CLONING AND STEM CELLSVolume 11, Number 1, 2009© Mary Ann Liebert, Inc.DOI: 10.1089/clo.2008.0072

Live Birth of Somatic Cell-Cloned Rabbits following Trichostatin A Treatment and Cotransfer

of Parthenogenetic Embryos

Qinggang Meng,1 Zsuzsanna Polgar,1 Jun Liu,1 and Andras Dinnyes1,2

Abstract

Somatic cell nuclear transfer (SCNT) efficiency is still low in rabbit. Previous studies indicated that trichostatinA (TSA) treatment could improve cloning efficiency and term development in the mouse, and cotransfer ofparthenogenetic (PA) embryos benefited the pregnancy of cloned embryos in porcine and the mouse. In thisstudy we investigated the effect of TSA treatment on the term development of the SCNT rabbit embryos, andthe possibility of the pregnancy maintenance of clones by cotransfer of PA embryos. The SCNT embryos wereproduced by fusing cumulus cells with enucleated cytoplasts before activation by electrical stimulation, andDimethylaminopurine (6-DMAP) and Cyclohexamide (CHX) treatments. They were cultured in EBSS-completemedium regardless of their treatment with or without TSA. In vitro developmental data showed no differencesin the cleavage and the blastocyst rates, and the blastocyst cell number between the TSA-treated and the un-treated SCNT embryos. Two of the six recipients became pregnant after the embryo transfer (ET) in the TSA-treated group, and one pregnant female delivered seven live and three stillborn pups. The death of all live pupsoccurred within an hour to 19 days. Four of the seven recipients became pregnant in the TSA-untreated group.Three of them gave birth to six live and eight stillborn pups. Four pups of the TSA-untreated group have growninto adulthood, and three of them produced progeny. Cotransfer of three to four PA embryos with 26–32 SCNTembryos to the same recipient resulted in pregnancy and birth rates statistically no different compared to thecontrol SCNT ET group. In conclusion, our results indicate that TSA treatment has a limited effect on the invitro development of the SCNT embryos; furthermore, both the TSA-treated and the untreated clones can de-velop to term in rabbits, but none of the offspring from TSA-treated embryos survived to adulthood in our ex-periment.

203

Introduction

CLONING ADULT RABBITS using somatic cell nuclear transfer(SCNT) technology could serve as a model for the re-

production of high value domestic or endangered mam-malians. Combined with transgenic technology, it could alsobe used to produce animal models for the study of some hu-man diseases. Although successful rabbit cloning with so-matic cell has been achieved (Chesne et al., 2002), the effi-ciency of the SCNT is still very low in rabbits. So far, only afew live births were reported, and most of the clones diedwithin the first 3 weeks after birth, limiting the further ap-plication of rabbit SCNT in biomedical research or animalreproduction. Recently, rabbits generated from adult fibro-blast have been produced using a protocol (Li et al., 2006)

modified from the previously published one (Challah-Jacques et al., 2003). However, among the 14 clones, most ofthe offspring died soon after birth; only three of the pupssurvived to adulthood.

Successful nuclear reprogramming from a somatic-state toan embryonic one is the key event in SCNT (Hochedlingerand Jaenisch, 2006; Rideout et al., 2001). It largely dependson the epigenetic state of the nuclei. Histone acetylation isone of the major epigenetic events. Recent studies haveshown that elevated levels of histone acetylation in donorcells or cloned embryos could improve their development,thus improving the efficiency of this technology. Treatmentof bovine fetal fibroblasts with sodium butyrate (NaBu), ahistone deacetylase (HDAC) inhibitor, increased the devel-opmental rate of cloned blastocysts (Shi et al., 2003). Re-

1Genetic Reprogramming Group, Agricultural Biotechnology Center, Godollo, Hungary.2Molecular Animal Biotechnology Laboratory, Szent Istvan University, Godollo, Hungary.

cently, live cloned progenies have been produced fromNaBu-treated cultured rabbit cumulus cells (Yang et al.,2007). Similarly, the treatment with TSA, another HDAC in-hibitor, improved cloning efficiency in cattle (Enright et al.,2003) and mice (Kishigami et al., 2006; Rybouchkin et al.,2006; Wang et al., 2007). Furthermore, recent reports indi-cated that TSA treatment could increase the cell number ofrabbit SCNT blastocysts (Xu et al., 2007), or the blastocystdevelopment rate (Shi et al., 2008a). Also, the histone acety-lation pattern of the TSA-treated rabbit SCNT embryos ap-peared to be more similar compared to those of the normalembryos (Shi et al., 2008b). However, term development ofthe TSA-treated cloned embryos in these species has not beenyet examined.

