Developmental Biology 364 (2012) 56–65

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Developmental Biology

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Abnormal chromosome segregation at early cleavage is a major cause of the full-termdevelopmental failure of mouse clones

Eiji Mizutani a,b,1, Kazuo Yamagata a,⁎, Tetsuo Ono a, Satoshi Akagi b, Masaya Geshi b, Teruhiko Wakayama a

a Laboratory for Genomic Reprogramming, RIKEN Center for Developmental Biology, Kobe, Japanb Research Team for Reproductive Biology and Technology, National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki, Japan

⁎ Corresponding author at: Research Institute for MicOsaka University, Osaka, Japan. Fax: +81 6 6879 8376.

E-mail address: yamagata@biken.osaka-u.ac.jp (K. Y1 Present address: Bioresource Engineering Division,

Tsukuba, Ibaraki, Japan.

0012-1606/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.ydbio.2012.01.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received for publication 11 October 2011Revised 27 December 2011Accepted 3 January 2012Available online 14 January 2012

Keywords:Nuclear transferEmbryo developmentLive-cell imaging

To clarify the causes of the poor success rate of somatic cell nuclear transfer (SCNT), we addressed the impactof abnormalities observed at early cleavage stages of development on further full-term development using‘less-damage’ imaging technology. To visualize the cellular and nuclear division processes, SCNT embryoswere injected with a mixture of mRNAs encoding enhanced green fluorescent protein coupled with α-tubulin (EGFP-α-tubulin) and monomeric red fluorescent protein 1 coupled with histone H2B (H2B-mRFP1) and monitored until the morula/blastocyst stage three-dimensionally. First, the rate of developmentof SCNT embryos and its effect on the full-term developmental ability were analyzed. The speed of develop-ment was retarded and varied in SCNT embryos. Despite the rate of development, SCNT morulae having morethan eight cells at 70 h after activation could develop to term. Next, chromosomal segregation was investigat-ed in SCNT embryos during early embryogenesis. To our surprise, more than 90% of SCNT embryos showedabnormal chromosomal segregation (ACS) before they developed to morula stage. Importantly, ACS per sedid not affect the rate of development, morphology or cellular differentiation in preimplantation develop-ment. However, ACS occurring before the 8-cell stage severely inhibited postimplantation development.Thus, the morphology and/or rate of development are not significant predictive markers for the full-term de-velopment of SCNT embryos. Moreover, the low efficiency of animal cloning may be caused primarily by ge-netic abnormalities such as ACS, in addition to the epigenetic errors described previously.

© 2012 Elsevier Inc. All rights reserved.

Introduction

Wilmut and Campbell first cloned a normal, fertile adult mammal –a sheep – by transplanting nuclei from adult mammary cells (Campbellet al., 1996; Wilmut et al., 1997). Soon after this breakthrough, cloningwas achieved by somatic cell nuclear transfer (SCNT) in mice(Wakayama et al., 1998) and bovines (Kato et al., 1998). Through care-ful technical refinements, it has now been achieved in manymammali-an species (Cibelli, 2007). However, the success rates of animal cloningby SCNT are consistently low, especially in the mouse: typically fewerthan 3% of cloned embryos develop to term (Thuan et al., 2010;Wakayama, 2007; Yang X. et al., 2007).

Recent analyses of SCNT embryos have revealed many abnormali-ties caused by incomplete epigenetic reprogramming of the donornucleus as being significant reasons for developmental failure. Duringthe development of naturally fertilized embryos, epigenetic marks are

robial Diseases,

amagata).RIKEN Bioresource Center,

rights reserved.

established over long periods during germ cell formation and fertili-zation. However in SCNT cloning, these processes might be bypassedand an adult somatic pattern of epigenetic modification that is nor-mally very stable must be reversed within a short period before zy-gotic genome activation (Fulka et al., 2004; Yang X. et al., 2007). Infact, abnormal DNA and histone modification patterns such asheavy methylation of the Sall3 locus (Ohgane et al., 2001), hyper-methylation pattern in the centromeres (Yamagata et al., 2007), orabnormal H3K9 methylation (Santos et al., 2003) has beenreported in SCNT embryos. Moreover, histone deacetylase inhibi-tors dramatically improved the success rate of mouse cloning(Kishigami et al., 2006b, 2007; Ono et al., 2010; Van Thuan et al.,2009). Thus, epigenetic alterations indeed exist at the zygotic stage ofSCNT embryo.

In addition to the epigenetic alterations, genetic abnormalitiesduring early cleavage stage, such as chromosomal abnormality mayalso be a reason for low success rate of cloning. Although not limitedin the SCNT embryos, it is now becoming apparent that the chromo-somal integrity is not secured more than we have thought. Inhuman, the clinical outcome data of preimplantation genetic diagno-sis revealed that surprisingly nearly 80–90% of embryos showednumerical and/or structural abnormalities in chromosomes at blasto-cyst stage, and this may be a reason for the low pregnancy rate in the

57E. Mizutani et al. / Developmental Biology 364 (2012) 56–65

assisted reproductive technologies (Santos et al., 2010; Vanneste etal., 2009). Especially, as evidenced by the high incidence of chromo-some mosaicism, a phenomenon that chromosome constitution differamong the blastomere, the abnormalities may mainly come from ab-normal chromosome segregation process during early cleavagestages. Also in mouse, the chromosome mosaicism was found in 25%of cleavage-stage embryos derived from naturally-mated female(Lightfoot et al., 2006). Furthermore, we and other group demon-strated that embryos produced by intracytoplasmic sperm injection(ICSI) frequently showed abnormal chromosomal segregation or con-stitution during first mitosis, and that this leads to early pregnancyloss (Tateno and Kamiguchi, 2007; Yamagata et al., 2009a). In fact,some reports document that the SCNT embryos from primates(Simerly et al., 2003), rabbits (Yin et al., 2002) and mice (VanThuan et al., 2006) show structural abnormalities of the nucleus,such as incorrect spindle formation. Improper distribution ofchromosomes at the metaphase plate has also been reported(Kawasumi et al., 2007; Yang J.W. et al., 2007; Yu et al., 2007). In ad-dition, structural and numerical aberrations of chromosomes weredemonstrated by spectral karyotyping analysis of SCNT 1-cell em-bryos (Osada et al., 2009). Taken together, it is possible that thechromosome instability during early cleavage may account for thelow success rate of the cloning.

