Architectural reorganization of the nuclei upon transfer into oocytes accompanies genome reprogramming

MOLECULAR REPRODUCTION AND DEVELOPMENT 73:1102–1111 (2006)

Architectural Reorganization of the NucleiUpon Transfer into Oocytes AccompaniesGenome ReprogrammingCATHERINE MARTIN,1,2 VINCENT BROCHARD,1 CAROLE MIGNE,1 DANIELE ZINK,2

PASCALE DEBEY,1,3 AND NATHALIE BEAUJEAN1*1UMR 13-1198 Biologie du Developpement, Institut National de Recherche Agronomique,Domaine de Vilvert, Jouy-en-Josas, France2Department Biologie II, Ludwig Maximillian Universitat Munchen, Planneg-Martinsried, Germany3UMR 8646 CNRS, UMR 5153 INSERM, Regulation et Dynamique des Genomes,Museum National d’Histoire Naturelle, Case Postale 26, Paris, France

ABSTRACT The ability of cloned embryos tosustain full-term development depends on the ability ofthe recipient ooplasm to reprogram the donor cellgenome. As the nuclear architecture has recentlyemerged as a key-factor in the regulation of geneexpression, we questioned whether early embryosobtained from transfer of ES metaphasic chromosomesinto mouse ooplasm would adopt the somatic orembryonic type of nuclear organization. We haveparticularly focused on the arrangement of chromoso-mal territories with respect to the nucleolar compart-ment, and the pericentric heterochromatin domainscalled chromocenters. We found that nuclear transfertriggers profound chromatin rearrangements includingthe dispersion of the donor cell chromocenters com-ponents. These rearrangements lead to a typical 1-cellpronuclear organization, namely a radial arrangementof the chromosome territories with centromeresattached to the nucleoli, which adopt the compactfibrillar structure of nucleolar precursor bodies (NPBs).Subsequently, during the second cycle, the clonedembryos undergo further reorganization with theestablishment of new chromocenters, clustered inone part of the nucleus, as during normal embryogen-esis. We could also establish that the adequatedistribution of chromosomal territories at the pro-nuclear stage seems important for the developmentuntil blastocyst. Mol. Reprod. Dev. 73: 1102–1111, 2006. � 2006 Wiley-Liss, Inc.

Key Words: nuclear transfer; chromatin; mouseembryo; nuclear ultrastucture; chromocenters

INTRODUCTION

Inmammals, gathering of two transcriptionally silentgenomes provided by maternal and paternal gametesupon fertilization produces a unique totipotent cell ableto direct the complete set of processes that give rise toviable offspring. The first steps in zygote formation

consist in a series of specific molecular events coupledwith dramatic changes in nuclear architecture andchromatin structure that play key role in gene regula-tion. Considering the fact that cloning often involves thetransfer of differentiated cell nuclei into enucleatedoocytes, the question emerges as to how the somaticgenome becomes functionally reorganized in the recon-structed embryos. Although transfer of differentiatednuclei has successfully led to full-term development ofviable and fertile offspring in several mammalianspecies, the reconstructed embryos still have a low rateof development. Thus, understanding how the donor cellgenome is reprogrammed could be helpful to improvenuclear transfer procedures.

At fertilization, transcription is not observed in bothgenomes. Subsequently zygotic gene activation (ZGA) isprogressively established following a species-dependenttemporal program (for review see Latham, 1999). In themouse, RNApolymerase II becomes activated by the endof the 1-cell stage (Bouniol et al., 1995) through anunspecific minor burst of transcription, apparentlylinked to modifications in chromatin structure (Schultzand Worrad, 1995; Adenot et al., 1997; Beaujean et al.,2000). Indeed, after entry of the spermatozoon, thehyper-compacted paternal chromatin rapidly initiatesthe replacement of protamines by oocyte-derived his-tones (for review see McLay and Clarke, 2003). More-over, most of the paternal genome is rapidlydemethylated, to an extent that depends on the species(Reik and Walter, 2001; Reik et al., 2001; Santos et al.,2002; Beaujean et al., 2004; Dean et al., 2005) and, at

� 2006 WILEY-LISS, INC.

Grant sponsor: Volkswagen-Stiftung (to DZ).

