Abstract
Aberrant patterns of DNA methylation are consistent events in SCNT derived embryos and mechanistically are believed to be related to abnormal development. While some epigenetic drugs have been used in attempts to improve SCNT efficiency but some concerns remained toward the safety of these drugs on the health of future offspring. Folate is an essential cofactor in one‐carbon cycle for conversion of homocysteine to methionine, thereby ensuring supply of SAM, the universal methyl donor for many biological methylation reactions including DNA methylation. Therefore, in vitro DNA hypo-methylation can be induced by folate deprivation and this study aims at deciphering the role of folic acid deprivation in culture medium of BFFs for 6 days on SCNT efficiency. Our data revealed that culture of fibroblast cells in folate− medium containing 0.5% FBS did not alter the cell cycle compared to other groups. Flowcytometric analysis revealed that DNA methylation (5-mC level) in folate deprived cells cultured in 0.5% serum was decreased compared to folate+ group. The result of bisulfite sequencing was in accordance with flowcytometric analysis, which indicated a decrease in DNA methylation of POU5F1 promoter. Gene expression analysis revealed an increase in expression of POU5F1 gene in folate− group. The nuclear area of the cells in folate− group was significantly larger than folate+ group. Induced DNA hypomethylation by folate deprivation in the folate− group significantly improved blastocyst rate compared to the folate+ group. DNA methylation level in POU5F1 promoter and ICR of H19 and IGF2 of SCNT derived embryos in the folate− group was similar to the IVF derived blastocysts. In conclusion, our results proposes a promising “non-chemical” instead of “chemical” approach using inhibitors of epigenetic modifier enzymes for improving mammalian SCNT efficiency for agricultural and biomedical purposes.
Highlights
SCNT embryos is the specific epigenetic characteristic of somatic donor cells, which is very far from the specialized epigenetic status of sperm and oocyte
Considering that in vitro folate deprivation, can induce DNA hypo-methylation this study aims at deciphering the role of folic acid deprivation in culture medium of bovine fibroblast donor cells (BFFs) for 6 days on SCNT efficiency
Since in this study we aimed to determine the effect of induced DNA hypo-methylation in fibroblast cells on SCNT efficiency by folate deprivation, mRNA expression of methylenetetrahydrofolate reductase enzyme (MTHFR) and betaine-homocysteine methyltransferase (BHMT) mRNA were assessed in both fibroblast and kidney cells to confirm that the only active pathway for methionine production in fibroblast cells is MTHFR
Summary
SCNT embryos is the specific epigenetic characteristic of somatic donor cells, which is very far from the specialized epigenetic status of sperm and oocyte. Many epigenetic drugs such as DNA methyltransferase inhibitors (DNMTis) and histone deacetylase inhibitors (HDACis) have been used to improve in vitro and in vivo development of SCNT embryos[6,7,8,9] These two categories of epigenetic modifiers by inducing DNA hypo-methylation and histone hyper-acetylation result in chromatin relaxation and thereby improves nuclear reprogramming. While some of these epigenetic drugs have remarkably improved the pre- and post-implantation development of SCNT derived embryos[6,7,8,9], but we have some concerns about the side effects of these drugs on the health of future offspring, which remained to be elucidated. In vitro folate deprivation result in a significant genomic DNA hypo-methylation in non-transformed cell lines[15]
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