Despite many efforts in the study of cloning, fundamental prob-lems remain. Mass analysis in which multiple cloned embryos areintermingled and extracted obscures the truth because the clonedembryos might differ individually, so we cannot know which ofthem is capable of developing to term. Moreover, staining methodsrequire fixation and/or denaturation steps to prevent the embryosfrom developing any further. Thus, even if we can find any differencein SCNT embryos compared with normally fertilized embryos, it isdifficult to link any effect to further development. For these reasons,although many reports have described abnormalities in SCNT embry-os during early embryogenesis, the central causes of the low cloningsuccess rate remain enigmatic.

Recently, we succeeded in developing a ‘less-damage’ live-cell im-aging system optimized for preimplantation mouse embryos. It in-volves microinjection with mRNAs encoding fluorescent proteinsand observations using a particular confocal microscope (Yamagataet al., 2009b). This system allows for long-term observations on mo-lecular dynamics during early embryo development in vitro, such aschanges in global DNA methylation status (Yamazaki et al., 2007b),heterochromatin constitution (Yamazaki et al., 2007a) and chromo-somal segregation patterns (Yamagata et al., 2009a). The most impor-tant point of our system is that embryos do not show any deleteriouseffects and can develop to term even after repeated fluorescent obser-vations for up to 4 days of culture. Thus, with this imaging technolo-gy, we can link the abnormalities in each embryo directly with theirdevelopmental ability for the first time. Furthermore, using thismethodology, the real causes for the low success rate of cloning andessential prerequisites for full-term development of SCNT embryoswill be clarified. Therefore, in the present study, we acquiredtime-lapse movies of cytogenetic dynamics during early cleavagesstages by labeling the spindle and chromosomes. We assessed theeffects of the rate of embryo development and of patterns of chro-mosomal segregation in SCNT embryos during early cleavage onfull-term development.

Materials and methods

Mice

All mice used in this study were purchased from SLC (Hamamatsu,Japan) or bred in our mouse colony. All animal experiments con-formed to our Guidelines for the Care and Use of Laboratory Animals

and were approved by the Institutional Committee for Laboratory An-imal Experimentation (RIKEN Kobe Institute).

Preparation of oocytes

Cumulus–oocyte complexes were collected from the oviducts of8- to 12-week-old BDF1 females that had been induced to superovulateby sequential injections of equine chorionic gonadotropin and humanchorionic gonadotropin (hCG) as described (Mizutani et al., 2008) andplaced in Hepes-buffered Chatot–Ziomek–Bavister (CZB) medium(Chatot et al., 1990) containing 0.1% hyaluronidase. The denudedoocyteswere placed in fresh CZBmediumand cultured until use. Cumu-lus cells for nuclear donation were prepared from all females used asoocyte donors. Theywere immediatelymixed in the collectionmedium,transferred to a drop of HEPES–CZB medium containing 10% polyvinyl-pyrrolidone (PVP), and used to provide donor nuclei for SCNT.

Preparation of mRNA

mRNAs encoding enhanced green fluorescent protein (EGFP)coupled with α-tubulin (EGFP-α-tubulin) and monomeric red fluo-rescent protein 1 (mRFP1) fused with histone H2B (H2B-mRFP1)were prepared as described (Yamagata et al., 2009b). Briefly, after lin-earization of the template plasmid at the Xba I sites, mRNA was syn-thesized using RiboMax™ Large Scale RNA Production Systems-T7(Promega, Madison, WI, USA). The 5′ end of each mRNA was cappedusing Ribo m7G Cap Analog (Promega). To circumvent the integrationof template DNA into the embryo genome, reaction mixtures from invitro transcription were treated with RQ-1 RNase-free DNase I(Promega). Synthesized RNAs were treated with phenol–chloroformfollowed by ethanol precipitation. After dissolution in RNase-freewater, mRNAs were subjected to gel filtration using a MicroSpin™G-25 column (Amersham Biosciences, Piscataway, NJ, USA) to removeunreacted substrates and then stored at −80 °C until used.

mRNA injection

mRNA injection into oocyteswas performed asdescribed (Yamagata,2010). Briefly, eachmRNAwas diluted to 5 ng/μL usingMilli-Q ultrapurewater (Millipore Corp., Madison,WI, USA) and an aliquot was placed ona micromanipulation chamber. Collected Metaphase II oocytes weretransferred to droplets of Hepes–CZB medium in the chamber and afew picoliters of mRNA solution were introduced into the oocyte cyto-plasm using a piezo-activated micromanipulator (Prime Tech, Ibaraki,Japan) with a glass micropipette (1–3 μm diameter).

SCNT procedure

SCNT were performed as described (Kishigami et al., 2006b;Wakayama et al., 1998). The chromosome spindle complex in meta-phase II oocytes previously injected with mRNAs was removed anda cumulus cell nucleus was injected into the ooplasm. The recon-structed oocytes were activated by incubation for 6 h in Ca2+-freeCZB containing 10 mM Sr2+, 5 μg/mL CB and 50 nM trichostatin A(TSA) (Kishigami et al., 2006b). After activation, they were furthercultured in CZB medium containing 50 nM TSA for 3 h.