*Correspondence to: Nathalie Beaujean, UMR 13-1198 Biologie duDeveloppement, Institut National de Recherche Agronomique,Domaine de Vilvert, 78352 Jouy-en-Josas, France.E-mail: [email protected]

Received 21 December 2005; Accepted 11 February 2006Published online 30 May 2006 in Wiley InterScience(www.interscience.wiley.com).DOI 10.1002/mrd.20506

least in the mouse, exhibits a high level of acetylationon histone H4 (Adenot et al., 1997; Santos et al., 2002).On the other hand, the maternal genome retains ahigh level of DNA methylation and methylatedhistone H3 binding properties (Arney et al., 2002;Santos et al., 2002). The major burst of transcriptionwill then occur later on, at the 2-cell stage, with therecruitment of specific transcription factors (Nothiaset al., 1995).These functional events appear intimately linked to

nuclear remodeling. For example, nucleoli with theusual tripartite ultrastructure are not present at the 1-cell stage. They are replaced by inactive compactfibrillar masses, called nucleolar precursor bodies(NPBs) (Takeuchi and Takeuchi, 1986; Flechon andKopecny, 1998), which persist until the 4/8-cell stage,that is, the time where RNA-polymerase I activity isfully established (Geuskens and Alexandre, 1984;Zatsepina et al., 2003). Furthermore, during the firstcell cycle, chromosomes from each pronucleus adopt aradial organization, termed ‘‘cartwheel’’, with most ofthe centromeres oriented towards the NPBs (Dozortsevet al., 2000;Martin et al., 2006).Wehave recently shownthat this organization is quickly disrupted at the 2-cellstage by large chromatin movements apparently linkedto the onset of transcription (Martin et al., 2006).In the past decade, numerous studies shed light on the

contribution of nuclei architecture to the functionalgenome regulation in somatic cells (Croft et al., 1999;Taddei et al., 2004; Parada et al., 2002; Cremer et al.,2003; Misteli, 2004). During interphase, each chromo-some occupies a fraction of the nuclear volume that isreferred to as ‘‘chromosome territory’’ (Manuelidis,1985; Cremer et al., 1993; Cremer and Cremer, 2001)with a nonrandom positioning mainly correlated to itsgene density (Croft et al., 1999; Tanabe et al., 2002;Cremer et al., 2003; Bolzer et al., 2005). The organiza-tion inside the territory, with regard to gene positioningand accumulations of various proteins such as tran-scription factors, also provides a framework wherebygene expression is favored in restricted areas (Zirbelet al., 1993; Dundr and Misteli, 2001). On the otherhand, heterochromatin tends to aggregate; especially,pericentric heterochromatin from several chromosomesforms clusters called chromocenters that appear to actas ‘‘silencing’’ domains (Manuelidis and Borden, 1988;Haaf and Schmid, 1991; Cerda et al., 1999). A function ofchromocenters in gene regulation is suggested by theobservation that the positioning of genes with respectto these compartments contributes to the regulation oftheir differential expression patterns (Brown et al.,1997, 1999; Francastel et al., 1999; Schubeler et al.,2000).In this article, we examined changes in chromatin

condensation and nuclear organization of the donor cellduring the two first cycles following nuclear transfer.We focused on chromosome positioning and the estab-lishment of chromocenters by tracking centromeres andpericentric heterochromatin to assess the ability ofproper gene expression in early embryos.

MATERIALS AND METHODS

Embryo Collection and Culture

Embryos were produced by natural fertilization ofC57/CBAmice. Superovulationwas induced by injectionof pregnant mare serum gonadotropin (PMSG, Inter-vert, 5 UI) followed by injection of human chorionicgonadotropin (hCG, Intervert, 5 UI) 48 hr later. Femalemice were then mated with C57/CBA males. Fertiliza-tion occurred at about 12 hr after hCG injection whichwas used as reference point for normal embryonicdevelopment (hours post-hCG i.e., hphCG). Fertilizedeggs were collected at the 1-cell stage from the ampullain M2 medium (Sigma-Aldrich, France) after a brieftreatment with 1 mg/ml of hyaluronidase in phosphate-buffered sodium (PBS, pH7.5) to separate them fromthesurrounding follicular cells. Two-cell stage embryoswere collected from the mice oviducts at 32 and48 hphCG. All experimental sets contained embryosfrom four to five different mice. All experiments wererepeated at least three to four times.