Similarly to SCNT in other species, only 1–3% of the trans-ferred SCNT rabbit embryos gave rise to live pups, despitethe fact that nearly 50% of the rabbit SCNT embryos couldhave developed to blastocyst stage (Challah-Jacques et al.,2003; Li et al., 2006). This indicates major losses duringpostimplantation development. It is possible that many ofthe defects in SCNT embryos occurring during the repro-gramming stage might attribute to disorders during placentadevelopment and with placenta function (Yang et al., 2007).

Previous reports indicated that the cotransfer of partheno-genetic embryos was more efficient for the SCNT embryopregnancy maintenance rather than using the timed admin-istration of estradiol or eCG, and it resulted in the birth of acloned piglet (De Sousa et al., 2002). Cotransfer of low num-ber of parthenotes with SCNT embryos could trigger thepregnancy establishment in porcine (Lai et al., 2002) and im-proved pregnancy and birth rates in mouse (Meng et al.,2008). The number of cotransfer of the SCNT embryo stud-ies is still low, and limited to pig and mouse studies. The ef-fects of embryo cotransfer on the term development of theSCNT embryos still remain unclear in other species, includ-ing rabbit, despite that this approach should improve the ef-ficiency of SCNT technology.

In this study we investigated the effect of TSA treatmentand cotransfer of parthenogenetic embryos on the term de-velopment of the SCNT rabbit embryos.

Materials and Methods

Animals and chemicals

The protocols for animal care and handling were approvedby the Animal Experiments Committee of ABC, Godollo,Hungary, and the Animal Health Authorities. Unless other-wise stated, all chemicals used were purchased from SigmaChemical Co.

Oocyte and donor cell collection

Mature (20–22-week-old) Hycole hybrid female rabbitswere superovulated by the injection of 120 IU pregnant mareserum gonadotropin (PMSG, Folligon, International B.V.,Boxmeer, Holland) intramuscularly and 180 IU human chori-onic gonadotropin (hCG; Choragon, Ferring GmbH, Kiel,Germany) was injected intravenously 72 h later. Matureoocytes were flushed from the oviducts 13–14 h after the hCGinjection with Medium 199 supplemented with 10% fetal calfserum (FCS) and 20 mM HEPES (M199). The cumulus cellswere removed from the oocytes by gentle pipetting in 5

mgmL�1 hyaluronidase M199 at 37°C and incubated inEarle’s balanced salt solution-complete (EBSS-complete)medium (Mitalipov et al., 1999) with 5% CO2 in air at 38.5°Cuntil use.

The cumulus cells were used as nuclear donors. They werecollected as described above from the cumulus–oocyte com-plexes of one donor female, and centrifuged at 3000 rpm for1 min and then kept in M199 at 4°C prior to cell insertion.

Nuclear transfer and TSA treatment

The somatic cell nuclear transfer procedure was carriedout essentially as described previously (Challah-Jacques etal., 2003) with minor modifications. The oocytes were stainedwith 5 �gml�1 Hoechst 33342 in EBSS-complete medium for20 min. Following the pretreatment in M199 with 7.5 �gmL�1

cytochalasin B for 10 min, the nuclei of oocytes were local-ized applying UV illumination for 1–2 sec and removed byan 18–20-�m outer diameter micropipette. Prior to cell in-sertion the enucleated oocytes were allowed to recover inEBSS-complete medium for 2 h.

