Low cloning efficiency is considered to be caused by the incomplete or aberrant epigenetic reprogramming of differentiated donor cells in somatic cell nuclear transfer (SCNT) embryos. Oxamflatin, a novel class of histone deacetylase inhibitor (HDACi), has been found to improve the in vitro and full-term developmental potential of SCNT embryos. In the present study, we studied the effects of oxamflatin treatment on in vitro porcine SCNT embryos. Our results indicated that the rate of in vitro blastocyst formation of SCNT embryos treated with 1 μM oxamflatin for 15 h postactivation was significantly higher than all other treatments. Treatment of oxamflatin decreased the relative histone deacetylase (HDAC) activity in cloned embryos and resulted in hyperacetylation levels of histone H3 at lysine 9 (AcH3K9) and histone H4 at lysine 5 (AcH4K5) at pronuclear, two-cell, and four-cell stages partly through downregulating HDAC1. The suppression of HDAC6 through oxamflatin increased the nonhistone acetylation level of α-tubulin during the mitotic cell cycle of early SCNT embryos. In addition, we demonstrated that oxamflatin downregulated DNA methyltransferase 1 (DNMT1) expression and global DNA methylation level (5-methylcytosine) in two-cell-stage porcine SCNT embryos. The pluripotency-related gene POU5F1 was found to be upregulated in the oxamflatin-treated group with a decreased DNA methylation tendency in its promoter regions. Treatment of oxamflatin did not change the locus-specific DNA methylation levels of Sus scrofa heterochromatic satellite DNA sequences at the blastocyst stage. Meanwhile, our findings suggest that treatment with HDACi may contribute to maintaining the stable status of cytoskeleton-associated elements, such as acetylated α-tubulin, which may be the crucial determinants of donor nuclear reprogramming in early SCNT embryos. In summary, oxamflatin treatment improves the developmental potential of porcine SCNT embryos in vitro.
Get full access to this article
View all access options for this article.
References
1.
BetthauserJ., ForsbergE., AugensteinM., ChildsL., EilertsenK., EnosJ., ForsytheT., GoluekeP., JurgellaG., KoppangR., LesmeisterT., MallonK., MellG., MisicaP., PaceM., Pfister-GenskowM., StrelchenkoN., VoelkerG., WattS., ThompsonS., and BishopM. (2000). Production of cloned piglets from in vitro systems. Nat. Biotechnol., 18, 1055–1059.
2.
BlackwellL., NorrisJ., SutoC.M., and JanzenW.P. (2008). The use of diversity profiling to characterize chemical modulators of the histone deacetylases. Life Sci., 82, 1050–1058.
3.
BuiH.T., WakayamaS., KishigamiS., ParkK.K., KimJ.H., ThuanN.V., and WakayamaT. (2010). Effect of Trichostatin A on chromatin remodeling, histone modifications, DNA replication, and transcriptional activity in cloned mouse embryos. Biol. Reprod., 83, 454–463.
4.
CerveraR.P., Marti-GutierrezN., EscorihuelaE., MorenoR., and StojkovicM. (2009). Trichostatin A affects histone acetylation and gene expression in porcine somatic cell nucleus transfer embryos. Theriogenology, 72, 1097–1110.
CharlesworthB., SniegowskiP., and StephanW. (1994). The evolutionary dynamics of repetitive DNA in eukaryotes. Nature, 371, 215–220.
7.
ChoiJ.H., MinN.Y., ParkJ., KimJ.H., ParkS.H., KoY.J., KangY., MoonY.J., RheeS., HamS.W., ParkA.J., and LeeK.H. (2010). TSA-induced DNMT1 down-regulation represses hTERT expression via recruiting CTCF into demethylated core promoter region of hTERT in HCT116. Biochem. Biophys. Res. Commun., 391, 449–454.
8.
Costa-BorgesN., SantaloJ., and IbanezE. (2010). Comparison between the effects of valproic acid and trichostatin A on the in vitro development, blastocyst quality, and full-term development of mouse somatic cell nuclear transfer embryos. Cell. Reprogram., 12, 437–446.