Nuclear Transfer

Superovulation of C57/CBA female mice was inducedwith PMSG and hCG as described above. Oocytes werecollected from oviducts at 14 hphCG and washed in M2.Subsequently, they were incubated in M2 containing5 mg/ml cytochalasin B and placed in a chamber on thestage of an inverted microscope (Nikon) equipped withmicromanipulators (Nikon-NarishigeMO-188, France).The chromatin spindle (visualized under differentialinterference contrast) was aspirated with a pipette asdescribed by Zhou et al. (2001). Donor chromosomeswere issued from ES cells (gift from Dr. Nagy, Toronto),previously cultured in DMEM and synchronized inmetaphase with demecolcin (Zhou et al., 2001). Donorchromosomes were then microinjected into the cyto-plasm of the enucleated oocytes. The nuclear transferembryos were activated by incubation for 3 hr in Ca2þ-free medium containing 10 mM Sr2þ. The time points ofembryos transfer into the activation medium was usedas reference time for embryonic development of theclones (hours post-activation i.e., hpa). At 4 hpa,embryos with visible nuclei, considered as activated,were either directly fixed for immunofluorescencestaining (first time-point) or transferred into Sr2þ-freeM16 medium and cultured at 378C in a humidifiedatmosphere enriched in 5% of CO2 for later analysis.

For the stepwise analysis of nuclear changes, eachexperimental set was divided into three groups fixed atdifferent times during thefirst cycle (4, 7, and 10hpa), orinto two groups fixed at 20 and 33 hpa during the secondcycle. The experiments were repeated 10 times for the 1-cell stages and 3 times for the 2-cell stages.

Immunofluorescence and Mounting

Embryoswere fixedwith 2%paraformaldehyde (PFA)in PBS for 20 min at room temperature (RT) andpermeabilized with 0.5% Triton X-100 (30 min, RT).Theywereblockedwith3%bovine serumalbumin (BSA)

Molecular Reproduction and Development. DOI 10.1002/mrd

NUCLEAR REORGANIZATION IN CLONED EMBRYOS 1103

in PBS for 45 min. Incubation with the primaryantibodies anti-HP1b (Euromedex, clone 1MOD 1A9)and CREST (human serum which recognizes bothCENP-A and B, gift fromDr. Paul Kalitsis, Melbourne),diluted in 3%BSA-PBS at 1:400 and 1:200, respectively,was performed overnight at 48C. After two washes with0.05% Tween-20 in PBS (30 min each, RT), embryoswere incubated with the secondary antibodies, coupledwith fluorescein or rhodamin (Jackson Immunore-search, West Grove, PA) and diluted in 3% BSA-PBSat 1:200, during 1 hr (RT). They were extensively rinsedagain to remove excess of antibodies and brieflypostfixed (2% PFA-PBS, 10 min, RT). Subsequentlychromatin was counterstained with 4 mg/ml Hoechst33342 (Calbiochem, San Diego, CA). The embryos werefinally deposited on slides and mounted under a cover-slip with citifluor (Citifluor Products, Canterbury, UK).

ES cells and 3T3 fibroblasts were processed similarly.

Fluorescence Microscopy and Image Analysis

Fluorescein and rhodamin signalswere recordedwitha Zeiss LSM 510 confocal laser scanning microscopeusing an oil-immersion objective (Plan Apochromatic63�n.a.1.4) and the488- and535-nmwavelengths of thelasers. The distance between two consecutive opticalsections was from 0.3 to 0.5 mm.

Hoechst staining was detected by epifluorescencemicroscopy performed with a Nikon inverted conven-tional microscope (Eclipse TE 300) using a standardfilter for Hoechst emission. Images were acquiredthrough a water-immersion objective (CFI Plan Apoc-hromatic 60� WI n.a.1.20 WD 0.22) with a cooled CCDcamera (Coolsnap HQ, Roper Scientific) driven by theMetamorph 6.0 software.

Three-dimensional reconstructions from imagesstacks were performed using Amira software.

Electron Microscopy

Cloned embryos at 4, 7, and 10 hpa were fixedovernight at 48C in a mixture of 2.5% glutaraldehydeand 0.7% paraformaldehyde dilued in 0.075 M Soren-

sen’s buffer pH 7.2 containing 0.2–0.5% ferricyanide.After three rinses in Sorensen’s buffer, samples werepostfixed inOsO4 2% in deionizedwater for 1 hr at roomtemperature. Then, after three rinses with deionizedwater, cloned embryos were dehydrated through anethanol series andfinally embedded inEMbed812 resin.The resin was polymerized for 4 days at 608C.

Ultrathin sections (90 mm) were cut with a Pabish topultra 170 ultramicrotome and collected on 200 meshgrids. Finally, sections were contrasted with uranylacetate plus lead citrate and were observed on atransmission electron microscope Philips CM12.

Three independent experiments were performedincluding at least 10 clones in each batch.

