Vitrification of Pronuclear Zygotes Perturbs Porcine Zygotic Genome Activation.

Tengteng Xu,Jun Jiao,Yunhai Zhang,Yangyang Ma,Zubing Cao,Yelian Yan,Qiuchen Liu,Chengxue Liu,Tong Yu,Anucha Sathanawongs,Xin Wang,Mengya Zhang

doi:10.3390/ani12050610

Abstract

Simple SummaryVitrification 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 (α-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.

Highlights

Vitrification is widely used to preserve animal genetic resources in agriculture and human gametes and embryos in reproductive medicine [1,2]
Results showed that the rates of 2-cell (82.6%), 4-cell (59.4%), and blastocyst (25.8%) from frozen-thawed pronuclear embryos were significantly reduced compared to control group (97.4%, 86.6%, and 48.8%) (Figure 1A,B) (p < 0.05)
We found that vitrification of pronuclear zygotes did not affect the total cell number of blastocysts, TE, and inner cell mass (ICM) cell numbers (Figure 1D)

Summary

Introduction

Vitrification is widely used to preserve animal genetic resources in agriculture and human gametes and embryos in reproductive medicine [1,2]. Vitrification has been successfully applied to mammalian embryos from different species, such as humans [3,4], mice [5], cattle [6], sheep [7], and pigs [8,9]. The effect of vitrification on zygotic genome activation (ZGA) during early embryonic development remains unclear. The developmental stage of ZGA varies among different species. It was reported that ZGA commonly occurs at the 2-cell stage in mice [19], 4–8 cell stage in pigs [20], 8–16 cell stage in cattle [21], sheep [22], and humans [23]. Accumulating evidence indicated that ZGA plays a critical role in early embryonic development. Mouse embryos, arrested at the 2-cell stage and displayed abnormal ZGA upon the exposure of α-amanitin, an RNA polymerase II inhibitor [24,25]. ZGA is an essential prerequisite for early embryonic development [20]

Results

Discussion

Conclusion