5 Gene expression analysis and DNA methylation patterns of porcine somatic cell nuclear transfer blastocysts with high and low incidence of apoptosis

Somatic cell NT (SCNT) efficiency remains poor, preventing the technology from being regularly used in the agricultural industry. It is believed that faulty epigenetic reprogramming of SCNT embryos leads to the low overall success. A clear apoptotic signature is associated with inappropriate gene expression and epigenomic aberrancies in many experimental cell culture systems, and we hypothesised that an apoptosis biomarker could be used to effectively separate properly reprogrammed porcine SCNT embryos from those that are destined to fail due to incomplete reprogramming. Therefore, our objective was to evaluate global gene expression and DNA methylation patterns in high- and low-apoptosis individual embryos in an effort to characterise the extent of genomic reprogramming that had taken place. Porcine SCNT blastocysts on Day 6 of development were stained with a nontoxic, noninvasive caspase activity reporter, and the top and bottom 20% of detected caspase activity were classified as high and low apoptosis, respectively (3 replicate cloning sessions; n = 13 embryos per group). Genomic DNA and total RNA were isolated from each individual blastocyst. The RNA sequencing libraries were prepared using the Ovation SoLo RNA-Seq system (NuGen, San Carlos, CA, USA). Reduced representation bisulfite sequencing libraries were prepared for DNA methylation analysis using a modification of the single-cell reduced representation bisulfite sequencing global DNA methylation analysis approach detailed by Guo et al. (2015 Nat. Protoc. 10, 645-59). The RNA sequencing analysis using EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) revealed 175 total differentially expressed genes (fold change ≥1.5; false discovery rate ≤0.05) between the high- and low-apoptosis SCNT embryos. This list of differentially expressed genes was used to perform enrichment analysis to identify overrepresented Gene Ontology (GO) terms or Kyoto Encyclopedia of Genes and Genomes pathways (DAVID Ease version 6.8 (https://david.ncifcrf.gov/) against the Sus scrofa background genome). However, no significantly enriched GO terms or pathways were identified (false discovery rate P > 0.05). Analysis of global DNA methylation patterns between high- and low-apoptosis SCNT embryos using MethylKit (Akalin et al. 2012 Genome Biol. 13, R87) revealed 335 differentially methylated 100-bp regions with at least 25% difference in methylation (adjusted P ≤ 0.01). Gene transcription start sites associated with these regions were used for enrichment analysis; again, no significant enrichment of GO terms or Kyoto Encyclopedia of Genes and Genomes pathways was identified. Principal component analysis of CpG methylation showed the low-apoptosis embryos clustering more tightly than the high-apoptosis embryos, which were highly scattered. Ongoing comparisons of high- and low-apoptosis cloned embryos with naturally fertilized embryos produced in vivo may provide more information about which embryos were properly reprogrammed. Although we are still pursuing a link between reprogramming and gene expression in high- and low-apoptosis embryos, we conclude that these data support a model of stochastic epigenetic reprogramming following SCNT and reinforce the necessity of identifying embryos most likely to be successful due to proper epigenetic reprogramming in order to increase SCNT efficiency.