Abstract
Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a differentiated somatic cell is reset to a state resembling totipotency, the efficiency of SCNT is likely to remain low.
Highlights
The process of cloning by somatic cell nuclear transfer (SCNT) requires the epigenetic signature of a differentiated somatic cell to be reset to a state resembling totipotency, capable of driving full development after fusion of the cell with an enucleated oocyte cytoplast
The decrease in the proportion of methylation at asatI-5 in SCNT embryos during the first seven days of development, from the donor cell state, followed by continued decrease in average methylation in extra-embryonic tissues, together with the lower levels of methylation found in sperm of SCNT bulls demonstrates that some epigenetic reprogramming is occurring following SCNT
Comparison of the methylation levels measured in SCNT blastocysts with those generated by in vitro fertilization (IVF) suggests that the seven day period from SCNT to blastocyst formation is either an insufficient period of time for complete reprogramming, or that SCNT embryos do not have the appropriate reprogramming factors to ensure that this repeat region is appropriately reprogrammed by the blastocyst stage
Summary
The process of cloning by SCNT requires the epigenetic signature of a differentiated somatic cell to be reset to a state resembling totipotency, capable of driving full development after fusion of the cell with an enucleated oocyte cytoplast. Because of the vital importance of correct chromosomal segregation during cell division [19], the CpG site found to be differentially methylated in sperm from control and SCNT bulls was examined during the development of embryos, fetuses, and post-natal animals generated by fertilization and SCNT. Of DNA methylation between SCNT and control embryos was the largest difference observed in this time course study Despite this very significant difference in DNA methylation, both IVF and SCNT blastocysts showed a lower proportion of DNA methylation than sperm used for fertilization and donor cells, respectively. All fetal and post-natal SCNT somatic tissues were hyper-methylated compared to those obtained from fetuses and cattle generated by AI; at an individual animal level a considerable overlap existed in the proportion of methylation at this sequence, with some SCNT individual animals exhibiting apparently normal DNA methylation levels. Methylation data from surrounding CpG sites able to be analyzed simultaneously by Sequenom analysis (Figure S1 and Table S1) showed that CpGs close to asatI-5 showed some similarity to the data reported in this manuscript, while more distant CpGs showed progressively less similarity to asatI-5
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