A cumulus cell was inserted in the perivitelline space ofthe enucleated oocyte using the same micropipette. The cy-toplast–cell constructs were induced to fuse by three 20 �sec3.2 kVcm�1 DC pulses in the activation medium (0.25M sor-bitol in water, supplemented with 0.5 mM HEPES, 0.1 mMCa(CH3COO)2, 0.5 mM Mg(CH3COO)2 and 1 mgmL�1

bovine serum albumin). The fused embryos were activatedapplying the previously described electrical pulses 1 h laterand treated for 1 h with 2 mM 6-dimethylaminopurine (6-DMAP) and 5 �gml�1 cycloheximide (CHX) in EBSS-com-plete medium. The cloned embryos were then subsequentlycultured in EBSS-complete medium with or without 5 nMTSA for 10 h long as described before (Kishigami et al., 2006;Xu et al., 2007).

Embryo culture

The SCNT embryos were cultured in EBSS-completemedium at 38.5°C in a humidified atmosphere of 5% CO2 inair either overnight until two- to four-cell stage was obtainedor for 4.5 days until they reached the expanded/hatchingblastocyst stage.

Parthenogenetic embryo production

Mature oocytes of 14–15 h post-hCG injection were artifi-cially activated and cultured in vitro as described above.

Cell counting of blastocysts

The blastocysts were incubated for 20 min in EBSS-com-plete medium containing 5 �gmL�1 Hoechst 33342, and cellnuclei were counted using reverse microscope applying UVexcitation.

Embryo transfer (ET) and cotransfer

Embryos were transferred through the infundibulum intoeach of the oviduct of the recipients using a laparoscopictechnique as described previously (Besenfelder and Brem,1993). Two- to four-cell stage nuclear transfer cloned em-bryos were transferred alone (SCNT) or cotransferred withthree to four parthenogenetic embryos (SCNT � PA) to the

MENG ET AL.204

recipients that were induced into pseudopregnancy by in-jection of 0.2 mL GnRH analog (Receptal®, Hoechst RousselVet) 22 h later following the injection of the oocyte donor fe-males (22 h asynchrony with SCNT two- to four-cell em-bryos). Recipients were operated by caesarean sections onday 30 post-ET. The birth and placental weights of progenywere measured, and the live pups were fostered to a femalewith pups of similar age. Some recipients were allowed togive birth naturally 32 days post-ET; the live pups wereraised thereafter by their recipient “mothers.”

Live cloned pups were fed once daily by the foster motherduring the first 3 weeks after birth and supplied with pelletfood following the fourth week. They were weaned 5 weeksafter their birth. The corpses were dissected to detect themorphologies of the main organs.

Statistical analysis

SSPS software was used for statistical analyses. Data wereanalyzed by ANOVA. Significance level was considered atp � 0.05.

Results

In vitro development of SCNT rabbit embryos with TSA treatment

The in vitro developmental data (Table 1) showed no dif-ferences between the TSA-treated and the untreated clonedembryos in the cleavage and blastocyst rates, and the blas-tocyst cell number.

Term development of TSA-treated and untreated SCNT embryos

Table 2 shows the term development of the cloned em-bryos treated with or without TSA. After embryo transfer,four of the seven recipients of the untreated group and twoof the six of the TSA-treated group became pregnant. Onefemale in the untreated group aborted 22 days post-ET, whileone in the TSA-treated group aborted 25 days post-ET. A fe-male of the TSA-treated group delivered seven live and threestillborn pups. Three females of the untreated group gavebirth to six live and eight stillborn pups. These results indi-cate that among SCNT rabbit embryos, both the TSA-treatedand untreated embryos can develop to term.

Postnatal development of SCNT rabbit pups

Most of the live cloned pups weighed in the ranges of53–67 g in the TSA-treated group and 61–73 g in the un-treated group, respectively, except for a small one (30 g) ofthe TSA-treated and two large pups (90 and 93 g) of the un-

treated groups. All of the seven pups in the TSA-treatedgroup died in an unknown reason within 1 h to 19 days af-ter birth, while one of the six pups in the TSA-untreatedgroup died in an unknown reason within 24 h and anotherone was sacrificed due to severe injuries in the belly causedaccidentally by her foster mother. Four of the six clones inthe untreated group have survived over 14 or 16 months,and three of them gave births to progenies after mating witha male rabbit, indicating fertility of the clones. The bodies ofthe dead clones were dissected to investigate the morpholo-gies of main organs. Although the autodigestion made thedetailed study of the organs difficult, no obvious abnormal-ities were observed in the heart, lung, liver, kidney, stom-ach, muscle, skin, bone, and brain of the clones.