RESULTS

Mitotic Chromosomes FromES Cells Transferredinto Oocyte Cytoplasm Are Rapidly Reorganized

into a Pronucleus-Like Structure

We first examined chromatin structure by stainingwith Hoechst, a nonintercalating DNA-specific dye thatbinds to theminor groove of theDNAdouble helixwith ahigher affinity for A-T rich sequences. Inmouse somaticcells, as well as in undifferentiated ES cells, Hoechstbrightly stains distinct domains that correspond topericentric heterochromatin assembled into chromocen-ters (Fig. 1A,B; Cerda et al., 1999). However, thechromatin organization is completely different in 1-cellstage embryos. In that case, Hoechst staining isrelatively uniform, except for a bright rim at theperiphery of the large pseudo-nucleoli termed NPB(Fig. 1C; Debey et al., 1989).

To address the question of whether chromatinorganization is modified upon nuclear transfer (NT)into the oocyte cytoplasm, we performed Hoechststaining in cloned embryos fixed at 4, 7, and 10 hrpostactivation (hpa) corresponding respectively to early,mid, and late stages of the first embryonic cycle. Notethat nuclear transfer was carried out with ES cellssynchronized inearlymitosis (Zhouetal., 2001) and thatonly one nucleus forms after activation.


Fig. 1. Chromatin structure as revealed byHoechst staining in somatic and embryonicmouse cells.NIH3T3 (A), ES cell (B), and a late 1-cell stage embryo (C) stained withHoechst and imaged by epifluorescencemicroscopy. Hoechst brightly stains distinct domains in 3T3 and ES nuclei and a ring of chromatin atthe periphery of NPBs (arrowheads) in both maternal (m) and paternal (p) PNs of 1-cell embryos. Scalebars: 5 mm.

1104 C. MARTIN ET AL.

Based on morphological criteria such as chromatintexture and distribution, we identified four differentpatterns in theNTnuclei, named types I to IV,which arepresented in Figure 2A. Their relative frequency isshown by the graph in Figure 2B. It should be noted thatthe heterogeneity between embryos at each time pointprobably results from the asynchrony due to the timenecessary for nuclear transfer.At 4hpa, that is, early 1-cell stage,mainly two types of

patterns were observed (n¼43). Type I nuclei (18%)presented a high concentration of large compact chro-matin clumps. Type II nuclei (82%) displayed smallerchromatin clumps spread in the nucleoplasm and someof these clumps were associated with the periphery ofNPBs.Three hours later, the nuclei had enlarged (average

diameters about 25 mm at 7 hpa vs. 15 mm at 4 hpa,n¼104) and 52% of them presented the type II pattern.The remaining 48% displayed the same pattern ofclumps but some NPBs were now encircled by a partialor complete rim of brightly stained chromatin (type III).

This progressive structural switch became obvious atthe last stage analyzed (10 hpa, n¼ 60): nuclei present-ing chromatin clumps (type III) were still observed buttheir proportion decreased (only 29%); the other nuclei(71%) had developed a pronucleus-like structure inwhich the chromatin was relatively homogeneouslydistributed and the NPBs were entirely surrounded bybrightly stained DNA (type IV; compare with Fig. 1C).

Taken together, these data indicate that the chroma-tin remodeling in the donor ES nucleus is a sequentialprocess extending over the whole first cycle aftertransfer, leading finally to a pronucleus-like structure(type IV).

NT Embryos Display Ultrastructural FeaturesSimilar to Normal Embryos

To analyze in more detail the ultrastructure of theremodeled nuclei, NT embryos were further examinedby electron microscopy. We first concentrated onstructures observed very early during the remodelingprocess, at least at the level of light microscopy, namely


Fig. 2. Identification and distribution of the four chromatin patterns during the 1-cell stage in NTembryos. A: Embryos reconstituted by nuclear transfer were cultured and fixed at 4, 7, or 10 hpa. Hoechststaining of DNA reveals four chromatin patterns named types I to IV. Scale bars: 5 mm.B: The graph showsthe frequency of each pattern at the different time points of fixation.


the NPBs. At the ultrastructural level, the NPBs fromnormal mouse embryos appear very soon during theformation of PNs as prominent spheres, composed ofhomogenous and densely packed fibrillar material,sharply delineated from the surrounding nucleoplasm(Fakan and Odartchenko, 1980; Adenot et al., 1991;for review Flechon andKopecny, 1998). They frequentlydisplay surface protrusions called ‘‘lenticles’’ enriched intypical nucleolar proteins of the nucleolus organizingregions (NORs; Takeuchi andTakeuchi, 1986). Remark-ably, the NPB-typical dense fibrillar core associatedwith characteristic lenticles was already formed in NTembryos at 4 hpa (Fig. 3A, arrow). At the same time, the