9.
DaiX., HaoJ., HouX., HaiT., YuY., JouneauA., WangL., and ZhouQ. (2010). Somatic nucleus reprogramming is significantly improved by m-carboxycinnamic acid bishydroxamide, a histone deacetylase inhibitor. J. Biol. Chem., 285, 31002–31010.
10.
DasZ.C., GuptaM.K., UhmS.J., and LeeH.T. (2010). Increasing histone acetylation of cloned embryos, but not donor cells, by sodium butyrate improves their in vitro development in pigs. Cell. Reprogram., 12, 95–104.
11.
DingX., WangY., ZhangD., WangY., GuoZ., and ZhangY. (2008). Increased preimplantation development of cloned bovine embryos treated with 5-aza-2′-deoxycytidine and trichostatin A. Theriogenology, 70, 622–630.
12.
EnrightB.P., KubotaC., YangX., and TianX.C. (2003). Epigenetic characteristics and development of embryos cloned from donor cells treated by trichostatin A or 5-aza-2′-deoxycytidine. Biol. Reprod., 69, 896–901.
13.
GidekelS., and BergmanY. (2002). A unique developmental pattern of Oct-3/4 DNA methylation is controlled by a cis-demodification element. J. Biol. Chem., 277, 34521–34530.
14.
HaoY., LaiL., MaoJ., ImG.S., BonkA., and PratherR.S. (2004). Apoptosis in parthenogenetic preimplantation porcine embryos. Biol. Reprod., 70, 1644–1649.
15.
HattoriN., NishinoK., KoY., HattoriN., OhganeJ., TanakaS., and ShiotaK. (2004). Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells. J. Biol. Chem., 279, 17063–17069.
16.
HochedlingerK., and JaenischR. (2002). Monoclonal mice generated by nuclear transfer from mature B and T donor cells. Nature, 415, 1035–1038.
17.
HuangY., TangX., XieW., ZhouY., LiD., YaoC., YaoC., ZhouY., ZhuJ., LaiL., OuyangH., and PangD. (2011). Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos. Cell. Reprogram., 14, 513–520.
18.
HubbertC., GuardiolaA., and ShaoR. (2002). HDAC6 is a microtubule-associated deacetylase. Nature, 417, 455–458.
19.
IagerA.E., RaginaN.P., RossP.J., BeyhanZ., CunniffK., RodriguezR.M., and CibelliJ.B. (2008). Trichostatin A improves histone acetylation in bovine somatic cell nuclear transfer early embryos. Cloning Stem Cells, 10, 371–380.
20.
InoueA., and ZhangY. (2011). Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos. Science, 334, 194.
21.
IsomS.C., LiR.F., WhitworthK.M., and PratherR.S. (2012). Timing of first embryonic cleavage is a positive indicator of the in vitro developmental potential of porcineembryos derived from in vitro fertilization, somatic cell nuclear transfer and parthenogenesis. Mol. Reprod. Dev., 79, 197–207.
22.
ItoS., D'AlessioA.C., TaranovaO.V., HongK., SowersL.C., and ZhangY. (2010). Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature, 466, 1129–1133.
23.
JanuchowskiR., DabrowskiM., OforiH., and JagodzinskiP.P. (2007). Trichostatin A down-regulates DNA methyltransferase 1 in Jurkat T cells. Cancer Lett., 246, 313–317.
24.
JinD., TanH.J., LeiT., GanL., ChenX.D., LongQ.Q., FengB., and YangZ.Q. (2009). Molecular cloning and characterization of porcine sirtuin genes. Comp. Biochem. Physiol. B Biochem. Mol. Biol., 153, 348–358.
25.
KangY.K., KooD.B., ParkJ.S., ChoiY.H., KimH.N., ChangW.K., LeeK.K., and HanY.M. (2001a). Typical demethylation events in cloned pig embryos. J. Biol. Chem., 276, 39980–39984.
