Reproductive Technology

Effect of Limited DNA Methylation Reprogramming in the Normal Sheep Embryo on Somatic Cell Nuclear Transfer

Abstract
Active demethylation of cytosine residues in the sperm genome prior to forming a functional zygotic nucleus is thought to be an important function of the oocyte cytoplasm for subsequent embryonic development in the mouse. Conversely, this event does not occur in the sheep or rabbit zygote and occurs only partially in the cow. The aim of this study was to investigate the effect of limited methylation reprogramming in the normal sheep embryo on reprogramming somatic nuclei. Sheep fibroblast somatic nuclei were partially demethylated after electrofusion with recipient sheep oocytes and undergo a stepwise passive loss of DNA methylation during early development, as determined by 5-methylcytosine immunostaining on interphase embryonic nuclei. A similar decrease takes place with in vivo derived sheep embryos up to the 8-cell stage, although nuclear transfer embryos exhibit a consistently higher level of methylation at each stage. Between the 8 cell and blastocyst stages DNA methylation levels in nuclear transfer embryos are comparable to those derived in vivo, but the distribution of methylated DNA is abnormal in a high proportion. By correlating DNA methylation with developmental potential at individual stages, our results suggest that somatic nuclei that do not undergo rapid reorganisation of their DNA before the first mitosis fail to develop within two to three cell cycles and that the observed methylation defects in early cleavage stages more likely occur as a direct consequence of failed nuclear reorganisation than in failed demethylation capacity. However since only embryos with reorganised chromatin appear to survive the 16 cell and morula stages, failure to demethylate the trophectoderm cells of the blastocyst is likely to directly impact on developmental potential by altering programmed patterns of gene expression in extra-embryonic tissues. Thus both remodelling of DNA and epigenetic reprogramming appear critical for development of both fertilised and nuclear transfer embryos.

Key words: Embryo • Developmental biology • Early development • Gene regulation

This article has been cited by other articles:

				C. Palmieri, P. Loi, G. Ptak, and L.D. Salda REVIEW PAPER: A Review of the Pathology of Abnormal Placentae of Somatic Cell Nuclear Transfer Clone Pregnancies in Cattle, Sheep, and Mice Vet. Pathol., November 1, 2008; 45(6): 865 - 880. [Abstract] [Full Text] [PDF]

				D J Kwon, C K Park, B K Yang, and H T Cheong Control of nuclear remodelling and subsequent in vitro development and methylation status of porcine nuclear transfer embryos Reproduction, May 1, 2008; 135(5): 649 - 656. [Abstract] [Full Text] [PDF]

				A. M. Giraldo, D. A. Hylan, C. B. Ballard, M. N. Purpera, T. D. Vaught, J. W. Lynn, R. A. Godke, and K. R. Bondioli Effect of Epigenetic Modifications of Donor Somatic Cells on the Subsequent Chromatin Remodeling of Cloned Bovine Embryos Biol Reprod, May 1, 2008; 78(5): 832 - 840. [Abstract] [Full Text] [PDF]

				L. H. Shi, J. S. Ai, Y. C. OuYang, J. C. Huang, Z. L. Lei, Q. Wang, S. Yin, Z. M. Han, Q. Y. Sun, and D. Y. Chen Trichostatin A and nuclear reprogramming of cloned rabbit embryos J Anim Sci, May 1, 2008; 86(5): 1106 - 1113. [Abstract] [Full Text] [PDF]

				H. Fulka, I. Barnetova, T. Mosko, and J. Fulka Epigenetic analysis of human spermatozoa after their injection into ovulated mouse oocytes Hum. Reprod., March 1, 2008; 23(3): 627 - 634. [Abstract] [Full Text] [PDF]

				K. Yamagata Capturing Epigenetic Dynamics During Pre-implantation Development Using Live Cell Imaging J. Biochem., March 1, 2008; 143(3): 279 - 286. [Abstract] [Full Text] [PDF]

				H. Niemann, X C. Tian, W A. King, and R. S F Lee Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning Reproduction, February 1, 2008; 135(2): 151 - 163. [Abstract] [Full Text] [PDF]

				F. Yang, R. Hao, B. Kessler, G. Brem, E. Wolf, and V. Zakhartchenko Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation Reproduction, January 1, 2007; 133(1): 219 - 230. [Abstract] [Full Text] [PDF]

				J. Yang, S. Yang, N. Beaujean, Y. Niu, X. He, Y. Xie, X. Tang, L. Wang, Q. Zhou, and W. Ji Epigenetic Marks in Cloned Rhesus Monkey Embryos: Comparison with Counterparts Produced In Vitro Biol Reprod, January 1, 2007; 76(1): 36 - 42. [Abstract] [Full Text] [PDF]

				T. Suteevun, R. Parnpai, S. L. Smith, C-C. Chang, S. Muenthaisong, and X. C. Tian Epigenetic characteristics of cloned and in vitro-fertilized swamp buffalo (Bubalus bubalis) embryos J Anim Sci, August 1, 2006; 84(8): 2065 - 2071. [Abstract] [Full Text] [PDF]

				N Cobbe Why the apparent haste to clone humans? J. Med. Ethics, May 1, 2006; 32(5): 298 - 302. [Full Text] [PDF]

				R. C. Ribas, J. E. Taylor, C. McCorquodale, A. C. Mauricio, M. Sousa, and I. Wilmut Effect of Zona Pellucida Removal on DNA Methylation in Early Mouse Embryos Biol Reprod, February 1, 2006; 74(2): 307 - 313. [Abstract] [Full Text] [PDF]

				L. Gioia, B. Barboni, M. Turriani, G. Capacchietti, M. G. Pistilli, P. Berardinelli, and M. Mattioli The capability of reprogramming the male chromatin after fertilization is dependent on the quality of oocyte maturation Reproduction, July 1, 2005; 130(1): 29 - 39. [Abstract] [Full Text] [PDF]

				H. D. Morgan, F. Santos, K. Green, W. Dean, and W. Reik Epigenetic reprogramming in mammals Hum. Mol. Genet., April 15, 2005; 14(suppl_1): R47 - R58. [Abstract] [Full Text] [PDF]

				H. Fulka, M. Mrazek, O. Tepla, and J. Fulka Jr DNA methylation pattern in human zygotes and developing embryos Reproduction, December 1, 2004; 128(6): 703 - 708. [Abstract] [Full Text] [PDF]