Genome activation in ICSI-derived horse embryos

GOSZCZYNSKI, D. E.; TINETTI, P.S.; CHOI, Y.H.; HINRICHS, K.; ROSS, P.J.

Conferencia; 46th Annual Conference of the International Embryo Technology Society (IETS); 2020

During preimplantation development, embryos go through a critical period of embryonic genome activation (EGA). The timing of EGA is species-specific, but little is known in horse embryos. Here, we aimed to characterize EGA in equine embryos produced by intracytoplasmic sperm injection (ICSI). Embryos were produced by ICSI of oocytes from three mares. Two embryos from each mare at each of eight developmental stages (MII, zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst) were individually analyzed by RNA-seq. Differential expression was evaluated using binomial Wald tests with an absolute logFC threshold of 1 in the DESeq2 R package. We found that EGA occurred in a two-step fashion. Minor EGA took place during the 2-cell to 4-cell transition, and featured upregulation of 751 genes and discrete downregulation of 60 genes in 4-cell embryos compared to 2-cell embryos. Differentially upregulated genes were enriched in gene ontology (GO) terms related to transcriptional activator activity, homeobox domains, and nucleosome assembly. Major EGA occurred during the 4-cell to 8-cell transition and included the largest number of differentially expressed genes (n=2,023) between consecutive stages. This period also featured the first massive transcript downregulation (n=816). Upregulated genes were enriched in GO terms related to ribosomal assembly, translation, and RNA modification. Additionally, we observed that the number of intronic sequences was significantly higher from the 4-cell stage onwards, indicating active transcription in comparison to oocytes, zygotes and 2-cell embryos. To evaluate the timing of paternal genome activation, we used whole genome sequencing data from the parents (average genome coverage of 19X) to quantify allele-specific expression. The average number of informative SNPs in exons, i.e. SNPs with alternative homozygous genotypes from the sire (AA mare ? BB sire), was 26,128 per mare, corresponding to 7,696 genes. Parental-specific transcript abundance was determined for each embryo, with an average of 1,911±865 informative SNPs detected per sample. Paternal alleles were considered expressed when they reached 10% of the maternal count. Across development, paternal transcripts became appreciable at the 4-cell stage, with 14.15±7.60 % of the informative SNPs exhibiting paternal expression, and increased thereafter until reaching a maximum of 96.34% at the blastocyst stage. Overall, this work demonstrates that EGA in horse embryos starts at the 4-cell stage and achieves its main activation at the 8-cell stage. Further analysis will be performed to detail paternal versus maternal gene expression at the different embryonic stages.