26.
KangY.K., KooD.B., ParkJ.S., ChoiY.H., KimH.N., ChangW.K., LeeK.K., and HanY.M. (2001b). Aberrant methylation of donor genome in cloned bovine embryos. Nat. Genet., 28, 173–177.
27.
KimE.Y., ParkM.J., ParkH.Y., NohE.J., NohE.H., ParkK.S., LeeJ.B., JeongC.J., RiuK.Z., and ParkS.P. (2012). Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system. Cell. Reprogram., 14, 305–311.
28.
KimY.B., LeeK.H., SugitaK., YoshidaM., and HorinouchiS. (1999). Oxamflatin is a novel antitumor compound that inhibits mammalian histone deacetylase. Oncogene, 18, 2461–2470.
29.
KumarB.M., MaengG.H., LeeY.M, LeeJ.H., JeonB.G., OckS.A., KangT., and RhoG.J. (2012). Epigenetic modification of fetal fibroblasts improves developmental competency and gene expression in porcine cloned embryos. Vet. Res. Commun., 37, 19–28.
30.
LaiL.X., and PratherR.S. (2003). Production of cloned pigs by using somatic cells as donors. Cloning Stem Cells, 5, 233–241.
31.
LiS., DuW., and LiN. (2004). Epigenetic reprogramming in mammalian nuclear transfer. Chin. Sci. Bull., 49, 766–771.
32.
LiY., LiuJ., DaiJ., XingF., FangZ., ZhangT., ShiZ., ZhangD., and ChenX. (2010). Production of cloned miniature pigs by enucleation using the spindle view system. Reprod. Domest. Anim., 45, 608–613.
33.
LivakK.J., and SchmittgenT.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(T) (−Delta Delta C) method. Methods, 25, 402–408.
34.
MaP., and SchultzR.M. (2008). Histone deacetylase 1 (HDAC1) regulates histone acetylation, development, and gene expression in preimplantation mouse embryos. Dev. Biol., 319, 110–120.
35.
Martinez-DiazM.A., CheL., AlbornozM., SenedaM.M., CollisD., CoutinhoA.R., El-BeirouthiN., LaurinD., ZhaoX., and BordignonV. (2010). Pre- and postimplantation development of swine-cloned embryos derived from fibroblasts and bone marrow cells after inhibition of histone deacetylases. Cell. Reprogram., 12, 85–94.
36.
MatsubaraK., LeeA.R., KishigamiS., ItoA., MatsumotoK., ChiH., NishinoN., YoshidaM., and HosoiY. (2013). Dynamics and regulation of lysine-acetylation during one-cell stage mouse embryos. Biochem. Biophys. Res. Commun., 434, 1–7.
37.
MiyamotoK., PasqueV., JullienJ., and GurdonJ.B. (2011). Nuclear actin polymerization is required for transcriptional reprogramming of Oct4 by oocytes. Genes Dev., 25, 946–958.
38.
MiyamotoK., and GurdonJ.B. (2013a). Transcriptional regulation and nuclear reprogramming: Roles of nuclear actin and actin-binding proteins. Cell. Mol. Life Sci., 70, 3289–3302.
39.
MiyamotoK., TeperekM., YusaK., AllenG.E., BradshawC.R., and GurdonJ.B. (2013b). Nuclear Wave1 is required for reprogramming transcription in oocytes and for normal development. Science, 341, 1002–1005.
40.
NicholsJ., ZevnikB., AnastassiadisK., NiwaH., Klewe-NebeniusD., ChambersI., ScholerH., and SmithA. (1998). Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell, 95, 379–391.
41.
OkanoM., BellD.W., HaberD.A., and LiE. (1999). DNA methyltransferases DNMT3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell, 99, 247–257.
42.
OnishiA., IwamotoM., AkitaT., MikawaS., TakedaK., AwataT., HanadaH., and PerryA.C. (2000). Pig cloning by microinjection of fetal fibroblast nuclei. Science, 289, 1188–1190.