chromatin itself displayed a diffuse and homogenoustexture and was delineated by a nuclear envelopedisplaying normal nuclear pores (Fig. 3B). Remarkably,we did not observe, at any time point, the layer ofcondensed chromatin at the nuclear periphery which istypical of somatic cell nuclei (Fig. 3C,E, small arrow-heads). Furthermore, the chromatin clumps observed atthe light microscopy level were not detected at theultrastructural level, suggesting that they represent ahigher local concentration of chromatin rather than ahigher condensation state. We also observed unusualinvaginations of the nuclear envelope (Fig. 3D) thatmight reflect physical constraints. Finally, a fraction ofthe NT embryos showed some vesicles apposed to thenuclear envelope or in the vicinity of the NPB. Wesuggest that they result from the nuclear transferprocedure that drives micro-quantities of culture med-ium simultaneously with the donor chromosomes intothe ooplasm.

Altogether, these results indicate that essentialfeatures of embryonic pronuclei, namely NPB display-ing a characteristic structure and the typical chromatintexture with a lack of perinuclear heterochromatin, arerapidly adopted by the transferred nuclei and main-tained during the whole 1-cell stage.

The Setting-Up of Centromeric and PericentricCompartments in Late One-Cell Cloned Embryos

Correlates With Developmental Potential

To address the question of how chromatin reorganiza-tion is orchestrated and which type of chromatin isinvolved, we next performed immunofluorescent detec-tion of CENP and HP1b.

These two proteins are well-characterized markers ofthe centric and pericentric compartments, respectively.HP1b binds to pericentric heterochromatin and, inmouse somatic cells, is particularly enriched in thebrightly Hoechst stained chromocenters (Fig. 4A, com-parewithFig. 1A) towhich the centromeres (revealed byimmunostaining of the CENP proteins) are apposed.Several centromeres are associated with one chromo-center, which represents pericentric regions from


Fig. 3. Ultrastructural features of nuclei from NT embryos duringthe 1-cell stage. NT embryos at 4 hpa (A,B), 7 hpa (C), or 10 hpa (D,E)were fixed and processed for electron microscopy. Dense and compactNPBs with ‘‘lenticle’’ protrusions (A, C arrows and enlargement) wereobserved at each stage. The nuclear envelope displays normal nuclearpores (B arrowheads and enlargement) and unusual large invagina-tions (D). Thenuclear peripherywas devoid of condensed chromatin (C,E small arrowheads). Nu, nucleus; Cy, cytoplasm. Scale bars: 1 mm (A,C, D, E) and 0.3 mm (B).

Fig. 4. Distribution of HP1b and centromeres in somatic andembryonic cells. NIH 3T3 (A), ES cells (B), and a late 1-cell stageembryo (C) were immunostained to detect HP1b (red) and CENP(green), and imaged by epifluorescence (A) or confocal (B, C) micro-scopy. Centromeres were associated with chromocenters enriched in

HP1b in both NIH-3T3 and ES nuclei. In 1-cell embryo after naturalfertilization, centromeres were associated with the NPBs periphery(arrowheads), which were also enriched in HP1b in the maternal (m)pronucleus (p: paternal). Scale bars: 5 mm.


different chromosomes (4 to 6; Cerda et al., 1999). Herewe show that the assembly of pericentric heterochro-matin and centromeres in chromocenters is preserved inES cells (Fig. 4B).In 1-cell mouse embryos obtained after natural

fertilization, the distribution of thesemarkerswas quitedifferent. HP1b was diffusely and homogeneouslydistributed in the nucleoplasm of both pronuclei. Inaddition, HP1b accumulated at the NPB peripheries inthe maternal PN (Fig. 4C, arrow). Furthermore, allcentromeres were associated with the NPBs (Fig. 4C,arrowheads), except for one or two located near thepronuclear envelope.We next analyzed the distribution of the same

markers in NT embryos fixed at 4 hpa (n¼43), 7 hpa(n¼ 126), and 10 hpa (n¼75). At 4 hpa, soon afternuclear transfer, HP1bwas uniformly distributed in thenucleoplasm. It did not show any particular accumula-tions in contrast to the highly heterogeneous texture ofthe chromatin at this stage (Fig. 5A, compare with typesI and II in Fig. 2A). At 7 hpa, 92% of embryos displayeddiscrete accumulations of HP1b around NPBs (Fig. 5D).Finally, at 10 hpa, the rim of peri-NPB chromatin washighly enriched in HP1b (in 85% of cases, Fig. 5G) as inthe maternal pronucleus of normal embryos.Inparallel, centromereswerealreadyorganizedat the

NPB periphery 4 hr after activation (Fig. 5B), adistribution that was maintained during the wholecycle. Surprisingly, and contrary to the situation during

normal development, centromeres lost transiently theircompact configuration: they became fuzzy and decon-densed in 82%of cloned embryos at 7 hpa (Fig. 5E). Theyseemed to recondense later since only 24% of the NTembryos had decondensed centromeres at the end of thefirst cycle (Fig. 5J). Surprisingly also, the decondensedcentromeres were almost never located near the HP1b-rich regions (Fig. 5F).

