The Effect Of KDM4A And GSK126 On The Somatic Reprogramming And In Vitro Developmental Potential Of Cloned Porcine Embryos

Animal cloning technology posses great advantages in accelerating genetic improvement of pig breeding,establishing animal models of human diseases and xenotransplantation,thus has made the pig become a hot pot in the biomedical field,but the cloning efficiency of pigs is extremely low,related experimental materials matured oocytes cannot be obtained in large quantities,the cloning technical system is not easy to master and still needs to be further improved,which greatly hinder the production and application of pig cloning.Till now,some reports on mice have confirmed that abnormal zygotic genome activation(ZGA)is one of the main reasons for the low cloning efficiency during the development of cloned embryos,but ZGA in pigs still remains unclear,therefore,it’s of great significance to preceed investigating the abnormal transcription and epigenetic patterns of cloned porcine embryos during the ZGA period and seek effective methods to improve the efficiency of pig cloning.By comparing the differences between normal in vivo fertilized embryos and cloned embryos at transcriptional levels and epigenetic modification levels,this study showed that the abnormal enrichment of H3K9me3 and H3K27me3 modifications contributed to the defected ZGA.And this study provided a strategy to promote somatic reprogramming and in vitro developmental potential through H3K27me3 modification inhibitor GSK126 incubation and KDM4 A overexpression,thus,it’s helpful for further research on the clinical application of pig cloning.Furthermore,matured oocytes will suffer severe oxidative stress damage after prolonged exposure to in vitro environments,leading to in vitro aging(IVA).Given that melatonin plays effective roles in anti-oxidative stress and its very few research on IVA in porcine oocytes,so we hope to find out an effective solution to improve the quality of IVA oocytes in pigs through exploring the mechanism of melatonin on alleviating IVA,hence,providing powerful support for subsequent acquisition of high-quality cloned embryos.The results in details are as follows:1)Cloned porcine embryos exhibited delayed ZGA and abnormal transcription profile during ZGA.Morever,some ZGA-initiated genes failed to activate,while some somatic genes were unsuccessful to be transcriptionally repressed.These abnormalities are closely related with inefficient reprogramming in porcine cloned embryos.2)H3K9me3 and H3K27me3 modifications were highly enriched in the genomic reprogramming-resistant regions(RRRs)and memory-preserved regions(MPRs)of porcine cloned embryos at 4-cell stage,resulting in abnormal gene expression during ZGA,which indicated that the abnormal enrichment of H3K9me3 and H3K27me3 modifications consisted of the main barriers to reprogramming during ZGA.3)H3K4me3 modification in porcine cloned embryos was less enriched in RRRs and MPRs,and was not a major barrier for reprogramming.Besides,in cloned porcine 4-cell embryos,RRRs and MPRs were relatively gene-poor regions but enriched for specific repeat sequences,such as LINE and LTR,implying that RRRs and MPRs mainly distributed in the low accessible heterochromatin regions.4)H3K9me3 and H3K27me3 modifications in porcine cloned 4-cell embryos were both effectively erased following the co-injection of KDM4 A and KDM6 A m RNAs.Overexpression of KDM4 A could significantly improve the developmental potential of cloned embryos in vitro,but overexpression of KDM6 A did not exert a positive effect.5)H3K9me3 and H3K27me3 modifications in porcine cloned 4-cell embryos were both effectively erased following GSK126 incubation for 48 h and KDM4 A m RNA injection,the expression of ten ZGA-related genes was significantly increased,and the in vitro developmental potential of cloned embryos were further improved,indicating an apparent synergistic effect of KDM4 A overexpression and GSK126 incubation.6)Melatonin effectively maintained the morphology,alleviated oxidative stress,decreased autophagy and early apoptosis levels,retarded the decline of mitochondrial membrane potential in IVA oocytes and significantly promoted subsequent embryonic development.