43.
OnoT., LiC., MizutaniE., TerashitaY., YamagataK., and WakayamaT. (2010). Inhibition of class IIb histone deacetylase significantly improves cloning efficiency in mice. Biol. Reprod., 83, 929–937.
44.
PalomequeT., and LoriteP. (2008). Satellite DNA in insects: A review. Heredity, 100, 564–573.
45.
ParkS.J., ParkH.J., KooO.J., ChoiW.J., MoonJ., KwonD.K., KangJ.T., KimS., ChoiJ.Y., JangG., and LeeB.C. (2012). Oxamflatin improves developmental competence of porcine somatic cell nuclear transfer embryos. Cell. Reprogram., 14, 398–406.
46.
PasqueV., JullienJ., MiyamotoK., Halley-StottR.P., and GurdonJ.B. (2011). Epigenetic factors influencing resistance to nuclear reprogramming. Trends Genet., 27, 516–525.
47.
PipernoG., LeDizetM., and ChangX.J. (1987). Microtubules containing acetylated alpha-tubulin in mammalian cells in culture. J. Cell. Biol., 104, 289–302.
48.
PolejaevaI.A., ChenS.H., VaughtT.D., PageR.L., MullinsJ., BallS., DaiY., BooneJ., WalkerS., AyaresD.L., ColmanA., and CampbellK.H.S. (2000). Cloned pigs produced by nuclear transfer from adult somatic cells. Nature, 7, 86–90.
49.
PratherR.S., HawleyR.J., CarterD.B., LaiL., and GreensteinJ.L. (2003). Transgenic swine for biomedicine and agriculture. Theriogenology, 59, 115–123.
50.
RideoutW.M.3rd, EgganK., and JaenischR. (2001). Nuclear cloning and epigenetic reprogramming of the genome. Science, 293, 1093–1098.
51.
RoddaD.J., ChewJ.L., LimL.H., LohY.H., WangB., NgH.H., and RobsonP. (2005). Transcriptional regulation of Nanog by Oct4 and Sox2. J. Biol. Chem., 280, 24731–24737.
52.
RybouchkinA., KatoY., and TsunodaY. (2006). Role of histone acetylation in reprogramming of somatic nuclei following nuclear transfer. Biol. Reprod., 74, 1083–1089.
53.
SchattenG., SimerlyC., AsaiD.J., SzukeE., CookeP., and SchattenH. (1988). Acetylated alpha-tubulin in microtubules during mouse fertilization and early development. Dev. Biol., 130, 74–86.
54.
SharifJ., MutoM., TakebayashiS., SuetakeI., IwamatsuA., EndoA., ShingaJ., Mizutani-KosekiY., ToyodaT., OkamuraK., TajimaS., MitsuyaK., OkanoM., and KosekiH. (2007). The SRA protein Np95 mediates epigenetic inheritance by recruiting DNMT1 to methylated DNA. Nature, 450, 908–912.
55.
ShiW., HoeflichA., FlaswinkelH., StojkovicM., WolfE., and ZakhartchenkoV. (2003). Induction of a senescent-like phenotype does not confer the ability of bovine immortal cells to support the development of nuclear transfer embryos. Biol. Reprod., 69, 301–309.
56.
SimonssonS., and GurdonJ. (2004). DNA demethylation is necessary for the epigenetic reprogramming of somatic cell nuclei. Nat. Cell Biol., 6, 984–990.
57.
SuJ., WangY., LiY., LiR., LiQ., WuY., QuanF., LiuJ., GuoZ., and ZhangY. (2011). Oxamflatin significantly improves nuclear reprogramming, blastocyst quality, and in vitro development of bovine SCNT embryos. PLoS One, 6, e23805.
58.
TahilianiM., KohK.P., ShenY., PastorW.A., BandukwalaH., BrudnoY., AgarwalS., IyerL.M., LiuD.R., AravindL., and RaoA. (2009). Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science, 324, 930–935.