Pregnancy maintenance after cotransferred of cloned andparthenogenetic embryos

All the SCNT embryos used in this experiment were with-out TSA treatment. All of the examined parameters, includ-ing pregnancy rate, number of fetuses formed and birth ratewere higher in the SCNT � PA group than in the SCNTgroup; however, with relatively low observation numbersnone of the differences were statistically significant. The livepups surviving to adulthood were found in both groups(Table 3). This result indicates that the cotransfer of PA em-bryos might benefit the pregnancy and term developmentrate but further studies are needed to confirm this.

Discussion

In the present study, we studied the term development ofthe TSA-treated rabbit SCNT embryos, which was an exten-sion of recent studies of the in vitro development of clonedrabbit embryos with the treatment of the HDAC inhibitors(Shi et al., 2008a, 2008b; Xu et al., 2007). We also investigatedthe improvement of the pregnancy maintenance of thecloned embryos with cotransfer of the parthenogenetic em-bryos. To the best of our knowledge, this is the second re-port of successful rabbit somatic cloning from cumulus cellssince the first one was reported 6 years ago (Chesne et al.,2002).

Our results indicate that the TSA treatment did not in-crease the blastocyst rate of rabbit SCNT embryos comparedto the untreated SCNT embryos. This is in agreement witha previous report (Xu et al., 2007). In our experiment we didnot find significant increase in the blastocyst cell number.Recent work has shown that the histone acetylation patternof the TSA-treated rabbit SCNT embryos seemed to be sim-ilar to a higher degree to those of the normal fertilized em-bryos compared to the untreated control group (Shi et al.,

TSA TREATMENT AND PA EMBRYO CO-TRANSFER IN RABBIT SCNT 205

TABLE 1. EFFECT OF TSA TREATMENT ON THE IN VITRO DEVELOPMENT OF SCNT RABBIT EMBRYOS

No.No. cleaved No. No. Cell No. of

Embryo cultured embryos blastocysts blastocysts blastocysttreatment embryos (%) (%) counted � SD

TSA-treated 87 66 (75.9) 52 (78.8) 42 202.9 � 41.0Untreated 119 93 (78.2) 74 (79.6) 58 171.5 � 49.2

Values were not significantly different by ANOVA.

2008b); furthermore, this phenomenon had also been foundin the TSA-treated mouse SCNT embryos (Wang et al., 2007),which may result in the improvements of the in vitro devel-opment of the TSA-treated cloned embryos. However, ourresults did not confirm the previous report, in which the blas-tocyst development rate of the TSA-treated rabbit SCNT em-bryos was significantly improved (Shi et al., 2008a). This con-flicting result may be attributed to the differences in the TSAtreatment, the cloning protocol, and the conditions of em-bryo culturing. Similar observations have been reported alsoin bovine. The blastocyst rate of bovine cloned embryostreated with 50 nM TSA was higher than that of the controlembryos (Iwamoto et al., 2007); furthermore, the TSA treat-ment reduced the DNA methylation level of the clonedbovine embryos compared to the IVF embryos (Iwamoto etal., 2008). However, other researches showed that the TSAtreatment did not affect the blastocyst rate, but increased thetotal cell number in bovine cloned embryos (Akagi et al.,2007); also no improvements were observed in the cleavage,the blastocyst rates and the blastocyst cell in the TSA treatedand the IVF control groups (Iager et al., 2008). These resultsindicate that the effect of the TSA treatment on the in vitrodevelopment of the SCNT embryos is related to specific ex-perimental environments and the conditions of the currenttreatment require further optimization.