These data show that the different components of thechromocenters from the donor cell are rapidly disruptedafter introduction into the recipient oocyte cytoplasm,with HP1b being relocated in the whole nucleoplasm,brightly Hoechst stained heterochromatin decondensedand centromeres associated with the NPBs (Fig. 5).Then the typical pronuclear organization of normalembryos is rapidly recapitulated as these componentsbecome re-associated by 10 hpa.

Tracking the centromeres via CENP detection givesalso indications about the global arrangement ofchromosome territories in the transferred nucleus. Innormal embryos, chromosome territories adopt by theend of the first interphase a typical cartwheel orienta-tion (Debey et al., 1989; Martin et al., 2006), in whichcentromeres associate with the NPBs while the rest ofthe chromosomes stretches out toward the pronuclearperiphery. In 82% of the normal embryos, all chromo-somes except on average three of them (two in thematernal and one in the paternal pronucleus) arearranged in the cartwheel, whereas the remaining


Fig. 5. Distribution ofHP1b and centromeres during the 1-cell stageinNTembryos.A–I: Light-optical sections ofNTembryos fixedat 4 (A–C), 7 (D–F), and 10 hpa (G–I) immunoprocessedwithHP1b (left-handpanels) andCRESTantibodies (centromeres,middlepanels).Right-hand panels are merged pictures with HP1b in red and CREST ingreen. The arrow in G indicates a centromere nonassociated with the

NPB periphery. Scale bars: 5 mm. J: Percentages of embryos displayingdecondensed centromeres at 7 and 10 hpa. K: Percentages of embryosdisplaying at maximum 3 centromeres not associated with the NPBperiphery (dark bars) and percentages of embryos developing until theblastocyst stage (light bars) in normal and NT development.


18% display inadequate repositioning with more thanthree centromeres not associated with NPBs. In clonedembryos, a similar cartwheel organization during thefirst interphase is suggested by the position of thecentromeres at the NPB periphery. However, thisorganization was not fully established in all nuclei sinceonly 59% of them displayed at maximum three centro-meres not associated to theNPBperiphery, whereas theother 41% displayed up to six centromeres not asso-ciated with the NPBs (Fig. 5K). Considering that therate of development to the blastocyst stage is approxi-mately 60% in NT embryos versus 85% in normalembryos (Fig. 5K), these findings suggest that thedevelopmental deficiency of the cloned embryos couldbe related to abnormalities in chromosomes arrange-ments.

Chromocenter Formation Takes Place Duringthe Two-Cell Stage in Normal Embryos

as well as in NT Embryos

We next wondered when and how chromocentersbecome established during development of clonedembryos. To address this question, the distribution ofCENPs and HP1b was analyzed during the 2-cell stagein NT embryos and compared with embryos obtainedafter natural fertilization.

In normal mouse embryos, the second cycle appearscrucial in terms of spatial restructuring of the genome(Martin et al., 2006). Briefly, in early 2-cell stageembryos (32 hphCG), the centromeres and HP1b areassociated with the periphery of NPBs as observed

during the 1-cell stage (Fig. 6A, compare with Fig. 4C).Then, they progressively relocate to the nucleoplasm,where they form chromocenter-like structures at thelate 2-cell stage (48 hphCG). These chromocenter-likestructures strongly resemble the chromocentersobserved in somatic nuclei (Fig. 6B, compare withFig. 4A).

In cloned embryos cultured in vitro until 20 hpa(corresponding approximately to 32 hphCG in normaldevelopment), HP1b displayed the peri-NPB localiza-tion characteristic of normal early 2-cell stage (Fig. 6E,compare with Fig. 6A). At 33 hpa (corresponding to 48hphCG innormal development),HP1bwasaccumulatedinto largenucleoplasmicpatches, as in thenormal late2-cell stage (Fig. 6F,B). This reorganization of HP1b wasaccompanied by the relocation of centromeres from theNPBs periphery to the nucleoplasmic HP1b patcheswith which they were associated at 33 hpa (Fig. 6E,F).

