Skip to main content Accessibility help

The development and expression of pluripotency genes in embryos derived from nuclear transfer and in vitro fertilization

  • Li-Bing Ma (a1) (a2), Xiao-Ying He (a3) (a2), Feng-Mei Wang (a4), Jun-Wei Cao (a5) and Teng Cheng (a3)...


Somatic cell nuclear transfer can be used to produce embryonic stem (ES) cells, cloned animals, and can even increase the population size of endangered animals. However, the application of this technique is limited by the low developmental rate of cloned embryos, a situation that may result from abnormal expression of some zygotic genes. In this study, sheep–sheep intra-species cloned embryos, goat–sheep inter-species cloned embryos, or sheep in vitro fertilized embryos were constructed and cultured in vitro and the developmental ability and expression of three pluripotency genes, SSEA-1, Nanog and Oct4, were examined. The results showed firstly that the developmental ability of in vitro fertilized embryos was significantly higher than that of cloned embryos. In addition, the percentage of intra-species cloned embryos that developed to morula or blastocyst stages was also significantly higher than that of the inter-species cloned embryos. Secondly, all three types of embryos expressed SSEA-1 at the 8-cell and morula stages. At the 8-cell stage, a higher percentage of in vitro fertilized embryos expressed SSEA-1 than occurred for cloned embryos. However, at the morula stage, all detected embryos could express SSEA-1. Thirdly, the three types of embryos expressed Oct4 mRNA at the morula and blastocyst stages, and embryos at the blastocyst stage expressed Nanog mRNA. The rate of expression of Oct4 and Nanog mRNA at these developmental stages was higher in in vitro fertilized embryos than in cloned embryos. These results indicated that, during early development, the failure to reactivate some pluripotency genes maybe is a reason for the low cloning efficiency found with cloned embryos.


Corresponding author

All correspondence to: Li-Bing Ma. School of Mathematics, Physics and Biological Engineering, Inner Mongolia University of Science & Technology, Aerding Avenue No. 7, Baotou, Inner Mongolia Autonomous Region 014010, China. Tel: +86 15848638996. Fax: +86 04725954358. e-mail address:


Hide All
Armstrong, L., Lako, M., Dean, W. & Stojkovic, M. (2006). Epigenetic modification is central to genome reprogramming in somatic cell nuclear transfer. Stem Cells 24, 805–14.
Aston, K.I., Li, G.P., Hicks, B.A., Sessions, B.R., Davis, A.P., Rickords, L.F., Stevens, J.R. & White, K.L. (2010). Abnormal levels of transcript abundance of developmentally important genes in various stages of preimplantation bovine somatic cell nuclear transfer embryos. Cell Reprogram 12, 2332.
Beaujean, N., Taylor, J., Gardner, J., Wilmut, I., Meehan, R. & Young, L. (2004). Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer. Biol. Reprod. 71, 185–93.
Beyhan, Z., Forsberg, E.J., Eilertsen, K.J., Kent-First, M. & First, N.L. (2007). Gene expression in bovine nuclear transfer embryos in relation to donor cell efficiency in producing live offspring. Mol. Reprod. Dev. 74, 1827.
Boiani, M., Eckardt, S., Schöler, H.R. & McLaughlin, K.J. (2002). Oct4 distribution and level in mouse clones: consequences for pluripotency. Genes Dev. 16, 1209–19.
Borowczyk, E., Caton, J.S., Redmer, D.A., Bilski, J.J., Weigl, R.M., Vonnahme, K.A., Borowicz, P.P., Kirsch, J.D., Kraft, K.C., Reynolds, L.P. & Grazul-Bilska, A.T. (2006). Effects of plane of nutrition on in vitro fertilization and early embryonic development in sheep. J. Anim. Sci. 84, 1593–9.
Brambrink, T., Foreman, R., Welstead, G.G., Lengner, C.J., Wernig, M., Suh, H. & Jaenisch, R. (2008). Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2, 151–9.
Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S. & Smith, A. (2003). Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–55.
Chen, Y., He, Z.X., Liu, A., Wang, K., Mao, W.W., Chu, J.X., Lu, Y., Fang, Z.F., Shi, Y.T., Yang, Q.Z., Chen, da Y., Wang, M.K., Li, J.S., Huang, S.L., Kong, X.Y., Shi, Y.Z., Wang, Z.Q., Xia, J.H., Long, Z.G., Xue, Z.G., Ding, W.X. & Sheng, H.Z. (2003). Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes. Cell Res. 13, 251–63.
Chung, Y., Bishop, C.E., Treff, N.R., Walker, S.J., Sandler, V.M., Becker, S., Klimanskaya, I., Wun, W.S., Dunn, R., Hall, R.M., Su, J., Lu, S.J., Maserati, M., Choi, Y.H., Scott, R., Atala, A., Dittman, R. & Lanza, R. (2009). Reprogramming of human somatic cells using human and animal oocytes. Cloning Stem Cells 11, 213–23.
Dattena, M., Chessa, B., Lacerenza, D., Accardo, C., Pilichi, S., Mara, L., Chessa, F., Vincenti, L. & Cappai, P. (2006). Isolation, culture, and characterization of embryonic cell lines from vitrified sheep blastocysts. Mol. Reprod. Dev. 73, 31–9.
Ferrer, F., Garcia, C., Villar, J. & Arias, M. (1995). Ultrastructural study of the early development of the sheep embryo. Anat. Histol. Embryol. 24, 191–6.
Fulka, J. Jr & Fulka, H. (2007). Somatic cell nuclear transfer (SCNT) in mammals: the cytoplast and its reprogramming activities. Adv. Exp. Med. Biol. 591, 93102.
He, S., Pant, D., Schiffmacher, A., Bischoff, S., Melican, D., Gavin, W. & Keefer, C. (2006). Developmental expression of pluripotency determining factors in caprine embryos: novel pattern of NANOG protein localization in the nucleolus. Mol. Reprod. Dev. 73, 1512–22.
Hong, S.G., Oh, H.J., Park, J.E., Kim, M.J., Kim, G.A., Koo, O.J., Jang, G. & Lee, B.C. (2012). Production of transgenic canine embryos using interspecies somatic cell nuclear transfer. Zygote 20, 6772.
Hosseini, S.M., Hajian, M., Forouzanfar, M., Moulavi, F., Abedi, P., Asgari, V., Tanhaei, S., Abbasi, H., Jafarpour, F., Ostadhosseini, S., Karamali, F., Karbaliaie, K., Baharvand, H. & Nasr-Esfahani, M.H. (2012). Enucleated ovine oocyte supports human somatic cells reprogramming back to the embryonic stage. Cell Reprogram 14, 155–63.
Hua, S., Zhang, Y., Song, K., Song, J., Zhang, Z., Zhang, L., Zhang, C., Cao, J. & Ma, L. (2008). Development of bovine–ovine interspecies cloned embryos and mitochondria segregation in blastomeres during preimplantation. Anim. Reprod. Sci. 105, 245–57.
Kühholzer, B., Baguisi, A. & Overström, E.W. (2000). Long-term culture and characterization of goat primordial germ cells. Theriogenology 53, 1071–9.
Kurosaka, S., Eckardt, S. & McLaughlin, K.J. (2004). Pluripotent lineage definition in bovine embryos by Oct4 transcript localization. Biol. Reprod. 71, 1578–82.
Lanza, R.P., Cibelli, J.B., Diaz, F., Moraes, C.T., Farin, P.W., Farin, C.E., Hammer, C.J., West, M.D. & Damiani, P. (2000). Cloning of endangered species (Bos gaurus) using interspecies nuclear transfer. Cloning 2, 7990.
Lee, E., Kim, J.H., Park, S.M., Jeong, Y.I., Lee, J.Y., Park, S.W., Choi, J., Kim, H.S., Jeong, Y.W., Kim, S., Hyun, S.H. & Hwang, W.S. (2008). The analysis of chromatin remodeling and the staining for DNA methylation and histone acetylation do not provide definitive indicators of the developmental ability of inter-species cloned embryos. Anim. Reprod. Sci. 105, 438–50.
Li, X., Kato, Y. & Tsunoda, Y. (2005). Comparative analysis of development-related gene expression in mouse preimplantation embryos with different developmental potential. Mol. Reprod. Dev. 72, 152–60.