Furthermore, the term development of the TSA-treatedrabbit SCNT embryos was observed in our study. Our re-sults showed that there were no difference in the pregnancyand the birth rate between the TSA-treated and untreatedgroups; also, development to term were obtained from bothgroups (Table 2). More interestingly, all of the seven clonesderived from the TSA-treated embryos died within 1 h to 19days after birth; however, four of the six clones from the con-trol group survived to adulthood. No obvious abnormalitieswere found in the main organs of the clones after death. Co-

incidently, early death of rabbits derived from the embryos,cloned with cultured fibroblasts and treated with NaBu, an-other HDAC inhibitor, has also been reported (Yang et al.,2007).

DNA methylation patterns are another important aspectof epigenetic states. They are closely linked to the chromatinstructure. There are approaches aimed at the reducing globallevels of methylation in an attempt to restore the develop-mental and the differentiation potential that are presumablybeneficial to improve the developmental competence of theSCNT generated reconstructs. It was found that altering thechromatin structure using exogenous molecules, adding thechromatin remodeling factor nucleoplasmin (NPL) tooocytes, improves the blastocyst development rate and thepregnancy of bovine-cloned embryos compared to the con-trol groups (Betthauser et al., 2006). The siRNA technologycan be used to knock down Dnmt1 gene expression in abovine donor cell line. It could improve the blastocyst de-velopment rates of the cloned embryos derived from thesedonor cells, suggesting that demethylation may be beneficialfor nuclear transfer-induced reprogramming prior to SCNT(Eilertsen et al., 2007). However, the treatment of the donorcells with 5-aza-cytidine or 5-aza-2-deoxycytidine, the clas-sic DNA methyltransferase inhibitors, prior to SCNT couldnot improve the blastocyst development rates (Enright et al.,2003, 2005; Jones et al., 2001). It is unclear whether the de-crease in blastocyst development rates was due to cytotoxi-city or to the demethylation of the genome. These results in-dicate that the effects of modification of epigenetic statususing exogenous chemicals might be long term, and it maynot be always positive. However, the treatment with theseHDAC inhibitors cannot be clearly linked to the deaths ofthese clones, due to the complicated characteristics of nu-clear reprogramming and shortage of data on this issue. Pre-vious report (Kishigami et al., 2006) and our study (Meng et

MENG ET AL.206

TABLE 2. EFFECT OF TSA TREATMENT ON THE TERM DEVELOPMENT OF RABBIT SCNT EMBRYOS

No. pupsNo. No. surviving

No. pregnant live toEmbryo embryo No. recipients No. stillborn pups adulthoodtreatment transferred recipients (%) fetuses (%) (%) (%)

TSA-treated 153 6 2 (33.3) 4 (2.6) 7 (4.6) 0 (0)a6.7Untreated 212 7 4 (57.1) 8 (3.8) 6 (2.8) 4 (66.7)b

a,bValues in the same column with different superscripts are significantly different.

TABLE 3. EFFECT OF COTRANSFER OF PA EMBRYOS ON THE PREGNANCY MAINTENANCE OF SCNT EMBRYOS

No. pupsNo. No. surviving

No. pregnant live toembryo No. recipients No. fetuses pups adulthood

Embryos transferred recipients (%) formed (%)a (%) (%)

SCNT 92 3 1 (33.3) 3 (3.3) 2 (2.2) 1 (50)SCNT � PAb 120 � 15 4 3 (75.0) 11 (8.1) 4 (3.3) 3 (75)

aIncluding stillbirth and live birth fetuses.bSCNT embryos � parthenogenetic embryos.Values were not significantly different by ANOVA.

al., 2008) show that the cloned mice derived from the TSA-treated SCNT embryos survived to adulthood, and they arefertile. The effects of epigenetic modifications are still un-clear, including histone acetylation on the postnatal devel-opment. These cloned rabbits might die because of someother reason rather than the TSA treatment, because the earlydeaths of SCNT clones are common in almost all the reportedspecies. Thus, the mechanism and long term effects of theTSA treatment on the postimplantation and postpartum de-velopments require further studies, which may include his-tone acetylation and its relationship with DNA replicationand global gene expression.

