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Biology of Reproduction 66, 534-543 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Nucleolar Changes in Bovine Nucleotransferred Embryos1

V. Barana,b, X. Vignona, D. LeBourhisa,c, J.P. Renarda, and J.E. Fléchon2,a

a Institut National de la Recherche Agronomique, Biologie du Développement et Biotechnologies, 78352 Jouy-en-Josas, France b Institute of Animal Physiology, 040 01 Kosice, Slovakia c UNCEIA, Services Techniques, 94750 Maisons-Alfort, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study focused on nucleolar changes in bovine embryos reconstructed from enucleated mature oocytes fused with blastomeres of morulae or with cultured, serum unstarved bovine fetal skin fibroblasts (embryonic vs. somatic cloning). The nucleotransferred (NT) embryos were collected and fixed at time intervals of 1–2 h (early 1-cell stage), 10–15 h (late 1-cell stage), 22–24 h (2-cell stage), 37–38 h (4-cell stage), 40–41 h (early 8-cell stage), 47–48 h (late 8-cell stage), and 55 h (16-cell stage) after fusion. Immunocytochemistry by light and electron microscopy was used for structure-function characterization of nucleolar components. Antibodies against RNA, protein B23, protein C23, and fibrillarin were applied. In addition, DNA was localized by the terminal deoxynucleotidyl transferase (TdT) technique, and the functional organization of chromatin was determined with the nick-translation immunogold approach. The results show that fully reticulated (active) nucleoli observed in donor cells immediately before fusion as well as in the early 1-cell stage after fusion were progressively transformed into nucleolar bodies displaying decreasing numbers of vacuoles from the 2- to 4-cell stage in both types of reconstructed embryos. At the late 8-cell stage, morphological signs of resuming nucleolar activity were detected. Numerous new small vacuoles appeared, and chromatin blocks reassociated with the nucleolar body. During this period, nick-translation technique revealed numerous active DNA sites in the periphery of chromatin blocks associated with the nucleolar body. Fully reticulated nucleoli were again observed as early as the 16-cell stage of embryonic cloned embryos. In comparison, the embryos obtained by fetal cloning displayed a lower tendency to develop, mainly during the first cell cycle and during the period of presumed reactivation. Correlatively, the changes in nucleolar morphology (desegregation and rebuilding) were at least delayed in many somatic NT embryos in comparison with the embryonic NT group. It is concluded that complete reprogramming of rRNA gene expression is part of the general nuclear reprogramming necessary for development after NT.

developmental biology, early development, gene regulation, nuclear reprogramming


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous experiments have provided evidence that successful cloning of mammals is possible with embryonic cells [1, 2], somatic fetal cells [3, 4], and various types of adult differentiated cells [5–7]. Essentially, two different strategies were used in the case of somatic cloning: one using quiescent cells as donors of nuclei [5, 6], and the other one favoring use of proliferating cells [7, 8]. However, the debate is not closed, because some proliferating cells may be in the G0/G1 phase. In the present study, we have compared the ability of blastomere nuclei (embryonic nucleotransfer [NT]), which were never observed to be in the G0 stage, to be reprogrammed with skin cell nuclei (somatic NT) from serum unstarved culture. The activity of transferred nuclei was tested by analyzing the structure-function parameters of the nucleoli. For reasons still unknown, the production rate of cloned liveborn animals is not efficient, whatever the technique. Only limited knowledge exists regarding the reprogramming processes in NT early embryos. Methods of NT are based on the introduction of a foreign nucleus (from blastomere of preimplantation embryos or somatic cultured cell) into a cytoplast (an enucleated mature oocyte) followed by in vitro culture before embryo transfer to a recipient female. In cleaving zygotes, the resumption of nuclear/nucleolar activity during species-specific stages of preimplantation development is controlled by small nuclear ribonucleoprotein (snRNP) complexes of maternal origin present in oocyte cytoplasm [9, 10]. These same factors are present in the cytoplasts, and the transferred nuclei seem to recapitulate the nuclear events that precede activation of the embryonic genome in normal embryos. A preliminary step would be a rapid and complete inhibition of gene expression of transferred nuclei [11] to come back to the status of inactive pronuclei and to avoid, as much as possible, precocious somatic-type gene expression in cleaving embryos [12]. Accordingly, already-active nuclei originating from morulae were observed to stop RNA synthesis after fusion [11, 13].