The proportion of CENPs dots associated with NPBperipheries is shown in the histograms of Figure 5 (bluebars). These data confirm the relocation of the centro-meres during the second cycle in normal embryos (68%of centromeres at 32 hphCG, n¼32, vs. 42% at 48hphCG, n¼ 45, were associated with NPB peripheries)as well as in cloned embryos (55% at 20 hpa, n¼18, vs.31% at 33 hpa, n¼28).

Finally, three-dimensional reconstructions weremade from late 2-cell nuclei of normal and clonedembryos (Fig. 6B,F). These reconstructions show thatthe chromocenters tend to accumulate in one part of thenuclear volume in both cases (Fig. 6D,H). Thus the


Fig. 6. Formation and distribution of chromocenters during the 2-cell stage in normal and NT embryos. The upper panels (A–D) showembryos obtained after natural fertilization, while the lower panelsshow NT embryos. Embryos were fixed at early (A, E) and late 2-cellstages (B, F) and immunostained for HP1b (red) and CENP (green);scale bars: 5 mm. The histograms in C and G show the percentages of

CENP dots associated with NPB peripheries (blue bars) and thepercentages of NPBs associated with HP1b (red bars), respectively. Dand H display the three-dimensional reconstructions of the wholenuclei shown in pictures B and F respectively (B and F display onlysingle light-optical sections).


polarity characteristic of 2-cell stage nuclei, alreadydescribed in normal mouse embryos by Mayer et al.(2000), is similarly established in cloned embryos.Altogether our data demonstrate that the introduc-

tion of ES cell mitotic chromosomes into an ooplasmtriggers processes leading to the formation of a pronu-cleus-like structure characteristic of the 1-cell stage,followed by the formation of chromocenters and thepolar organization characteristic of the 2-cell stage.Although nuclear reorganizations observed duringnormal development appear to be recapitulated, cen-tromeres positioning is less restrained in NT embryosand might be related to their reduced developmentalpotential.

DISCUSSION

Nuclei of ES cells or differentiated somatic cells can bereprogrammed and reach totipotency after transfer intoan oocyte as demonstrated by the birth of viable clonedembryos. However, the low efficiency of cloning afternuclear transfer suggests that reprogramming is oftenincomplete. In order to improve this procedure, it isnecessary to understand the processes underlying the‘‘reprogramming’’ events during normal and clonedevelopment. While most studies address nucleargenome organization in somatic cells, we asked herewhether specific reorganizations of the genome areassociated with reprogramming after NT, whether suchre-organizationsmight be different from those observedin normal embryos, and whether differences in genomereorganization might explain developmental failure ofcloned embryos. Therefore, we compared the chromatintexture combined with the distribution of centric andpericentric markers, respectively CENPs and HP1b, inboth cloned and normal embryos. Our results show thatthe genome organization of mouse ES cells is dramati-cally remodeled after NT. This remodeling involves (i)the disruption of the nucleolar compartment and of thechromocenters, (ii) the formation, at 1-cell, of the typicalpronuclear structure with compact fibrillar NPB sur-rounded by centromeres, (iii) the highly dynamicreformation of chromocenter-like structures during the2-cell stage. This process is rapidly engaged afternuclear transfer and could be divided into five stepsschematized on Figure 7.Most intriguing is the disconnection between chro-

matin condensation and HP1b accumulation during theearliest phases of chromatin remodeling (type I to III).Duringmitosis, HP1b does not seem to remain attachedto metaphasic chromosomes (Minc et al., 1999; Haya-kawa et al., 2003), however HP1b-enriched chromocen-ters are rapidly restored at G1 in somatic (Hayakawaet al., 2003) as well as in ES cells (this report). This isclearly not the case during the first G1 after NT into theoocyte. This absence ofHP1baccumulationmight bedueto either a lower concentration of HP1b in the embryos,or a lower affinity of HP1b for pericentric chromatin.Another alternative suggested by the ultrastructuraldata is that the clumps of condensed chromatin observedby Hoechst staining represent a higher local concentra-

tion of euchromatin rather than condensed heterochro-matin per se. As the nuclear volume is at that time quitesmall and contains several largeNPBs, it is possible thatphysical constraints lead to a higher compaction ofeuchromatin.

