Effect Of Vitrification On The Development And Genomic Activation Of Porcine Early Embryos

itrification to preserve oocytes and embryos,would facilitate international exchange of genetic resources,reduce the risk of disease transmission,and benefit animal conservation and human fertility preservation,and therefore plays an increasingly important role in livestock reproduction and human assisted reproductive technology.However,the effect of vitrification,as a strong external stimulation,on the degradation of maternal transcripts and the activation of embryonic genome during the transformation of maternal zygote is not clear.In order to investigate the effect and mechanism of vitrification on porcine embryo development,we conducted the following studies.Firstly,effect of vitrification on the in vitro development of PAEs and IVFEs before implantation was studied.PAEs and IVFEs were cultured in vitro for 16h and then vitrified and were selected for resuscitation culture.Untreated PAEs served as control.The results showed that the developmental efficiency of 2-cells and blastocysts in the vitrification group was significantly lower than that in the control group(2-cell of PAEs:97.40%Vs.82.60%,blastocyst:48.80%Vs.25.80%;2-cell of IVFEs:60.18%Vs.35.74%,4-cell:11.92%Vs.3.00%.vitrification group Vs.control group,P<0.05).The results of indirect immunofluorescence staining showed that the blastocyst quality(total cell number,trophectderm cell number and inner cell mass)of PAEs in vitrification group was similar to that in the control group.Then,effect of vitrification on transcriptional activity of porcine embryo genome was studied.Protocol for evaluating transcriptional activity of embryonic genome was established.The porcine PAEs was treated with α-amanitin(α-amanitin,RNA polymerase Ⅱ inhibitor)at 16h.It was found that the developmental efficiency of PAEs decreased significantly after treatment.EU staining was performed on the treated 4-cell and 8-cell PAEs.It was found that fluorescence intensity of α-amanitin treated PAEs was significantly decreased in 4-cell and 8-cell stage.EU staining of vitrified embryos was carried out,and results showed that the fluorescence intensity of EU staining in vitrification group was not only similar to that in α-amanitin treatment group,but also significantly weaker than that in non-vitrification group at 2-cell,4-cell and 8-cell stages.It is suggested that vitrification obstruct the activation of porcine embryo genome.Finally,expression of certain important genes in porcine 4-cell embryos was detected by single-cell real-time fluorescence quantitative PCR,and the effect of vitrification on embryo genome activation was explored at the molecular level.The results show that,vitrification not only causes up-regulation(CK1,PKA,HASPIN and GPX4)or down-regulation(HDAC8,KDM2B,SMYD3,TET2,ALPL,TEAD2,ESSRB,CDH2,SIRT1,FGFR2 and HSD3B1)of multiple maternal genes in porcine 4-cell embryos,but also causes abnormal expression of some zygote genes(CCL24,EPHA4,GATA2,KRT8,TCF23,BMP4 and IL4).In conclusion,results of the present study indicate that vitrification of porcine pronuclear embryos can lead to decreased early embryo development efficiency and abnormal genome activation,and cause partial maternal transcript and zygote gene expression disorder.The results of this study provide a preliminary basis for further revealing the molecular mechanism of vitrification affecting the activation of porcine embryo genome and improving the efficiency of vitrification embryo development.