Typical changes in nucleolar ultrastructure are good morphological markers of rRNA synthesis. Resumption of nucleolar activity is accompanied by a well-characterized transformation of nucleolar architecture during the 8-cell stage of bovine embryos collected in vivo [14]. The nucleolus very rapidly reacts to cell metabolic changes. In bovine embryonic NT embryos, a reversible inhibition of nucleolar activity was recorded at the 2-cell stage, followed by reinitiation one cell cycle earlier (at the 4-cell stage) than in control (in vitro-produced) embryos [15]. Four papers [15–18] presenting ultrastructural changes of nucleolar morphology in NT embryos after fusion showed that originally active nucleoli became vacuolized and lost their granular component before the 8-cell stage, in which a nucleolar reconstruction occurred. So, it seems to be generally accepted that a transient nucleolus transcriptional silencing occurs. The rRNA synthetic machinery would be reactivated as development proceeds in developing NT embryos.

The present study focused on nucleolar changes in bovine embryos reconstructed from mature enucleated and interphasic oocytes fused with blastomeres of bovine morulae (embryonic NT) or with bovine fetal skin fibroblasts (somatic NT). The aim of the study was to compare embryonic versus somatic NT embryos and to provide a detailed analysis of nucleologenesis in NT embryos after fusion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Production of NT Embryos

The NT embryos were reconstituted from in vitro-matured oocytes by fusion with bovine morula blastomeres (embryonic NT embryos) or fetal bovine skin fibroblasts (somatic NT embryos). The detailed protocols have already been described [7, 19, 20]. Embryos used to provide nuclei for embryonic NT were routinely produced in vitro; they were cultured for 5 days in B2 medium (CCD Laboratories, Paris, France) supplemented with 2.5% (v/v) fetal calf serum (FCS; Sigma-Aldrich, St. Quentin Fallavier, France) up to the morula stage (40–60 cells). Cells cultured for somatic NT were derived from skin dissected from the back of a 2-mo-old bovine fetus. Skin explants were directly cultured in plastic Petri dishes in Dulbecco modified Eagle medium supplemented with 2 mM glutamine, 100 U/ml of penicillin, 100 µg/ml of streptomycin (Life Technologies, Cergy Pontoise, France), and 10% FCS. The outgrowths were then passaged every 3 days at a 1:3 dilution. After three passages, the cell culture appeared as a homogeneous layer of fibroblast-like cells. Eighty percent confluent cells were used between passages 4 and 15. The cell-cycle phase of the donor cells was at least 80% in S phase for embryonic cells according to Barnes et al. [21] and approximately 40% in S phase, <30% in the G2/M phase, and >30% in the G1 phase for the subconfluent somatic cells (unpublished results). Bovine oocytes were obtained by aspiration from ovaries collected at a slaughterhouse. The oocytes were matured in vitro, stained with 0.5 µg/ml of Hoechst 33342 (Sigma-Aldrich), and enucleated by micromanipulation under an inverted microscope equipped with epifluorescence to control chromosome removal. The cytoplasts were aged in vitro for 8–10 h and stored at 10°C for 6–8 h until fusion or treated with 2 mM 6-dimethylaminopurine (6-DMAP; Sigma-Aldrich) for 4 h before fusion according to the method of Loi et al. [22].

Dissociated donor cell was inserted under the zona pellucida and fused with the recipient cytoplast (activated at the same time) by electrostimulation with two electric pulses of 1.2 kV/cm for 50 µsec. Reconstituted embryos were cultured in B2 medium microdrops with 10% FCS on a Vero cells (Rhône-Mérieux, Lyon, France) monolayer for in vitro development.

Collection of NT Embryos

For microscopy, the cultured reconstituted embryos were harvested at different time intervals after fusion and distributed according to cell stage: early or late 1-cell, 2-cell, 4-cell, early or late 8-cell, and 16-cell embryos (Table 1). Each group of embryos was processed and evaluated separately. To check development, separate batches of embryos, which were not processed for microscopy, were cultured until the blastocyst stage (Table 2).


