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RNA sequencing revealed abnormal gene expression and benefits of vitamin C treatment in cloned bovine embryos

ABSTRACT: Somatic cell nuclear transfer (SCNT) in mammals has been mostly unsuccessful. In this study, we measured and compared transcription profiles of cloned and fertilized embryos using RNA sequencing. Before zygotic genome activation (ZGA), fewer genes were activated in the cloned embryos than in vitro fertilization (IVF), leading to insufficient activation of protein transport and degradation functions. In blastocysts, terms of “embryo development” and “adherens junction” represented major differences for repressed genes in SCNT compared to IVF. Vitamin C (Vc) was found to relax donor cell chromatin and promote cloned embryo development. RNA sequencing indicated that Vc treatment restored some abnormal genes and processes in the cloned embryos. Processes of autophagy, RNA-editing and cell junction were not fully established, but they were improved by Vc treatment. Overexpression of ZNF641, one of the completely restored genes by Vc treatment, improved the cloned embryos’ potential. Finally, Vc treatment rescued some long non-coding RNAs that were aberrantly expressed in the cloned embryos. Collectively, many important genes and biological processes were abnormal in the cloned embryos, some of which can be improved by Vc treatment. This investigation provides several practical aspects that could be useful for improving cloning efficiency and its future applications.

ORGANISM(S): Bos taurus

PROVIDER: GSE123705 | GEO | 2020/12/12

REPOSITORIES: GEO

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Project description:The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization (IVF) were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (> 2-fold vs. controls) than did the extraembryonic tissues (P < 1.0 M-CM-^W 10M-bM-^@M-^S26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1M-bM-^@M-^S5% per embryo transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages, although these alterations were not always statistically significant for all three SCNT groups because of variability. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT. Comparative gene expression analyses using post-implanted E6.5 cloned embryos were performed by microarray. Cloned embryos were produced with three different types of donor cells (cumulus cells, neonatal Sertoli cells and fibroblasts). As controls, sex- and genotype-matched embryos produced by in vitro fertilization were used. Each embryos were mechanically dissected into the embryonic and extraembryonic parts at E6.5 and were subjected to gene expression microarray.

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RNA sequencing revealed abnormal gene expression and benefits of vitamin C treatment in cloned bovine embryos

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