Intracytoplasmic sperm injection (ICSI)

Sperm were collected from the cauda epididymidis of BDF1 males(>12 weeks) in 0.2 mL drops of TYH medium (Toyoda et al., 1971)and capacitated by incubation for 2 h at 37 °C under 5% CO2 in air.ICSI was performed according to the original procedures (Kimura andYanagimachi, 1995). Briefly, oocytes preinjected with mRNAs weretransferred to a droplet (about 10 μL) of Hepes–CZB medium in thechamber of a microscope stage. One microliter of capacitated sperm

58 E. Mizutani et al. / Developmental Biology 364 (2012) 56–65

suspension in TYH medium was mixed with 9 μL of Hepes–CZB medi-um containing 12% (w/v) PVP in a micromanipulation chamber. Thehead of each spermatozoon was separated from the tail by applyingpiezo-pulses and injected into each oocyte.

Imaging procedures

According to our previous report (Yamagata et al., 2009a,b),mRNAs were injected into the metaphase II oocytes from sexuallymatured B6D2F1 females. After the SCNT, the embryos forming pseu-dopronuclei (PN) were transferred to drops of CZB medium on aglass-bottomed dish (12 embryos per drop), placed in the incubator(MI-IBC, Tokai Hit, Shizuoka, Japan) on the microscope stage and in-cubated at 37 °C under 5% CO2 in air. The devices for imaging wereas described (Yamagata et al., 2009b). Briefly, an inverted microscope(IX-71, Olympus, Tokyo, Japan) was equipped with a Nipkow diskconfocal unit (CSU-10, Yokogawa Electric Corp., Tokyo, Japan) andan electron multiplying charge-coupled device (EM-CCD) camera(iXon BV-887, Andor Technology, Belfast, UK). The system has a Zmotor (Ludl Electronic Products, Hawthorne, NY, USA) and an autoX–Y stage (Sigma Koki Co., Ltd., Tokyo, Japan). Images were acquiredin two or three channels (light microscope, green and red fluores-cence) using the auto shutter (Ludl Electronic Products). The powersettings of the 488 nm and 561 nm lasers (CVI Melles Griot, Albu-querque, NM, USA) fired from the tip of lens were set at 0.1 mWusing an optical power meter (TB-200, Yokogawa Electric). This sys-tem was housed in a dark room at 30 °C. Device control and imageanalysis were performed using MetaMorph software (MolecularDevices, Sunnyvale, CA, USA). For time-lapse observations, imageswere taken over 59 h at 15 min intervals. At each time point, 51 fluo-rescent images were taken 2 μm apart in the z-axis for opticalsectioning.

Analysis of live-cell imaging data

Before embryo transfer, the raw fluorescent images were trans-formed to movies using the MetaMorph software, and the details ofearly cleavage in individual embryos were analyzed retrospectively.The number of nuclei and the growth rates of embryos were analyzedwith stereoscopically presented images from the ‘4D viewer’ functionof the MetaMorph software. The numbers of nuclei at each time pointin individual embryo were entered in Microsoft Office Excel softwareand graphed against time. Any abnormal chromosomal segregation(ACS) occurring in embryos was detected by analyzing the recon-structed movies and listed in same column of the Excel sheet.

Embryo transfer

After live-cell imaging, SCNT embryos were screened into fourgroups using a glass needle under the stereomicroscope based on thenumbers of blastomere nuclei at 70 h after activation: ≥16, 12–15,8–11 and ≤7. And as another line of experiment, SCNT embryos weredivided into four groups based on the timing of occurrence of ACS.Each group of embryos was then transferred separately to the oviductof a pseudopregnant ICR female that had been mated with a vasecto-mized ICR male at one day postcopulation (dpc) or to the 3-dpc uteri.All recipient females were euthanized at 19.5 dpc and examined forthe presence of fetuses.

When we transferred single cloned embryos, four to ten ICR strainembryos produced by routine ICSI or in vitro fertilization (IVF) wereco-transferred as carrier embryos to maintain pregnancy in the recipi-ent females. In such cases, these control embryos were transferredinto the contralateral 1-dpc oviducts or 3 dpc uteri. To avoid naturalbirths, recipient females were euthanized at 17.5 or 18.5 dpc for evalu-ating the full-term developmental ability of each cloned embryo. Mice

generated from SCNT were distinguished by their eye colors, briefly,cloned mice had black eyes and ICSI and IVF mice had red eyes.

Immunofluorescence staining

The live-cell imaged SCNT embryos reaching the blastocyststage were washed three times in phosphate buffered saline(PBS) containing 0.5% PVP, fixed in 4% paraformaldehyde in PBSfor 40 min and permeabilized with 0.25% Triton X-100 in PBS.The embryos were washed twice in the PBS-PVP, stored overnightin PBS with 1% bovine serum albumin and 0.1% Triton X-100 at4 °C for blocking. They were then incubated with primary anti-bodies dissolved in the blocking buffer at room temperature forat least 3 h. The primary antibodies used were rabbit polyclonalanti-Oct3/4 (Cat. No. sc-9081, Santa Cruz Biotechnology, SantaCruz, CA, USA) and mouse monoclonal anti-cdx2 (Cat. No.MU392A-UC, BioGenex, CA, USA). After being washed threetimes in blocking buffer, the embryos were further incubatedwith the following secondary antibodies: Alexa Fluor 488-labeledgoat anti-rabbit IgG and Alexa Fluor 546-labeled goat anti-mouseIgG (both from Molecular Probes, Eugene, OR, USA). Followingthree washes with PBS-PVP, DNA was stained with 4′,6-diamino-2-phenylindone (DAPI; 2 μg/mL; Molecular Probes). They werethen observed using the same imaging system as described above.