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  		TABLE 1. Time periods, developmental stages, and numbers of embryos used for microscopic analysis


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  		TABLE 2. In vitro developmental potential of embryos reconstructed from embryonic or somatic cells

The zona pellucida was removed with 0.5% pronase. After washing, the embryos were fixed for electron-microscopic labeling (with 3.7% paraformaldehyde and 0.2% glutaraldehyde mixture) or light-microscopic immunocytochemistry (with 3.7% paraformaldehyde) for 30 or 60 min at 4°C. The aldehydes were prepared in 0.1 M phosphate buffer or Sörensen buffer (pH 7.3–7.4). The same buffer was used for the washing step after fixation.

Immunofluorescence

Fixed and extensively washed embryos were permeabilized with 0.5% (v/v) Triton X-100 in PBS for 15–30 min at room temperature. Whole embryos were processed by the indirect immunofluorescent staining method for detection of the nucleolar proteins fibrillarin and B23/nucleophosmin. Primary (Table 3) and secondary antibodies diluted in PBS containing 0.25% (w/v) BSA were applied for overnight incubation at 4°C or 1-h incubation at room temperature. As a final step, the washed embryos were mounted on glass slides using Mowiol (Hoechst, Frankfurt am Main, Germany). For immunostaining control, the primary antibody was omitted.


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  		TABLE 3. Panel of applied primary antibodies

Actinomycin D-Treated Donor Cells

Bovine fetal skin fibroblasts exponentially growing in monolayers were treated with 5 µg/ml of actinomycin D (Sigma-Aldrich) for 15, 30, and 60 min to inhibit rRNA transcription. The cell cultures with or without drug treatment were fixed in situ for 30 min at 4°C with 3.7% paraformaldehyde prepared in PBS or Sörensen buffer (pH 7.3).

Immunofluorescent staining was performed on cells grown on coverslips. After fixation, the cells were permeabilized with 0.5% Triton X-100 in PBS containing 0.5% BSA. Primary antibodies (against fibrillarin or protein B23/nucleophosmin) were applied overnight at 4°C. Secondary antibodies conjugated with fluorescein isothiocyanate or Texas red were applied for 60 min at room temperature. For immunostaining control, the primary antibody was omitted.

Electron-Microscopic Immunolabeling

Fixed and washed embryos were pre-embedded in agar blocks (2% [w/v] in PBS), dehydrated with ethanol, and embedded in LR White (Sigma, St. Louis, MO) or Lowicryl K4M (Chemische Werke Lowi, Waldkraiburg, Germany) resin by polymerization at -20°C under ultraviolet light.

Ultrathin sections were labeled by the indirect immunogold method. For preincubation, 10% normal FCS or 5% (v/v in PBS) normal goat serum (NGS) was used. Primary and secondary antibodies were diluted in PBS (pH 7.3) containing 5% FCS or 2.5% (v/v) NGS. After washing, bound primary antibodies were revealed with species-specific secondary antibody coupled with 10-nm colloidal gold particles (Amersham, Saclay, France). All incubations were performed at room temperature or, for some primary antibodies, overnight at 4°C. Labeling technique was tested by omitting the primary antibody or incubating the sections with gold particles free of secondary antibody. After the immunolabeling procedure, the sections were contrasted with 5% uranyl acetate in water for 15 min.

For the most sensitive detection of DNA in situ, the terminal deoxynucleotidyl transferase (TdT) immunotechnique was used [28]. Ultrathin sections were incubated in TdT medium containing 20 µM 5-bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP; Sigma-Aldrich) for 10 min at 37°C, followed next by incubation in the same medium supplemented with dCTP, dATP, and dGTP for 15 min at 37°C. Incorporated BrdUTP was then visualized by the indirect immunogold labeling method. In control experiments, TdT was omitted in the medium or BrdUTP was replaced by 5-bromouridine monophosphate.

Ultrastructure and immunolabeling of nucleoli were analyzed on serial sections.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The data collected in this study represent standard morphological features observed in most of the samples prepared at each time interval after fusion (see Table 1 for the chronology of normal in vitro development). The embryos cleaving at a lower speed than normal for in vitro development were not used for microscopic analysis, except for 1-cell stage-arrested embryos. As shown in Table 3, somatic NT embryos displayed a lower tendency to develop at all cleavage stages. Their nucleoli were more variable in morphology compared with embryonic NT embryos. This fact was mostly observed during the period of presumed nucleolar reactivation (at the 8-cell stage). Development from the 8- to 16-cell to the blastocyst stage was at least delayed in almost all somatic NT embryos.

