| Cloned animals have been generated from somatic cells in several species in recent years. However, cloning is very inefficient and characterized by high embryonic lethality associated with severe abnormalities of cloned fetuses and placentas in rodents and ruminants. Erasure of differentiation memory from somatic cells used as donor nuclei to an embryonic-like totipotent state requires epigenetic reprogramming. Faulty epigenetic reprogramming is highlighted as the major cause of developmental failure and abnormal phenotypes in cloned animals. In the present work, genome-wide epigenetic reprogramming was evaluated in cloned fetuses and adults, through measurement of DNA 5-methylcytosine content with reverse-phase liquid chromatography (HPLC). Aborted cloned fetuses, cloned fetuses from interrupted pregnancies and those affected by hydroallantois displayed significant DNA methylation losses compared to in vivo controls. Cloned adult cows, however, had similar levels of DNA methylation as in vivo derived adult cows, suggesting that there is an epigenetic reprogramming threshold that is met by a subset of cloned animals. Moreover, DNA methylation was significantly different between cloned fetuses and corresponding placentomes, but not in vivo controls. This finding denotes that the inner cell mass, which gives rise to the fetus, and the trophectoderm, the precursor of the placenta, are subject to different reprogramming pathways in cloned animals. A novel EST (expressed sequence tag) was identified in the bovine genome, termed SICH-1 (silenced in cloned hydrops-1). SICH-1 transcripts were identified in several tissues, including in vivo placentomes. Interestingly, no expression was detected in hydrops cloned placentomes or oocytes. Additionally, expression of imprinted genes (IGF-2, IGF-2r), genome-wide methyltransferases (Dnmt-1, Dnmt-3a) and critical genes for placental function and differentiation (placental lactogen and Gcm-1 ) were investigated in abnormal cloned placentomes and/or fetuses by RT-PCR. Transcripts for all these key genes were identified in abnormal cloned placentomes in the same manner as in vivo placentomes. The data show here that cloned fetuses are indeed subject to genome-wide epigenetic reprogramming errors, and are somewhat tolerant to DNA methylation losses. Additionally, the identification of a novel genomic sequence, silenced in abnormal cloned placentomes, reveals a potential diagnostic marker for placental alterations after nuclear transfer. |
