Abstract
Cryopreservation of porcine cloned zygotes has important implications for biotechnology and biomedicine research; however, lower embryo developmental potential remains an urgent problem to be resolved. For exploring the sublethal cryodamages during embryo development, this study was designed to acquire the mRNA and long non-coding RNA (lncRNA) profiles of 2-cells, 4-cells and blastocysts derived from vitrified porcine cloned zygotes using transcriptome sequencing. We identified 167 differentially expressed (DE) mRNAs and 516 DE lncRNAs in 2-cell stage, 469 DE mRNAs and 565 lncRNAs in 4-cell stage, and 389 DE mRNAs and 816 DE lncRNAs in blastocyst stage. Functional enrichment analysis revealed that the DE mRNAs during embryo development were involved in many regulatory mechanisms related to cell cycle, cell proliferation, apoptosis, metabolism and others. Moreover, the target genes of DE lncRNAs in the three embryonic stages were also enriched in many key GO terms or pathways such as “defense response”, “linoleic acid metabolic process”, “embryonic axis specification”, “negative regulation of protein neddylation”, etc., In conclusion, the present study provided comprehensive transcriptomic data about mRNAs and lncRNAs for the vitrified porcine cloned zygotes during different developmental stages, which contributed to further understand the potential cryodamage mechanisms responsible for impaired embryo development.
Highlights
In addition to being important livestock, pigs are ideal large animal models for biological and biomedical research because they share similar anatomy, genetics, physiology, and pathology with humans (Tang et al, 2007; Wilkinson et al, 2013)
We found that the DE mRNAs were enriched in large numbers of significant GO terms such as “negative regulation of transcription, DNA-templated”, FIGURE 5 | GO and KEGG enrichment analysis of differentially expressed (DE) mRNAs and long non-coding RNA (lncRNA) targets in 2-cells derived from vitrified porcine cloned zygotes. (A) Histogram and (B) scatter plot of GO enrichment for DE mRNAs; (C) histogram and (D) scatter plot of GO enrichment for target genes of DE lncRNAs; (E) scatter plot of KEGG enrichment for DE mRNAs; (F) scatter plot of KEGG enrichment for target genes of DE lncRNAs
11 target genes of DE lncRNAs were significantly enriched in a “defense response” GO term, suggesting 2-cell embryo competence to respond to the impact of vitrification
Summary
In addition to being important livestock, pigs are ideal large animal models for biological and biomedical research because they share similar anatomy, genetics, physiology, and pathology with humans (Tang et al, 2007; Wilkinson et al, 2013). The generation of genetically modified pigs has great promise in livestock industry and human disease treatment including regenerative medicine, xenotransplantation, stem cell therapy and others (Yang and Wu, 2018; Yue et al, 2021). The cloned embryos obtained using these technical procedures are indispensable to result in genetically identical offspring (Keefer, 2015). For research and therapeutic purposes, the production of embryonic stem cells with the same genetic characteristics requires cloned embryos by SCNT technique (Gouveia et al, 2020). It is very essential to effectively cryopreserve porcine cloned embryos in order to genetic resource preservation as well as convenient research and utilization in related fields
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