Statistical analysis

The significance of the number of nuclei at 70 h after activationand the frequency of ACS at each division were evaluated using theχ2 test. The data for the durations of 2-cell and 4-cell stage and thetimes required for transition to the next developmental stage ineach embryo were evaluated using Tukey–Kramer multiple compari-son test followed by F-test. Pb0.05 was regarded as statisticallysignificant.

Results

Quantitative analysis of cell division dynamics during early cleavage

Fig. 1 shows the experimental flowchart of this study. Werecorded movies of cellular and chromosomal division three-dimensionally in early cleavage, from 10 h after activation (pseudo-pronuclear stage) to about 70 h after activation (morula stage). Tolabel the spindle and nucleus, a mixture of mRNAs encoding EGFP-α-tubulin and H2B-mRFP1 (Yamagata et al., 2009b), was injectedinto metaphase II oocytes. As controls, ICSI embryos produced usingthe same females were also imaged simultaneously. SupplementaryFig. 1 shows examples of time-lapse images of embryonic developmentamong ICSI- and SCNTembryos (Fig. S1A) and three-dimensional repre-sentation at morula stage (Fig. S1B). The dynamics of cytogenesis werequantified by image analysis based on these four-dimensional imagesinformation. Althoughourmethodology as presentedherewas relative-ly complex, the results were reproducible whenwe repeated the exper-iments. Moreover, the rate of development of SCNT embryos tomorula/blastocyst stage (79% from 2-cell stage embryos, Table 2) was compara-ble to that of our previous study (77%; Kishigami et al., 2006b). Thus,we considered that our methodology was valid and that the phe-nomena observed in this study were not caused by potential var-iability between experiments arising from factors such as differentbatches of oocytes and donor cells.

Firstly, when we counted nuclei number of SCNT and ICSI embryosat 70 h after activation, SCNT embryos showed less nuclei numbercompared with ICSI embryos (Fig. 2A). To analyze the detail ofcleavage speed, we counted the time-dependent changes in num-bers of nuclei during early cleavage in 108 embryos produced by

Selection of embryos

Embryo transfer

B

mRNA injection

Enucleation and nuclear transfer Multi-dimensional

imagingEGFP-tubulinH2B-mRFP1

A

Analysis of image data

ActivationPN Morula

TSA 9h

Cloned pups?

Duration: 60 hoursTime interval: 15 minZ-axis: 2 µm interval x

51 images (totally 100 µm)

Channels: Bright field, 488 nm, 561 nmLens used: UPlanApo x20 oilLaser power: 0.1 mW (from lens tip)

Fig. 1. Scheme of the live-cell imaging experiments. (A) Metaphase II oocytes were injected with mRNAs and then enucleated and injected with donor cumulus cell nuclei. Theactivated reconstructed oocytes were set on the live-cell imaging system and fluorescent images were acquired from the pseudopronuclear (PN) stage to the morula stage. Condi-tions for imaging were listed in box. (B) After live-cell imaging, the SCNT embryos were selected and grouped based on analyses of reconstructed time-lapse movies. Groups ofembryos were transferred separately to pseudopregnant females and assessed their full-term developmental ability.

59E. Mizutani et al. / Developmental Biology 364 (2012) 56–65

ICSI and 108 embryos generated by SCNT, respectively (Fig. 2B).Just before the onset of anaphase of the first mitotic divisionwas defined as ‘time zero’ and the numbers of nuclei of each em-bryo were plotted until 53 h later. The cloned embryos showed awider range of division timing in each embryo than ICSI embryos,especially from the 2- to 4-cell transition and from the 4- to 8-cellstage (Fig. 2B). Thus, the speed of development was slightly re-tarded in SCNT embryos (Fig. 2B). The final numbers of nuclei at53 h after first division were slightly lower in SCNT embryos(12.5±3.96) than in ICSI embryos (14.2±4.48).

We then analyzed the rate of cell division to determinewhat causedthis variance and retardation in the speed of growth of SCNT embryos.First, the times taken (in hours) at the 2-cell and 4-cell stages (named‘intrastage duration’; Fig. 3A) were compared between SCNT and ICSIembryos. The SCNT embryos took slightly longer at each developmentalstage (2-cell, 21.81±3.21 h; 4-cell, 15.52±4.01 h) compared with ICSIembryos (2-cell, 20.23±2.11 h; 4-cell: 13.07±2.11 h, Fig. 3B). Next,we analyzed the times required for transition to the next stage(named ‘interstage duration’; Fig. 3A). This timewas defined as the du-ration from the onset of anaphase in the leading blastomere to that ofthe last blastomere to divide during each cleavage stage. Thus, it corre-sponded to the synchrony of division among blastomeres in each em-bryo. The interstage durations of SCNT embryos (2- to 4-cell, 2.16±2.14 h; 4- to 8-cell, 5.97±3.24 h) were significantly longer than inICSI embryos (2- to 4-cell, 1.13±1.25 h; 4 to 8-cell, 3.61±3.02 h;Fig. 3C). These data suggest that therewasweaker synchrony of divisionamong the blastomeres of SCNT embryos than in ICSI embryos. Thus,the retardation and variance in speed of development seen in SCNT

embryos arose from cumulative delays in the timing of cleavageamong their constituent blastomeres.