Li, Y., Dai, Y., Du, W., Zhao, C., Wang, H., Wang, L., Li, R., Liu, Y., Wan, R. & Li, N. (2006). Cloned endangered species takin (Budorcas taxicolor) by inter-species nuclear transfer and comparison of the blastocyst development with yak (Bos grunniens) and bovine. Mol. Reprod. Dev. 73, 189–95.
Loh, Y.H., Wu, Q., Chew, J.L., Vega, V.B., Zhang, W., Chen, X., Bourque, G., George, J., Leong, B., Liu, J., Wong, K.Y., Sung, K.W., Lee, C.W., Zhao, X.D., Chiu, K.P., Lipovich, L., Kuznetsov, V.A., Robson, P., Stanton, L.W., Wei, C.L., Ruan, Y., Lim, B. & Ng, H.H. (2006). The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat. Genet. 38, 431–40.
Loi, P., Ptak, G., Barboni, B., Fulka, J. Jr, Cappai, P. & Clinton, M. (2001). Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells. Nat. Biotechnol. 19, 962–4.
Lorthongpanich, C., Laowtammathron, C., Chan, A.W., Ketudat-Cairns, M. & Parnpai, R. (2008). Development of interspecies cloned monkey embryos reconstructed with bovine enucleated oocytes. J. Reprod. Dev. 54, 306–13.
Ma, L.B., Yang, L., Hua, S., Cao, J.W., Li, J.X. & Zhang, Y. (2008a). Development in vitro and mitochondrial fate of interspecies cloned embryos. Reprod. Domest. Anim. 43, 279–85.
Ma, L.B., Yang, L., Zhang, Y., Cao, J.W., Hua, S. & Li, J.X. (2008b). Quantitative analysis of mitochondrial RNA in goat–sheep cloned embryos. Mol. Reprod. Dev. 75, 33–9.
Murakami, M., Otoi, T., Wongsrikeao, P., Agung, B., Sambuu, R. & Suzuki, T. (2005). Development of interspecies cloned embryos in yak and dog. Cloning Stem Cells 7, 7781.
Niemann, H., Tian, X.C., King, W.A. & Lee, R.S. (2008). Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning. Reproduction 135, 151–63.
Park, P.J., Colletti, E., Ozturk, F., Wood, J.A., Tellez, J., Almeida-Porada, G. & Porada, C. (2009). Factors determining the risk of inadvertent retroviral transduction of male germ cells after in utero gene transfer in sheep. Hum. Gene Ther. 20, 201–15.
Pivko, J., Grafenau, P. & Kopecný, V. (1995). Nuclear fine structure and transcription in early goat embryos. Theriogenology 44, 661–71.
Solter, D. & Knowles, B.B. (1978). Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc. Natl. Acad. Sci. USA 75, 5565–9.
Srirattana, K., Imsoonthornruksa, S., Laowtammathron, C., Sangmalee, A., Tunwattana, W., Thongprapai, T., Chaimongkol, C., Ketudat-Cairns, M. & Parnpai, R. (2012). Full-term development of gaur–bovine interspecies somatic cell nuclear transfer embryos: effect of trichostatin A treatment. Cell Reprogram 14, 248–57.
Stadtfeld, M., Maherali, N., Breault, D.T. & Hochedlinger, K. (2008). Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2, 230–40.
Sugawara, A., Sugimura, S., Hoshino, Y. & Sato, E. (2009). Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes. Zygote 17, 195202.
Takahashi, K. & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–76.
Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. & Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–72.
Vassilieva, S., Guan, K., Pich, U. & Wobus, A.M. (2000). Establishment of SSEA-1- and Oct-4-expressing rat embryonic stem-like cell lines and effects of cytokines of the IL-6 family on clonal growth. Exp. Cell Res. 258, 361–73.
Wakayama, T., Tabar, V., Rodriguez, I., Perry, A.C., Studer, L. & Mombaerts, P. (2001). Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science 292, 740–3.
Wang, K., Out, H.H., Chen, Y., Lee, Y., Latham, K. & Cibelli, J.B. (2011). Reprogrammed transcriptome in rhesus–bovine interspecies somatic cell nuclear transfer embryos. PLoS One 6, e22197.
Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. & Campbell, K.H. (1997). Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–3.
Wilmut, I., Beaujean, N., de Sousa, P.A., Dinnyes, A., King, T.J., Paterson, L.A., Wells, D.N. & Young, L.E. (2002). Somatic cell nuclear transfer. Nature 419, 583–6.
Xing, X., Magnani, L., Lee, K., Wang, C., Cabot, R.A. & Machaty, Z. (2009). Gene expression and development of early pig embryos produced by serial nuclear transfer. Mol. Reprod. Dev. 76, 555–63.
Yan, L., Lei, L., Yang, C., Gao, Z., Lei, A., Ma, X. & Dou, Z. (2008). Improved isolation and culture of embryonic germ cells from Guanzhong dairy goat. Sheng Wu Gong Cheng Xue Bao 24, 1670–6.
Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I. & Thomson, J.A. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–20.
Zhao, Z.J., Ouyang, Y.C., Nan, C.L., Lei, Z.L., Song, X.F., Sun, Q.Y. & Chen, D.Y. (2006). Rabbit oocyte cytoplasm supports development of nuclear transfer embryos derived from the somatic cells of the camel and tibetan antelope. J. Reprod. Fertil. 52, 449–59.


The development and expression of pluripotency genes in embryos derived from nuclear transfer and in vitro fertilization

  • Li-Bing Ma (a1) (a2), Xiao-Ying He (a3) (a2), Feng-Mei Wang (a4), Jun-Wei Cao (a5) and Teng Cheng (a3)...


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed