Submitted October 22, 2003
Returned for revision November 18, 2003
Accepted March 12, 2004
Embryo
Methylation Reprogramming and Chromosomal Aneuploidy in In Vivo Fertilized and Cloned Rabbit Preimplantation Embryos
Wei Shi ,
Fatma Dirim ,
Eckhard Wolf ,
Valeri Zakhartchenko ,
and
Thomas Haaf *
* To whom correspondence should be addressed. E-mail: haaf{at}humgen.klinik.uni-mainz.de.
Abstract
Active demethylation of the paternal genome but not of the maternal genome occurs in fertilized mouse, rat, pig and bovine zygotes. To study whether this early demethylation wave is important for embryonic development, we have analyzed the global methylation patterns of both in vivo fertilized and cloned rabbit embryos. Anti-5-methylcytosine immunofluorescence of in vivo fertilized rabbit embryos revealed that the equally high methylation levels of the paternal and maternal genomes are largely maintained from the zygote up to the 16-cell stage. The lack of detectable methylation changes in rabbit preimplantation embryos suggests that genome-wide demethylation is not an obligatory requirement for epigenetic reprogramming. The methylation patterns of embryos derived from fibroblast and cumulus cell nuclear transfer were similar to those of in vivo fertilized rabbit embryos. Fluorescence in situ hybridization with chromosome-specific BACs demonstrated significantly increased chromosomal aneuploidy rates in cumulus cell nuclear transfer rabbit embryos and embryos derived from nuclear transfer of rabbit fibroblasts into bovine oocytes, compared to in vivo fertilized rabbit embryos. The incidence of chromosomal abnormalities was correlated with subsequent developmental failure. We propose that postzygotic mitotic errors are one important explanation, why mammalian cloning often fails.
Key words:
Embryo •
Assisted Reproductive Technology •
Cumulus cells •
Early development •
Fertilization