Effect of the speed of early embryogenesis on the full-term developmentof SCNT embryos

To examine whether the retardation in cleavage rate affected full-term development, SCNT morulae were divided into four groupsbased on the numbers of blastomere nuclei at 70 h after activation(Fig. 2A): ≥16, 12–15, 8–11 and ≤7. Each group of SCNT embryoswas transferred separately to a pseudopregnant female mouse(Fig. 1B). Interestingly, SCNT embryos with more than 8 nuclei at70 h after activation could develop to term; 1.7% from embryos hav-ing more than 16 nuclei, 0.8% from embryos with 12–15 nuclei and1.3% from embryos having 8–11 nuclei, respectively. On the otherhand, SCNT embryos with fewer than 7 nuclei could not develop toterm (Table 1). These data suggested that the speed of developmentis not necessarily a key factor for the full-term development of SCNTembryos except for very slow-growing ones. In addition, it appearsthat weak synchrony of blastomere division in SCNT embryos mightbe not a fatal cause of developmental failure.

Abnormal chromosomal segregation (ACS) during early embryogenesisand interactions with the rate of development

When we monitored mitotic divisions during early cleavage, someembryos showed abnormal chromosomal behaviors in mitotic

B

<7

8-11

12-15

>16

ICSI

SCNT

0 20 40 60 80 100 (%)

9 4a 19 68a

17 17b 31 35b

A

048

1216202428

0 5 10 15 20 25 30 35 40 45 50

048

1216202428

0 5 10 15 20 25 30 35 40 45 50

Nu

mb

er o

f n

ucl

eiN

um

ber

of

nu

clei

ICSI

SCNT

(h)

(h)

0

4

8

12

16

20

0 5 10 15 20 25 30 35 40 45 50

SCNT ave.ICSI ave.

Nu

mb

er o

f n

ucl

ei

(h)

14.212.5

Average

1 to 2 cell2 to 4 cell

4 to 8 cell

8 to 16 cell

1 to 2 cell2 to 4 cell

4 to 8 cell

8 to 16 cell

Fig. 2. Comparison of the rate of development between ICSI and SCNT embryos.(A) Comparison of the numbers of nuclei in ICSI and SCNT embryos at 70 h after acti-vation. Embryos were categorized to four groups based on the numbers of nuclei:≤7, 8–11, 12–15 and ≥16. Embryos with different numbers of nuclei are shown in dif-ferent colored bars. The percentages in each group are also shown on the bars. Valueswith different superscripts (a and b) in the same category of nuclei number differ sig-nificantly between ICSI and SCNT at Pb0.05. (B) Time-dependent increases in the num-bers of nuclei of ICSI and SCNT embryos are shown. The top graph indicates ICSIembryos, the middle graph indicates SCNT embryos and the bottom graph shows thepooled mean values. For comparing the timing of division exactly, the time point atanaphase onset just before the first mitosis was defined as 0 h. Differently coloredlines indicate individual embryos.

A

0

10

20

30

(h)

2-cell 4-cell

ICSI ICSI SCNTSCNT

40

0

5

10

15

20(h)

2 to 4-cell 4 to 8-cell

ICSI ICSI SCNTSCNT

B

20.23 21.81

13.0715.52

1.13 2.163.61

5.97

* <0.05*

* <0.05*

*

C

0

2

4

6

8

0 5 10 15 20 25 30 35 40 (h)

2-cell intrastage duration

4-cell intrastage duration

4-8 cell interstageduration

2-4 cell interstagedurationN

um

ber

of

nu

clei

*

Fig. 3. The detail of cleavage timing in each ICSI and SCNT embryo. (A) The model of‘intrastage duration’ (black arrows) and ‘interstage duration’ (red arrows) duringearly cleavage. (B) Intrastage duration at the 2-cell and 4-cell stages by ICSI andSCNT embryos. Different marks indicate different embryos. The black bars and nu-merals indicate the mean time spent at each developmental stage in hours. (C) Com-parison of the interstage duration required for the 2- to 4-cell and 4- to 8-celldivisions between ICSI and SCNT embryos. Bars indicate the average time in hours re-quired for completing each mitotic division.

Table 1Generation of offspring from cloned embryos with different rates of development.

No. of culturedembryos

No. of 2-cellembryos

Numbers of nuclei at70 h after activation

No. oftransferredembryos

No. of clonedoffspring (%)a

397 394 ≥16 114 2 (1.7)12–15 121 1 (0.8)8–11 78 1 (1.3)≤7 42 0

a Based on the no. of transferred embryos.

60 E. Mizutani et al. / Developmental Biology 364 (2012) 56–65

blastomere. In detail, a part of the chromosomes were misalignedwith the metaphase plate, and/or the lagging chromosomes werefound at the anaphase stage. As a result of these abnormal behaviors,ectopic micronuclei formation was frequently found in SCNT embryos(Fig. 4A). We defined all of such unusual movement of chromosomesat mitotic division during early embryogenesis as “abnormal chromo-some segregation (ACS)”. Time-lapse images of typical patterns ofACS and the formation of micronuclei are shown in Fig. 4A. ACSoccurred at all mitoses, 2- to 4-cell, 4- to 8-cell and 8- to 16-cell(Fig. 4B, Supplemental movie 1). The incidence of ACS in SCNT embry-os was significantly higher than those of ICSI and IVF embryos, espe-cially at the first cell division (Fig. 4C). Surprisingly, the incidence ofSCNT embryos without any ACS (we defined these as showing normalchromosomal segregation, or NCS embryos) was only 8.9% (versus37.8% in ICSI embryos and 45.9% in IVF embryos). We also analyzedthe detail of cleavage speed of NCS and ACS embryos (Fig. S2). Inter-estingly, the resulting speed of development to the morula/blastocyststage of ACS embryos was almost same as that of NCS embryos(Fig. S2), and morula-stage embryos with ACS were indistinguishablemorphologically from those with NCS (Fig. 4B, Supplemental movie

1). Moreover, embryos were cultured continuously after live-cell im-aging until the expanded blastocyst and immunostained using anti-Oct3/4 and cdx2 antibodies (Fig. S3). Regardless of the chromosomesegregation type, the proper cell allocation to inner cell mass (ICM)and trophectoderm (TE) was found (Fig. S3). These results indicatethat ACS might not affect embryonic growth and differentiation upto the blastocyst stage.

