Vitrification of Pronuclear Zygotes Perturbs Porcine Zygotic Genome Activation

Simple Summary Vitrification is a commonly used cryopreservation technique that has played an important role in the preservation of animal genetic resources and assisted reproduction in humans. However, the effect of vitrification on embryonic genome activation remains to be elucidated. This study found that vitrification reduced the developmental efficiency of pig blastocysts. Further studies found that vitrification reduced transcriptional activity during embryonic genome activation and disrupted gene expression. Our findings suggest that vitrification impairs genome activation in pig embryos, and our results can provide new insight into improving the developmental efficiency of vitrified embryos. Zygotic genome activation (ZGA) plays an essential role in early embryonic development. Vitrification is a common assisted reproductive technology that frequently reduces the developmental competence of embryos. However, the effect of vitrification on porcine ZGA and gene expression during ZGA remains largely unclear. Here, we found that vitrification of pronuclear zygotes derived from parthenogenetic activation (PA) and in vitro fertilization (IVF) resulted in a significant reduction in the rates of 2-cell, 4-cell, and blastocysts, but did not affect the quality of blastocysts. Functional research revealed that RNA polymerase II Inhibitor (alpha-amanitin) treatment significantly reduced global transcriptional activity and developmental efficiency of both 4-cell and 8-cell embryos, implying an essential role of ZGA in porcine early embryonic development. Furthermore, vitrification did not affect the synthesis of nascent mRNA of 2-cell embryos, but significantly inhibited global transcriptional activity of both 4-cell and 8-cell embryos, suggesting an impaired effect of vitrification on porcine ZGA. Correspondingly, the single-cell analysis showed that vitrification caused the downregulation or upregulation expression of maternal genes in 4-cell embryos, also significantly decreased the expression of zygotic genes. Taken together, these results indicated that vitrification of pronuclear zygotes impairs porcine zygotic genome activation.