Light Microscopy

In donor cells, light-microscopic immunostaining of protein B23 (Fig. 1a) and fibrillarin (Fig. 1d) exhibited the typical appearance of fully active nucleoli displaying a branching structure. The same staining pattern was observed in morula blastomeres, as already shown [29], immediately before insertion under the zona pellucida of recipient cytoplasts. Treatment of the cycling cells with actinomycin D resulted in loss of the branched pattern of active nucleoli. After the 15-min treatment (Fig. 1, b and e), the nucleoli became rounded. The 60-min treatment induced ring-shaped nucleoli (Fig. 1, c and f).



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  		FIG. 1. Confocal immunofluorescence of protein B23 (a–c) and fibrillarin (d–f) on cultured bovine skin fibroblasts (donor cells). a and d) Normally growing fibroblasts. b and e) After 15-min treatment with actinomycin D. c and f) After 60-min treatment with actinomycin D. Compared to controls, the nucleoli have already lost their reticulated structure within 1 h of treatment. Bars = 10 µm

Because cytoplasts, after aging in vitro or 6-DMAP treatment, were interphasic [30], the transferred nuclei did not lose their envelope and grew, in favorable cases, to the size of pronuclei.

After light-microscopic immunostaining of protein B23 (Fig. 2, a–d) and fibrillarin (Fig. 2, e–h), somatic NT embryos were analyzed in parallel with skin cells. They had already lost the branching nucleolar structure early during the 1-cell stage and, in addition, revealed migration of the transferred nucleus from the periphery toward the cytoplast center. After that time, the nucleoli further compacted and rounded, keeping a heterogeneous fluorescent staining. During the 2-cell stage (Fig. 2, d and h), the nucleoli were denser and smaller in comparison with those of the 1-cell period. At the 4-cell stage, the protein B23 immunostaining pattern became more diffuse throughout the nucleoplasm (data not shown). These same changes were recorded in embryonic NT embryos fixed at the same time after fusion (data not shown). In embryos uncleaved at 22–26 h after fusion, propidium iodide staining revealed a variable appearance of chromatin, which was very often fragmented (Fig. 3a) and condensed (Fig. 3b).



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  		FIG. 2. Confocal immunofluorescence of protein B23/nucleophosmin (a–d) or fibrillarin (e–h) on somatic NT embryos at 30 min (a and e), 60 min (b and f), and 120 min (c and g) after fusion and the 2-cell stage (d and h). Bars = 10 µm



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  		FIG. 3. Fluorescence microscopy after propidium iodide staining of chromatin structure in degenerated somatic NT embryos at the 1-cell stage. Nuclear fragmentation (a) and extreme chromatin condensation (b) are observed. Bars = 10 µm

Electron Microscopy

The description of nucleologenesis and of the different components of the nucleolus in the bovine embryo has been given previously by Kopecny et al. [14].

Early 1-cell stage During the early 1-cell stage, fully reticulated (active) nucleoli observed in donor cells immediately before fusion progressively changed their morphology (Fig. 4, a–d). Although the reticulated character of nucleoli was preserved, the global shape of nucleoli became more regular and roundish, as illustrated by the aspect of the B23-positive dense fibrillar component (Fig. 4d). The DNA was intensely labeled in close association with the nucleolus as well as inside (fibrillar centers) in both types of NT embryos (Fig. 4, a and c). The RNA was regularly localized in the dense fibrillar component (Fig. 4b).



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  		FIG. 4. Ultrastructure and immunolabeling (arrows) of nucleoli in early 1-cell stage NT embryos. DNA was labeled with TdT technique inside and/or around the nucleolus (a and c). Localization of RNA (b) and protein B23 (d) within dense fibrillar component of nucleolar body was observed. a and b) Embryonic NT embryos. c and d) Somatic NT embryos. Bars = 0.5 µm

Late 1-cell stage During the late 1-cell stage, fully reticulated nucleoli were no longer observed. Instead, round nucleoli were visible and composed only of compact fibrillar material arranged around electron-lucid areas (vacuoles) of different sizes. The vacuoles were free of DNA in the case of embryonic NT embryos and in the majority of somatic NT embryos (Fig. 5, a and c). However, in some somatic NT embryos, a few gold particles revealing DNA by the TdT technique were visible inside the vacuoles (data not shown). The reduced dense fibrillar component was labeled with anti-B23 in both cases (Fig. 5, b and d).



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  		FIG. 5. Electron-microscopic morphology and immunolabeling (arrows) of nucleoli in late 1-cell stage NT embryos. DNA labeled using the TdT technique was found only within the periphery of the vacuolated nuclear body (a and c). Localization of protein B23 (b and d) within the dense fibrillar component of the nucleolar body was observed. a and b) Embryonic NT embryos. c and d) Somatic NT embryos. Bars = 0.5 µm

2-Cell stage At the 2-cell stage, round nucleoli contained fewer, but relatively larger, vacuoles in comparison with the previous stage. Chromatin was labeled outside the nucleoli in embryonic NT embryos (Fig. 6a) and in the majority of somatic NT embryos, except for a few in which small gold clusters were still visible inside the vacuoles (Fig. 6d). Occasionally, in both types of embryos, nucleoli were observed displaying segregation of fibrillar and granular nucleolar components (data not shown). The dense fibrillar component was slightly labeled with anti-B23 (Fig. 6b).



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  		FIG. 6. Electron-microscopic morphology and immunolabeling (arrows) of nucleoli in 2-cell stage (a, b, and d) and 4-cell stage (c, e, and f) NT embryos. The DNA was localized with the TdT technique inside or outside the nucleolus body (a and d). Localization of protein B23 (b and f) showed an exceptionally dense labeling in somatic NT embryos (f). RNA (c) and protein C23 (e) were localized within the nucleolar body. a–c) Embryonic NT embryos. d–f) Somatic NT embryos. Bars = 0.5 µm

4-Cell stage At the 4-cell stage, vacuolated nucleoli became ring-shaped with occasional dense inclusions (Fig. 6c). The DNA was no longer detected inside the vacuole, except for a few cases in somatic NT embryos (data not shown). The dense fibrillar component was generally labeled with anti-B23, anti-C23, and anti-RNA in both types of embryos (Fig. 6, c, e, and f).

Early 8-cell stage During the early 8-cell stage, nucleoli maintained their round and vacuolated appearance. In embryonic NT embryos, the vacuoles were more numerous and smaller than those observed at the 4-cell stage. Chromatin was closely associated with the nucleolar bodies as a continuous surrounding layer (Fig. 7a). In somatic NT embryos, the nucleolar bodies were round with small vacuoles and, in some cases, were partially associated with chromatin (Fig. 7b).



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  		FIG. 7. Electron-microscopic morphology and immunolabeling (arrows) of nucleoli in early 8-cell stage embryos. DNA labeled using the TdT technique is associated with the periphery of the vacuolized dense nucleolus body. a) Embryonic NT embryo. b) Somatic NT embryo. Bars = 0.5 µm

Late 8-cell stage During the late 8-cell stage, embryonic NT embryos contained multivacuolated nucleoli displaying a tendency toward reticulation. The DNA was detected in the periphery of nucleoli as well as in several foci with an appearance like fibrillar centers (Fig. 8a). In somatic NT embryos, round, compact nucleolar bodies displayed a large vacuole and/or a few small vacuoles surrounded by a dense fibrillar component, and chromatin blocks were distributed either close to or in partial association with the nucleoli (Fig. 8c). A small amount of RNA was localized in the dense fibrillar component in both cases (Fig. 8, b and d).



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  		FIG. 8. Electron-microscopic morphology and immunolabeling (arrows) of nucleoli in late 8-cell stage embryos. DNA was labeled with the TdT technique (a), and immunolocalization of RNA was observed (b and d). Chromatin blocks (arrows) associate with vacuolized nucleolus, not labeled (c). a and b) Embryonic NT embryos. c and d) Somatic NT embryos. Bars = 0.5 µm

16-Cell stage At the 16-cell stage, embryonic NT embryos possessed typical reticulated nucleoli with a branched dense fibrillar component. These active nucleoli were very intensely penetrated by DNA (Fig. 9a). Somatic NT embryos had nucleolar bodies very similar to those of embryonic NT embryos at the late 8-cell stage, that is, multivacuolated with signs of ongoing reticulation (Fig. 9b). The DNA was penetrating the nucleolar body in embryos reaching this cell stage.



