Abstract
Somatic cell nuclear transfer (scNT) embryos suffer from damage caused by micro-operation (manipulation) and inefficient genome reprograming that hinder their normal development at different levels and in distinct ways. These two effects are inseparable in the nature of the scNT embryo, although methods to separately measure them are needed to improve scNT technology and evaluate incoming reprogramming tools. As an attempt to meet these demands, we made bovine sham nuclear-transfer (shNT) blastocysts, special embryos made with a standard nuclear-transfer procedure at the zygote stage, while retaining an intact genome. We compared their transcriptomes with those of other blastocysts derived by in-vitro fertilization (IVF) or scNT. Correlation analysis revealed a singularity of shNT blastocysts as they separately gathered from the others. Analysis of developmentally important genes revealed that, in shNTs, the stemness-associated differentially expressed genes (DEGs), including OCT4, were mostly underrepresented. Overrepresented epi-driver genes were largely associated with heterochromatin establishment and maintenance. By multilateral comparisons of their transcriptomes, we classified DEGs into three groups: 561 manipulation-associated DEGs (MADs) common to shNTs and scNTs, 764 donor genome-associated DEGs (DADs) specific to scNTs, and 1743 zygote manipulation-associated DEGs (zMADs) specific to shNTs. GO enrichment analysis generated various terms involving “cell-cell adhesion,” “translation,” and “transcription” for MADs and “cell differentiation” and “embryo implantation” for DADs. Because of the transcriptomic specificity of shNTs, we studied zMADs in detail. GO enrichment analysis with the 854 zMADs underrepresented in shNTs yielded terms related to protein and mitochondria homeostasis, while GO enrichment analysis of 889 shNT-high zMADs yielded terms related to endoplasmic reticulum stress and protein transport. We summarized the DEGs, which, with further investigation, may help improve our understanding of molecular events occurring in cloned embryos and our ability to control clonal reprogramming.
Highlights
Somatic cell nuclear transfer is a powerful technique to produce genetically modified animals that can be used as industrial bioreactors or models for biomedical research (Kang et al, 2003; Niemann and Lucas-Hahn, 2012)
We examined the gene expression profiles, of sham nuclear-transfer (shNT) blastocysts, to determine how the transcriptome differed from in-vitro fertilization (IVF) and Somatic cell nuclear transfer (scNT)
We analyzed global gene expression pattern in bovine blastocysts generated by various methods, i.e., IVF, scNT, and shNT techniques and compared their transcriptomes to identify the factors induced by manipulation of oocytes or zygotes
Summary
Somatic cell nuclear transfer (scNT) is a powerful technique to produce genetically modified animals that can be used as industrial bioreactors or models for biomedical research (Kang et al, 2003; Niemann and Lucas-Hahn, 2012). Some repressive epigenetic marks such as DNA methylation and histone H3 lysine 9 or lysine 27 methylations (H3K9me and H3K27me) showed a very limited epigenetic reprogramming This is why the developmental ability of cloned embryos are improved by the treatment of small chemicals that serve as HDAC (trichostatin A, sodium butyrate, scriptaid, and valproic acid) or DNMT (5-azacytidine) inhibitors (Kwon et al, 2017). Likewise, these factors can hinder correct reprogramming and normal development in scNT embryos. Using existing embryo samples derived from standard scNT, it is impossible to discriminate one effect from the other because the two are intertwined
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