cofactors with the targeting genes.
Spatial Interactions Between NPBs and rDNA
The NPBs are intranuclear entities, which full homologues have not been described in somatic cells so far. In contrast to prenucleolar bodies (PNBs), which serve as precursors of the mature nucleoli in somatic cells in mitosis [5], embryo NPBs remain intact during interphase, but become dissolved during zygotic cleavage [16]. Other essential differences between NPBs and PNBs are the precursor sizes (up to 10 µm in NPBs and 0.2 µm in PNBs) and their fine organization: NPBs are tight and homogenous fibrillar bodies [13, 16, 23], whereas PNBs are loose and fibrillogranular structures [38–40]. Based on the commonly accepted idea that NPBs serve as a structural support for reactivated rRNA genes, one could expect that all rRNA genes, including those not transcribed, also associate with NPBs. However, the results of our work show that this is not entirely true. As shown in Figures 3, 4, and 5c, from one to several NPBs in almost every nucleus examined do not interact with rDNA. The rDNA-free NPBs were present in nuclei of one-cell as well as late two-cell embryos, even though zygotic pronuclei remained transcriptionally silent, whereas late two-cell embryos actively synthesized rRNA (Figs. 3, a, b, and d, and 4a, inactive embryos; Figs. 4b, and 5c, active embryos). These observations confirm the recently obtained data that demonstrate that in late two-cell mouse embryos a portion of NPBs does not recruit the RNA polymerase I transcription machinery and the nucleolar protein fibrillarin, which are involved in rDNA transcription and processing of the nascent rRNA transcripts, respectively [18]. Taken together, these observations argue in favor of a novel idea that NPBs can persist without direct associations with rDNA and therefore might be heterogeneous in their ability to nucleate the assembly of the functional nucleoli. Nevertheless, one cannot exclude the possibility that spatial interactions between rRNA genes and NPBs are dynamic and could temporally be established or lost during embryo development. Thus, reduction of the DNA-free NPB number might occur because of NPB fusion. The structural and molecular peculiarities of rDNA-bound and rDNA-free NPBs merit further investigation. Why rRNA genes have a tendency to associate with NPBs is also an open question.
NOR Polymorphism
Finally, our data showed that, as in mouse somatic cells, embryo chromosome NORs detected in the first cleavage mitosis by FISH vary in size and/or fluorescence intensity (Fig. 2, a–c). In somatic cells of various mammalian species, chromosome-to-chromosome NOR morphological variability detected by Ag-NOR staining or FISH is considered as an indicator of unequal distribution of rDNA unit copies between NOR-bearing chromosomes [29, 30, 32, 41, 42]. Therefore, we concluded that rRNA genes are unequally distributed between mouse embryo chromosomes as well. Unexpectedly, variations in size and fluorescence intensity were also observed in NORs of sister chromatids of a single metaphase chromosome. These observations provide evidence in favor of the novel view that in mouse early embryo chromosomes rRNA gene copies might be unequally distributed between sister chromatids (Fig. 2, a'''–c'''). In the literature, differences in banding pattern between adjacent regions of sister chromatids, including NORs, are referred as lateral asymmetry [33]. Despite the fact that the origin of lateral asymmetry still remains unclear, this phenomenon has been described in the constitutive (C-bands) and intercalated (G-bands) heterochromatin of mouse embryo chromosomes [43, 44]. Lateral asymmetry has also been shown in Ag-NORs of somatic metaphase chromosomes after cell incubation with an analogue of thymidine, BrdU [45, 46]. Asymmetry of sister Ag-NORs was interpreted in terms of their asymmetrical transcription activity, caused by unequal incorporation of BrdU to rDNA of the two chromatids. However, in our work the lateral asymmetry of NORs was observed in chromosomes of zygotic embryos, which remained transcriptionally inert and Ag-negative [14, 16]. Embryos were not incubated with BrdU before the hybridization in situ procedure. We assume, therefore, that lateral asymmetry in sister chromatid NORs might reflect differences in their number of rRNA gene copies that arise because of recombination between rDNA repeats.
Recombination events between rDNA sequences are known for many years and have been described by genetic approaches in various species including yeasts, flies, and mice. It has been proposed that in Drosophila rDNA genetic recombination would lead to the production of new sister chromatid strands, one containing a greater number and the other a lesser number of rDNA tandem repeats than originally contained in either parental chromatid [47]. On the other hand, it has been experimentally shown that in mice rDNA recombination between nonhomologous chromosomes are rare [48]. It is tempting to speculate, therefore, that the lateral asymmetry of NORs detected by FISH in the first cleavage mitosis is a morphological manifestation of unequal genetic exchanges among rDNA sequences on sister chromatid NORs, which occur in one-cell mouse embryos. If so, this event could lead to unequal distribution of rRNA genes between blastomeres during the first mitotic cleavage, thus providing them with different developmental potencies. Indeed, spontaneous reduction of rRNA gene copy number in chicken embryos by a factor of two affects their growth and finally arrests embryo development [49]. It would be interesting, therefore, to study the variability of chromosome NORs in mouse oocytes as well as in two-cell and later mouse embryos to shed more light on the relationship between rRNA gene copy number, embryo viability, and blastomere potencies for differentiation.
ACKNOWLEDGMENTS
We are thankful to Dr. I. Grummt (German Cancer Research Center, Heidelberg, Germany) for the original rDNA coding plasmids; Drs. F. Wachtler and K. Schoefer (Histologisch-Embryologisches Institut der Univ. Wien, Austria) for help with assimilation of a FISH technique; and Dr. M. Olson (University of Mississippi Medical Center, Jackson, MS).
FOOTNOTES
1 Supported by the Russian Scientific Foundation for Basic Researches (projects 02-04-49373, 03-04-48951, and 06-04-49392). 
2 Correspondence: Olga Zatsepina, Schemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, Moscow, Russia 117997. FAX: 7 095 335 71 03; zatsepin{at}ibch.ru
Received: 13 September 2005.
First decision: 5 October 2005.
Accepted: 16 January 2006.
REFERENCES
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