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  		FIG. 9. Electron-microscopic morphology and immunolabeling (arrows) of nucleoli in 16-cell stage embryos. DNA was labeled in presumed fibrillar centers using the TdT technique. Bars = 0.5 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Morphological Changes of the Nucleolus after NT

Our experiments extend previous electron-microscopy observations by King et al. [17], Lavoir et al. [18], and Kanka et al. [15] describing nucleolar changes in embryonic NT embryos during the first cleavage stages. As already observed by Lavoir et al. [18] and Kanka et al. [15], loss of the granular component and vacuolization clearly are the most marked ultrastructural features of the nucleolar changes. This remodeling can be interpreted as a gradual decrease of transcriptional activity. In fact, the morphological evidence of decreasing rRNA synthetic activity is paralleled by the temporary decline of all nuclear RNA synthetic activity as shown by autoradiography [13].

Parallel Effects of Actinomycin D and NT as Shown with Protein Markers

Protein B23 and protein C23 are major nucleolar proteins located in the granular and dense fibrillar components [25, 26]. These proteins are involved in activation of rDNA transcription (protein C23) and in processing of pre-rRNA (protein B23) during assembly of preribosomes [26, 31]. During mitosis, they are redistributed into the cytoplasm [25], where they temporarily associate with the chromosome periphery before being associated with prenucleolar bodies [27, 32–34]. From a structural point of view, these proteins are components of the nucleolar/nuclear matrix [35–37].

Fibrillarin, a nucleolar protein marker localized in the dense fibrillar component [24], has been shown to be associated with U3 snRNA [23] and with U8 snRNA and U13 snRNA [38] in the snRNP complex involved with the early processing of pre-rRNA molecules [39]. Fibrillarin was found in nucleolus remnants and prenucleolar bodies [40], contributing to nucleolar reformation during interphase [41]. The amount of fibrillarin increases or decreases depending on the nucleolar activity [42].

Actinomycin D blocks pre-rRNA transcription and/or processing of preformed pre-rRNA transcripts and modifies nucleolar morphology depending on the dose and duration of treatment with this drug [43, 44]. The high concentration we used probably blocked both pre-rRNA synthesis and processing of preformed pre-rRNA molecules. Under these conditions, the silver-stained proteins, B23 and C23 [45, 46], were observed during interphase in the fibrillar, but not in the granular, component at the periphery of segregated nucleoli and in dense fibrillar bodies scattered in the cytoplasm [47], and the granular component was translocated from the inactivated nucleolus to the nucleoplasm [48].

Our results on donor cells show that loss of the reticulated appearance of the active nucleolus is very rapid after rRNA transcription block by actinomycin D. Compact, round nucleoli were already observed after 15 min and fully segregated nucleoli after 1 h of treatment. The decreasing immunofluorescent signal observed with 1-h treatment of somatic cells corresponds to the loss of granular component (decrease of B23 staining) and to a tendency of small prenucleolar bodies to assemble (fibrillarin staining).

Similar changes were observed in immunofluorescent localization of B23 and fibrillarin during the first two cell cycles of NT embryos, suggesting a progressive silencing of the rRNA transcription machinery. At the electron-microscopic level, only small amounts of both proteins B23 and C23 were found in the compact fibrillar structure of vacuolized nucleoli. So, the nucleolus obviously loses the granular component after fusion, although marker proteins are still found in the nucleolar remnants, as in mitotic somatic cells (see above). In the case of protein C23, the incidence of immunogold signal was constant at all developmental stages after NT.