Similarly, in later typeII/type III NT embryos, HP1baccumulation on NPBs periphery is observed in 95% ofthe embryos (Fig. 4), whereas condensation of chro-matin around NPBs is only visible in approximately50% of the embryos (Fig. 2B). One possible explanationis that chromatin within the clumps is progressivelyrecruited to form the chromatin ring at NPBs. Alter-natively, it is possible that chromatin clumps only


Fig. 7. Scheme illustrating the step-wise chromatin remodelingduring the two first cycles after nuclear transfer. (1) Before 4 hpa, thenucleus first swells while large NPBs, to which centromeres areassociated, are formed (type I). In the same time, HP1b is diffuselydistributed in the whole nucleoplasm and does not accumulate oncondensed chromatin, although large nucleoplasmic clumps can beseen. (2) From 4 to 7 hpa, the nuclear size is increasing so that thepacked chromatin gains space and decondenses (type II). However,smaller chromatin clumps persist in the nucleoplasm. (3) At 7 hpa, acondensed chromatin ring forms at NPBs periphery while thecentromeres transiently decondense (type III). (4) From 10 hpaonwards, most of the transferred nuclei adopt a pronucleus-likeorganization (type IV) with a condensed chromatin ring, partiallystained with anti-HP1, around the NPB and associated with centro-meres. (5) By the 2-cell stage, chromocenters are formed and clusteredin one part of the nucleus, forming a Rabl-like orientation.


represent euchromatin and that pericentric hetero-chromatin, bound by HP1b and already associatedwith the centromeres around the NPBs, is still in aquite ‘‘decondensed’’ state. Both mechanisms mightinvolve HP1b auto-recruitment as well as small RNAs(for review see Grewal and Rice, 2004). Strikingly, atthe same time centromeres transiently decondenseand are completely spatially disconnected from theHP1b-rich regions, two features which have neverbeen observed in fertilized embryos. The coincidence ofthese events suggests that they are in close relation.Indeed, the transient elongation of the centromeres,from a compact configuration to a decondensed form,could help the restructuration of the chromatin aroundthe NPBs.

At the 2-cell stage, the nuclear organization is similarto that of normal embryos, and presents originalfeatures characteristic of that stage never observed inmammalian differentiated cells, namely a polaritywhich recalls the Rabl polarization of chromosomes inplant cells (Shaw et al., 2002 and references therein).Mayer et al. (2000) were the first to describe thechromocenters polarity in mouse 2-cell stage nuclei byin situ hybridization with centromeric specific probes.Here we find a similar nuclear partitioning in bothnormal and cloned embryos using whole-mount immu-nodetection of CENPs and HP1b and three-dimensionreconstructions. This means that the transferrednucleus is able to initiate and complete the same large-scale movements of centromeric and pericentric hetero-chromatin as in normal embryos during the secondcycle. It also suggests that transferred nuclei proceedthrough normal genome reorganization, that is, apronucleus-like structure at the 1-cell stage and furtherchromosome territories movements leading to the Rabl-like orientation at the 2-cell stage.

Nevertheless, a detailed examination of the reconsti-tuted pronuclear configurations reveals that half of thecloned embryos displayed an aberrant nucleoplasmicdistribution of centromeres suggesting an abnormallocalization of the corresponding chromosomes in thenuclear volume. In parallel, the development rate ofcloned embryos was also reduced and only half of themreached theblastocyst stage, althoughalmost all of thempassed to 2-cell where they adopted the Rabl-likeconfiguration. This suggests that restructuring eventsoccurring at the 1-cell stage are the most important forfurther development to blastocyst.

It is worth noting that in both normal and clonedembryos, chromocenters genesis is achieved by the endof the 2-cell stage, a stage that corresponds to the majorZGA during mouse normal development (Clegg andPiko, 1983; Latham et al., 1991a; Bouniol et al., 1995;Aoki et al., 1997). Although the exact temporal pat-tern of gene expression in NT embryos is not known, ithas been shown that 2-cell specific genes are activated in2-cell NT embryos (Latham et al., 1991b). It is thereforepossible that the components (either proteins or RNAs)involved in the formation of chromocenters are presentsimilarly in 2-cell normal andNT embryos. Finally, only

3.1% of the embryos reconstructed with ES metaphasesdevelop to term (Zhou et al., 2001). This suggests thatthe observed nuclear remodeling leading to the 2-cellspecific chromocenters spatial organization is notsufficient and that further modifications of the donorgenome, for example, epigenetic modifications, arerequired to support full development.

ACKNOWLEDGMENTS

We thank Paul Kalitsis for his generous gift ofantibodies, the CeMIM (Centre de Microscopie etd’Imagerie du Museum) and the MIMA2 platform(Microscopie et Imagerie des Microorganismes, Ani-maux et Aliments) for access to confocal and electronmicroscopy. We thank Linda Maulny and the UEAR foranimal care. This work was supported by a grant fromthe Volkswagen-Stiftung to D.Z.

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Architectural reorganization of the nuclei upon transfer into oocytes accompanies genome reprogramming