We also studied the effect of the cotransfer of additionalparthenogenetic embryos on the pregnancy maintenance ofthe rabbit SCNT embryos. Our results suggested that all theinvestigated parameters, including pregnancy, number of fe-tus formed, and live birth, were higher in the cotransfergroup than in the SCNT embryo transfer control group; how-ever, no statistical differences were demonstrated. Previousreports indicated that the cotransfer of the additionalparthenogenetic embryos could be beneficial to the implan-tation and the pregnancy of the cloned embryos in porcine(De Sousa et al., 2002) and mouse (Meng et al., 2008). Themechanism of the in vivo developmental improvement of theSCNT embryos by cotransfer is unclear. In the normal in vivodevelopment, the establishment and the maintenance of thepregnancy require signaling by the conceptus (embryo/fe-tus and associated extraembryonic membranes) and recip-rocal interactions between the conceptus and the endome-trium (Spencer et al., 2004). Trophoblast giant cells cansecrete protein factors (Lee et al., 1988; Linzer and Fisher1999), acting as paracrine regulators within the endometriumto maintain pregnancy. A recent study showed that thepostimplantation mouse conceptus is, at least in part, able toregulate the expression of specific genes (Angpt1/2, Dtprp,G1p2, and Prlpa) in the endometrium undergoing decidual-ization. Furthermore, certain genes’ expression in the uterusdepends on the presence of a healthy conceptus (Bany andCross, 2006). The SCNT clones commonly have a high occur-rence of placental defects (Yang et al., 2007). We found thatthe parthenogenetic embryos manifest better in vitro devel-opmental capacity in rabbit than the SCNT ones. Therefore,it is possible that the parthenogenetic embryos could implantin the endometrium more efficiently and trigger a good preg-nant status, which might be a better receptive status for theSCNT embryos to implant and to maintain pregnancy. Pre-vious reports had shown that aggregation of blastomeres ofcloned embryos with fertilized ones improved the develop-ment of the resulted embryos and achieved live chimera(Matsuda et al., 2002; Skrzyszowska et al., 2006); also, thecloned embryos showed better term development when theywere combined with one or two parthenogenetic blastomeresrather than with the regular ones (Yang et al., 2007).

In this experiment, we chose parthenogenetic embryos as“helper embryos” instead of the fertilized ones. Unlike thefertilized embryos, rabbit parthenotes could merely developup to 11.5 dpc (Ozil and Huneau, 2001), thus avoiding thecompetition effects on the clones during the later postim-plantation development. Furthermore, we found that the co-transferred mouse parthenogenetic embryos served better forthe improvement of the implantation and the pregnancy ofthe SCNT embryos than the fertilized ones (data not shown).

In porcine, the cotransferred fertilized embryos could outcompete the developmentally inferior SCNT ones, indeedamong the several hundreds of the SCNT embryos cotrans-ferred with the fertilized ones only one was derived from theSCNT embryos (Onishi et al., 2000; Verma et al., 2000).

In summary, our results indicate that the TSA treatmenthas limited effect on the in vitro development of SCNT rab-bit embryos and both the TSA-treated and the untreatedclones could develop to term. However, the effects of theTSA treatment on the overall health condition of the clonesneed further investigation. In rabbits, further studies areneeded to clarify, whether cotransfer of parthenogenetic em-bryos would increase significantly the postimplantation de-velopment of cloned rabbit embryos, similar to pig (De Sousaet al., 2002) and mouse (Meng et al., 2008).

Acknowledgments

This study was supported by Wellcome Trust (Grant No.070246), EU FP6 (Teamoholic, MEXT-CT-2003-509582;CLONET, MRTN-CT-2006-035468; MED-RAT, LSHG-CT-2006-518240), and Chinese–Hungarian Bilateral projects(TET CHN-28/04, CHN-41/05).

Author Disclosure Statement

The authors declare that no conflicting financial interestsexist.

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Address requests for reprints to:Prof. Andras Dinnyes

Szent Istvan UniversityPater K. u. 1.Godollo 2100

Hungary

E-mail: andrasdinnyes@yahoo.com

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