Characterization and comparative analyses of transcriptomes of cloned and in vivo fertilized porcine pre-implantation embryos

Abstract

Somatic cell nuclear transfer (SCNT) is the only method known to rapidly reprogram differentiated cells into totipotent embryos. Most cloned embryos become arrested before implantation and the details of the underlying molecular mechanism remain largely unknown. Dynamic regulation of the transcriptome is a key molecular mechanism driving early embryonic development. Here, we report comprehensive transcriptomic analysis of cloned embryos (from Laiwu and Duroc pigs) and in vivo fertilized embryos (from Duroc pigs) using RNA-sequencing. Comparisons between gene expression patterns were performed according to differentially expressed genes, specific-expressed genes, first-expressed genes, pluripotency genes and pathway enrichment analysis. In addition, we closely analyzed the improperly expressed histone lysine methyltransferases and histone lysine demethylases during cell reprogramming in cloned embryos. In summary, we identified altered gene expression profiles in porcine cloned pre-implantation embryos in comparison to normal in vivo embryos. Our findings provide a substantial framework for further discovery of the epigenetic reprogramming mechanisms in porcine SCNT embryos.

TDG is a pig-specific epigenetic regulator with insensitivity to H3K9 and H3K27 demethylation in nuclear transfer embryos

“…Large-scale analyses of the embryonic transcriptomes have reported different MZT timing between in vivo -fertilized (IVO) embryos (four-cell stage) and SCNT embryos (eight-cell stage) ( Cao et al., 2014a ; He et al., 2019 ). Here, we investigated the RNA-seq profiles of two- to eight-cell IVO embryos (IVO2c, IVO4c, IVO8c), four- to eight-cell SCNT embryos (SCNT4c, SCNT8c), and the donor pig fetal fibroblasts (PFF) ( Table S1 ).…”

“…Among the EGA-OFF genes, we surprisingly found that the deficient TDG expression could not be rescued by KDM4A + GSK126 treatment ( Figure 6 A). Notably, TDG is specifically transcribed during pig MZT ( He et al., 2019 ; Kong et al., 2020 ) and is barely detected at the MZT stage in mouse ( Deng et al., 2014 ; Liu et al., 2016 ), human ( Hendrickson et al., 2017 ; Yan et al., 2013 ), and cattle embryos ( Graf et al., 2014 ; Jiang et al., 2014 ) ( Figures 6 B and S4 C). TDG is a well-known enzyme for excising thymine, 5-hydroxymethyluracil (5hmU), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), which are generated from either 5-methylcytosine (5mC) deamination by AICDA/APOBECs or 5mC hydroxylation by TET proteins ( Figure 6 C) ( Cortellino et al., 2011 ; Maiti and Drohat, 2011 ).…”

“…∗∗ p < 0.01; n.s., not significant; two-tailed Student's t test. (B) Line plots illustrating the dynamic transcriptional changes of TDG in pig IVO embryos ( He et al., 2019 ; Kong et al., 2020 ). (C) Schematic illustration of regulators related to the DNA methylation (C→5mC) and demethylation (5mC→C) process.…”

“… (B) Line plots illustrating the dynamic transcriptional changes of TDG in pig IVO embryos ( He et al., 2019 ; Kong et al., 2020 ). …”

Assessment of the Growth and Reproductive Performance of Cloned Pietrain Boars

“…A fibroblast cell was inserted into the perivitelline space of enucleated oocytes with a smooth surface through the same slit [24]. After culture in PZM3 medium [26] at 39 • C for 1.5 h, the fusion of oocyte-donor cell complexes was performed via two successive DC pulses at 1.2 kV/cm for 30 µs [27].…”