Nucleolar Nucleic Acids

The immunocytochemical labeling of DNA revealed total (embryonic NT embryos) or partial (somatic NT embryos) disassociation of chromatin from vacuolated nucleolus bodies followed by DNA reintroduction inside the rebuilding nucleoli of late 8- or 16-cell embryos, respectively. These changes can be the manifestation of rDNA chromatin remodeling as part of the general nuclear reprogramming [49, 50]; they are absolutely analogous with progressive DNA penetration into the nucleolar precursor body of the normal embryo during activation of rRNA synthesis after fertilization [14, 29, 51]. The RNA labeling in compact or vacuolated nuclei is not as informative as the DNA labeling, because RNA may be present in inactive nucleolus precursor bodies of bovine embryos [52]. In the present case, the low level of RNA observed in nucleolar bodies before rebuilding of reticulated nucleoli may correspond to that found in embryos after fertilization and/or to pre-rRNA resulting from inhibition of processing after NT, as after treatment with actinomycin D [44, 48].

From our study, it appears that rRNA expression is generally completely repressed after embryonic NT in an enucleated and activated oocyte. It is probably a necessary, but not a sufficient, condition for correct reprogramming, as shown by possible defects in ribosomal synthesis detected later [10]. Incomplete inhibition of either rRNA or mRNA synthesis, as detected in the present study and that of Chastant-Maillard et al. [53], respectively, may disturb the onset of development. It may also be the case for mRNA transcripts of the donor cell [54]. If so, then nuclear injection [55] would be preferable to cell fusion; however, no improvement was observed in a preliminary experiment [56].

Nucleologenesis after NT

In this report, the reinitiation of nucleolar activity was observed at the late 8-cell stage, and fully reticulated nucleoli were formed in 16-cell morulae of embryonic NT embryos. In comparison, the somatic NT embryos displayed a lower rate of development, mainly during the period of presumed nucleolar reactivation between the 8- and 16-cell stages (11% vs. 50%, P < 0.001, see Table 2). Observation of normally cleaving somatic NT embryos revealed that nucleolar reticulation was delayed and appeared less frequently in somatic NT embryos than in embryonic NT embryos obtained under the same experimental conditions. Decreased viability of somatic versus embryonic NT embryos was also observed, not only in our laboratory and not only in the bovine species [57]. However, this is not a valid rule in all cases, because many other factors may influence the development of NT embryos [49]. To obtain comparative materials, we used the same procedure for embryonic and somatic NT, that is, fusion of a donor cell with an aged or preactivated oocyte. This procedure yielded, in our hands, better results with embryonic than with somatic cloning [7, 20]; the reasons for this are not yet known. In both cases, donor cells were not induced into a quiescent stage. In these conditions, a large portion of the somatic cells were likely in the G0 /G1 stage, and some of them, as well as most of the embryonic ones, were in the S phase [21]. However, the cell-cycle stage of the donor cells is not likely to account for the different results, because the technique involved use of recipient cytoplasts in the interphasic stage [30], which are compatible with all phases of the transplanted nucleus [21]. When using fusion into enucleated MII oocytes followed by activation instead of the former procedure, somatic cloning proved to be as efficient as embryonic cloning (30% blastocysts obtained in our laboratory with skin fibroblasts; see also [5, 8]). It would be of interest to analyze how the nucleus and nucleolus are remodeled in such recipient oocytes to gain a better understanding of nuclear reprogramming.

Conclusion

The present study compared nuclear (nucleolar) reprogramming in two populations of NT embryos differing in cleavage rate, and interestingly, a possible link between delay in nucleolar changes and cleavage rate was found. Although an improvement in efficiency of development after NT can be expected, mainly by reducing pre- and perinatal mortality [58], the early stages after fusion also represent a clue period for NT embryos [59], because early hidden defects may have long-term effects [60].


   ACKNOWLEDGMENTS
 
The authors thank E. Laloy for preparation of the donor cells and E.M. Tan, P.K. Chan, R.L. Ochs, and M. Thiry for the kind gift of antibodies. The authors also thank R. Hunter for improving the style of the manuscript.


   FOOTNOTES
 
First decision: 21 May 2001.

1 Grant support from INRA, Centre International des Etudiants et Stagiaires, and grant VEGA 2/7027/20 of Slovak Academy of Sciences. Back

2 Correspondence. FAX: 33 01 34 65 22 41; flechon{at}jouy.inra.fr Back

Accepted: September 27, 2001.

Received: April 20, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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