Effect of ACS on the full-term development of SCNT embryos

We assessed the full-term developmental ability of SCNT em-bryos with ACS. Each morula was grouped based on the stagewhen it first showed ACS. These groups of SCNT morulae were

B

A

PN 2-cell 4-cell 8-cell Morula

1-2 ACS

2-4 ACS

C

H2BTubulinH2B

NCS

4-8 ACS

8-16 ACS

BF

1-2

2-4

4-8

8-16

NCS

ICSI

SCNT

7.1a 15.3b 22.4a 17.3ab 37.8a

20.9b 23.4b 34.8b 12.0b 8.9b

(%)0 20 40 60 80 100

ACS

IVF 3.5a3.5a 20.5a 26.6a 45.9a

Misalignedchromosome

Laggingchromosome

2-cell2-cellanaphasemetaphasePN

H2BTubulin

Fig. 4. Abnormal chromosomal segregation found in SCNT embryos. (A) Typically abnormal chromosomal behaviors. As the example, time-lapse images of chromosome segregationat first mitosis are shown. The upper and lower panels indicate the patterns of misaligned chromosome and lagging chromosome, respectively. Arrows indicate the ectopic chro-mosomes during mitosis and arrow heads suggest the resulting micronuclei at the 2-cell stage. We defined all of such abnormal movement of chromosomes as abnormal chromo-somal segregation (ACS). (B) Variety of types of chromosomal segregation in SCNT embryos. ACS occurred at each division during early cleavage. Arrows indicate the chromosomalfragments appearing during the division. Merged and bright field (BF) images of 16-cell stage embryos are also shown. Green, EGFP-α-tubulin; Red, H2B-mRFP1. (C) The percent-ages of embryos with ACS at each division in IVF, ICSI- and SCNT embryos. Values with different superscripts (a and b) in the same category, i.e., 1–2 ACS, 2–4 ACS, 4–8 ACS, 8–16ACS and NCS embryos, differ significantly between IVF, ICSI and SCNT at Pb0.05.

61E. Mizutani et al. / Developmental Biology 364 (2012) 56–65

transferred separately to pseudopregnant female mice. Three livecloned pups were obtained from totally 330 morulae transferred(0.9%, Table 2). Importantly, SCNT embryos in which ACS occurredbefore the 8-cell stage could not generate any live clones. On theother hand, 8–16-cell ACS embryos and NCS SCNT embryos coulddevelop to term (8–16-cell ACS embryos 2.5%; NCS embryos 7.1%;Table 2). Thus, ACS occurring during chromosomal segregation inearly cleavage stages of SCNT embryos can strongly affect theirdevelopmental potential. In other words, as the proportion ofSCNT embryos which do not cause any ACS until 8-cell stagewas quite low (20.9%, Fig. 4) and cloned mice could only be

derived from these embryos, it indicated the failure of SCNT em-bryos to survive appears to have been mainly caused by chromo-somal instability arising during successive cell divisions.

Retrospective confirmation of prerequisites for the full-term developmentof cloned embryos by individual imaging and embryo transfer

From our results, it appears that the SCNT embryo with the poten-tial to develop to term must bear more than eight nuclei at 70 h afteractivation and also needs to demonstrate NCS, at least until the 8-cell

Table 2Relationship between full-term development and chromosome abnormalities in SCNTembryos.

Type of chromosomeabnormality

No. of transferredembryos

No. of live clonedoffspring (%)a

1- to 2-cell ACS 86 02- to 4-cell ACS 64 04- to 8-cell ACS 112 08- to 16-cell ACS 40 1 (2.5)NCS 28 2 (7.1)Total 330 3 (0.9)

SCNT, somatic cell nuclear transfer; ACS, abnormal chromosomal segregation; NCS,normal chromosomal segregation.

a Based on the no. of transferred embryos.

62 E. Mizutani et al. / Developmental Biology 364 (2012) 56–65

stage. However, it is difficult to get statistically significant answers inanimal cloning studies because of the extremely low overall successrate of SCNT. Therefore, we added another line of experiments tostrengthen our conclusions. We carried out single SCNT embryotransfer after imaging and retrospectively analyzed only those em-bryos showing full-term development (Fig. 5). In total, 328 activatedSCNT embryos were monitored for 60 h; 72 morphologically normalmorulae were selected randomly and cotransferred singly with carri-er embryos into pseudopregnant females. Three cloned mice (wenamed them in birth order Teru, Kazu and Eiji) were obtained bythis technique (Table 3, Fig. 6A). The body and placental weightswere almost same as in previous reports on mouse cloning(Table 3). Then, the corresponding movies of early cleavage of thesethree embryos were analyzed retrospectively (Fig. 6A, Supplementalmovie 2). First, their speed of development (Fig. 6B) and the synchro-ny of blastomere division (Fig. S4A, B) were analyzed. None of thesethree embryos diverged from the average speed of development, al-though the Eiji embryo showed slightly delayed development com-pared with the Teru and Kazu embryos. The numbers of nuclei inthe Teru, Kazu and Eiji embryos at 70 h after activation were 17, 16and 16, respectively, and they were not necessarily ‘top speed’ em-bryos in this assay (the three fastest embryos showed 20, 22 and23 nuclei at this point). These values confirmed our conclusionthat a viable SCNT embryo should have more than eight nuclei at themorula stage for full-term development. And the weak synchrony of

ICR morul

A

B

C

A

B C

DE

. . .

MorulaPN

SCNTembryos

Retrospeanalys

Fig. 5. Generating clonedmice by single SCNT embryo transfer. Scheme of the single SCNT embtime-lapse movies. One SCNT embryo and four to ten fertilized ICR strain embryos were group

blastomere division itself may not be a reason for developmental failureof SCNT embryos, because although the 4- to 8-cell division of Kazuwashighly synchronous (taking approximately 1 h), Teru showed asyn-chronous blastomere division (taking nearly 8 h) (Fig. S4B). Next, thechromosomal segregation patterns of these embryos were assessed.Two of the three successfully cloned mice developed from embryoswith NCS (Fig. 6A; Teru and Kazu) but one was from an 8- to 16-cellACS embryo (Fig. 6A; Eiji, Table 3). Taken together, these results suggestthat the speed of development and synchrony during early cleavage arenot important for the full-term development of SCNT embryos, howev-er, normal chromosomal segregation at least up to the 8-cell stage isneeded for producing cloned mice successfully.

Discussion

Prerequisites for the full-term development of cloned embryos

In present study, we found two prerequisites for the full-term de-velopment of cloned embryos as follows. We clarified that SCNT em-bryos vary in their rate of development compared with ICSI embryosat early cleavage stages. Although rapid growth per se was not neces-sary for the successful full-term development of SCNT embryos, em-bryos with fewer than 7 nuclei at 70 h after activation could notdevelop to term. We also demonstrated that ACS occurred in mostSCNT embryos during their early cleavage stages, even though theyappeared normal morphology through normal light microscopy. In-terestingly, no cloned mice were generated from SCNT embryos bear-ing ACS before the 8-cell stage, suggesting that the embryo has toshow normal chromosomal segregation at least until this stage if itis to be viable.

Reasons for the low success rate of cloning

Our data suggest that much of the full-term developmental failureof SCNT cloned embryos is caused by cytogenetic abnormalities suchas ACS occurring during early cleavage. This conclusion is surprisingbecause epigenetic incompatibility and/or incomplete reprogram-ming of the somatic genome have been thought to be the majorcauses for the low success rates in animal cloning so far. In theSCNT technique, the clone's genome is derived from a terminally

Embryotransfer

a

Clonedmouse

ctiveis

ryo transfer experiment. Morula stage SCNT embryos were identified individually from theed and transferred to pseudopregnant ICR females. PN, pseudopronuclear stage.

Table 3The results of single SCNT-generated cloned embryo transfer.

No. of single embryo-transferSCNT embryos

No. of live clonedoffspring (%)

Name of viable embryo Chromosomal status up to70 h after activation

Number of nuclei Live body weight(placental weight) in g

72 3 (4) Teru NCS 17 1.15 (0.33)Kazu NCS 16 0.74 (0.43)Eiji 8- to 16-cell ACS 16 1.43 (0.30)

NCS, normal chromosomal segregation; ACS, abnormal chromosomal segregation.

63E. Mizutani et al. / Developmental Biology 364 (2012) 56–65

differentiated donor somatic cell nucleus. These nuclei have a differ-ent epigenetic status from germ cells and must undergo reprogram-ming in the recipient ooplasm. Actually, abnormal global DNAmethylation and histone modification patterns have been found inSCNT embryos (Yang X. et al., 2007). Moreover, although SCNT em-bryos show normal genomic imprinting pattern (Inoue et al., 2002),altered X-inactivation (Inoue et al., 2010) and abnormal Oct3/4 ex-pression (Boiani et al., 2002; Kishigami et al., 2006a) in SCNT embryoshave been also reported.

Teru

Kaz

uE

iji

PN 2-c

H2B

Merge

H2B

Merge

H2B

Merge

PupsA

B

Nu

mb

er o

f n

ucl

ei

0

4

8

12

16

20

0 5 10 15 20 25 3

Fig. 6. Retrospective analysis of cloned mice generated by single SCNT embryo transfer. (A) Lblack-eyed pups were cloned mice (arrows) and the other pups were derived from fertilizeddpc and Kazu was obtained at 17.5 dpc. The chromosomal behavior of the original SCNT embproducing clones Teru and Kazu had NCS. ACS occurred in the embryo producing clone Eijinumbers of nuclei between successful SCNT embryos. The mean rate of division of SCNT em

However, our findings here suggest that most SCNT embryos ex-hibit severe genetic aberrations during chromosomal segregation be-fore they develop to morula stage and such embryos fail to produceviable clones. Taken together, it is probable that the SCNT embryosin which such epigenetic reprogramming occurred correctly and si-multaneously revealed normal chromosomal segregation could de-velop to term. To our knowledge, this is the first demonstration ofone important reason for the low success rate of cloning, based on di-rect evidence using ‘less-damage’ live-cell imaging technology.

ell 4-cell 8-cell Morula

0 35 40 45 50 55

SCNT ave.

(h)

Teru

Kazu

Eiji

ive cloned mice produced by single SCNT embryo transfer and their origin embryos. TheICR embryos gestated in the same recipient female. Teru and Eiji were obtained at 18.5ryos that successfully produced cloned mice was presented in right panels. The embryosat the 8- to 16-cell division (boxed image, arrow). (B) Comparison of the increases inbryos is shown in the blue line.

64 E. Mizutani et al. / Developmental Biology 364 (2012) 56–65

In present study, we succeeded to improve the cloning rate byscreening the embryo with NCS (0.9% to 7.1%). However, unexpected-ly, the full-term development rate of SCNT embryos in this study waslower (0.9%) than previous results from our laboratory (5.3%, (VanThuan et al., 2009)). This was probably caused by the difference in ex-perimental conditions, namely the different culture system and/orprolonged handling time of embryos before the transfer might affectthe developmental ability of SCNT embryos.

Chromosomal abnormalities and their effects on full-term development

Surprisingly, the SCNT embryos showed a high rate of ACS at earlycleavage stages (Fig. 4C). We reported previously (Yamagata et al.,2009a) that ACS at first division caused serious damage to the devel-opmental ability of ICSI embryos. Here, none of the SCNT embryosshowing ACS up to the 8-cell stage could develop to term. However,there was no difference between the appearance of morulae fromembryos with NCS or ACS at the light microscopy level. ICM–TE cellsegregation at the blastocyst stage, highlighted by Oct3/4 and cdx2immunostaining, was unaffected (Fig. S3). Moreover, the rate of de-velopment at cleavage stage was similar in these two groups of em-bryos (Fig. S2). These results demonstrated that, regardless ofembryonic morphology, ACS is a critical factor leading to failed devel-opment. However, ACS occurring after the 8-cell stage enabled SCNTembryos to develop to term because cloned mice including ‘Eiji’were obtained from an embryo in which ACS occurred at the 8- to16-cell division. One possible reason for this result is that the numberof blastomeres with normal chromosomes may be sufficient to sup-port full-term development even if some of them show ACS in laterdivisions. Another possible reason is that blastomeres showing ACSmay have the potential to differentiate into TE cells. For example,the giant trophoblast cell contains an amplified genome that arisesby endoreduplication (Varmuza et al., 1988) and a functional placentacan be formed from cells having abnormal chromosome numberssuch as tetraploid blastomeres (Ohta et al., 2008). Thus if ACS oc-curred in the blastomere which is destined to differentiate to TE,they may be able to form functional placentae. To elucidate this, it isnecessary to track the each cell from early cleavage stage to ICMand TE cells of expanded blastocyst. However, unfortunately, probesustainability and image resolution still insufficient at present toachieve such purpose. We will re-evaluate this possibility after theimprovement of our technology in future.

Causes of ACS

It is uncertain what causes such chromosome abnormality at latercleavage stages in SCNT embryos. It has been reported that the chro-mosomal damage of SCNT or ICSI embryos was caused by some tech-nical reasons such as micromanipulation procedure (Yu et al., 2007)or chemical reaction during sperm preincubation time (Tateno,2008; Tateno and Kamiguchi, 2007). In addition, Osada et al. analyzedthe chromosomal integrity of the SCNT embryos immediately afternuclear transfer by spectral karyotyping, and found that structuraland numerical abnormalities occurred frequently and that their inci-dence differed between donor cell types (Osada et al., 2009). Thus,it is also possible that incorrect constitution of the chromosomes inthe donor nucleus may directly reflect the chromosomal abnormali-ties in the SCNT embryo. However, these chromosomal abnormalitiesmainly appeared at the first mitotic event, and thus the abnormalchromosome behavior at later stages seems to be caused by inherentdefects in the mitotic machinery rather than technical reasons. In mi-totic chromosomes of mouse cells, the kinetochore is assembled onthe distal ends of chromosomes (centromeric region). This is a hugeand complicated protein–DNA structure composed of multiproteincomplexes and centromeric satellite DNA. Yamagata et al. reportedthat the centromeric DNA of injected somatic cell nuclei was more

highly methylated than that of the germ cell lineage and that thishypermethylation status was maintained during preimplantation de-velopment (Yamagata et al., 2007). This particular epigenetic status ofthe centromere might lead to abnormal construction of the kineto-chore and further ACS in SCNT embryos. Another possibility is thatthe spindle assembly itself or the spindle assembly checkpoint(SAC) in SCNT embryos is abnormal. In primate SCNT embryos,some molecules required for spindle pole assembly, such as nuclear-mitotic apparatus (NuMA) and centrosomal kinesin HSET, were lack-ing (Simerly et al., 2003). Tani et al. (2007) reported that bovineoocytes used for SCNT have aberrant spindle morphology and ACSat MII caused by insufficient SAC signals from somatic cell nuclei.Aurora kinases also have important roles in cell division. In particular,Aurora B is a component of the chromosomal passenger complex re-quired for correct spindle–kinetochore attachments during chromo-somal segregation and cytokinesis (Hauf et al., 2003; Zeitlin et al.,2001). Cells lacking Aurora B activity showed aberrant cytokinesisand multinucleation (Adams et al., 2000; Terada et al., 1998). In thepresent study, some multinuclear blastomeres were observed in de-velopmentally arrested SCNT embryos but not in ICSI embryos. There-fore, the activity of such mitotic kinases in SCNT embryos may beweak or abnormal.

Perspectives

Here, we were able to evaluate the embryogenesis of SCNT embry-os having full-term developmental ability using a single embryotransfer technique and live-cell imaging analysis. By applying thismethod, it will be possible to find other developmental markers andto observe in detail epigenetic changes in successful SCNT embryos.This will help in determining the essential criteria for successful ani-mal cloning. These new fluorescent probes and live-cell imaging sys-tems will help in establishing the prerequisites for the full-termdevelopment of mammalian SCNT cloned embryos and in under-standing the mechanisms of donor cell genomic reprogramming.

Supplementary materials related to this article can be found on-line at doi:10.1016/j.ydbio.2012.01.001.

Acknowledgments

We are grateful to the Laboratory for Animal Resources and GeneticEngineering of RIKEN Center for Developmental Biology in Kobe, forhousing the mice. Financial support for this research was providedby a grant under the Japanese programon Scientific Research in PriorityAreas (15080211) and by a Grant-in-Aid for Scientific Research onInnovative Areas (21114521).

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