59.
TamashiroK.L.K., WakayamaT., AkutsuH., YamazakiY., LacheyJ.L., WortmanM.D., SeeleyR.J., D'AlessioD.A., WoodsS.C., YanagimachiR., and SakaiR.R. (2002). Cloned mice have an obese phenotype not transmitted to their offspring. Nat. Med., 8, 262–267.
60.
WangL.J., ZhangH., WangY.S., XuW.B., XiongX.R., LiY.Y., SuJ.M., HuaS., and ZhangY. (2011). Scriptaid improves in vitro development and nuclear reprogramming of somatic cell nuclear transfer bovine embryos. Cell. Reprogram., 13, 431–439.
61.
WeeG., ShimJ.J., KooD.B., ChaeJ.I., LeeK.K., and HanY.M. (2007). Epigenetic alteration of the donor cells does not recapitulate the reprogramming of DNA methylation in cloned embryos. Reproduction, 134, 781–787.
62.
WhitworthK.M., ZhaoJ., SpateL.D., LiR., and PratherR.S. (2011). Scriptaid corrects gene expression of a few aberrantly reprogrammed transcripts in nuclear transfer pig blastocyst stage embryos. Cell. Reprogram., 13, 191–204.
63.
WilmutI., SchniekeA.E., McWhirJ., KindA.J., and CampbellK.H.S. (1997). Viable offspring derived from fetal and adult mammalian cells. Nature, 385, 810–813.
64.
XiongY., DowdyS.C., PodratzK.C., JinF., AttewellJ.R., EberhardtN.L., and JiangS.W. (2005). Histone deacetylase inhibitors decrease DNA methyltransferase-3B messenger RNA stability and down-regulate de novo DNA methyltransferase activity in human endometrial cells. Cancer Res., 65, 2684–2689.
65.
YamanakaK., SugimuraS., WakaiT., KawaharaM., and SatoE. (2009). Acetylation level of histone H3 in early embryonic stages affects subsequent development of miniature pig somatic cell nuclear transfer embryos. J. Reprod. Develop., 55, 638–644.
66.
YangF., HaoR., KesslerB., BremG., WolfE., and ZakhartchenkoV. (2007). Rabbit somatic cell cloning: Effects of donor cell type, histone acetylation status and chimeric embryo complementation. Reproduction, 133, 219–230.
67.
YangX., SmithS.L., TianX.C., LewinH.A., RenardJ.P., and WakayamaT. (2007). Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning. Nat. Genet., 39, 295–302.
68.
ZhangY., KwonS.H., YamaguchiT., CubizollesF., RousseauxS., Kneisse,lM., CaoC., LiN., ChengH.L., ChuaK., LombardD., MizerackiA., MatthiasG., FrederickW.A., KhochbinS., and MatthiasP. (2008). Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally. Mol. Cell. Biol., 28, 1688–1701.
69.
ZhaoJ., HaoY., RossJ.W., SpateL.D., WaltersE.M., SamuelM.S., RiekeA., MurphyC.N., and PratherR.S. (2010). Histone deacetylase inhibitors improve in vitro and in vivo developmental competence of somatic cell nuclear transfer porcine embryos. Cell. Reprogram., 12, 75–83.
70.
ZhaoJ., RossJ.W., HaoY., SpateL.D., WaltersE.M., SamuelM.S., RiekeA., MurphyC.N., and PratherR.S. (2009). Significant improvement in cloning efficiency of an inbred miniature pig by scriptaid treatment after somatic cell nuclear transfer. Biol. Reprod., 81, 525–530.
71.
ZhaoM., RiveraR.M., and PratherR.S. (2013). Locus-specific DNA methylation reprogramming during early porcine embryogenesis. Biol. Reprod., 88, 1–10.
72.
ZuccottiM., GaragnaS., and RediC.A. (2000). Nuclear transfer, genome reprogramming and novel opportunities in cell therapy. J. Endocrinol. Invest., 23, 623–629.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
