Effect Of Transcription Factor ELF4 On Porcine Oocyte Maturation And Preimplantation Embryo Development Potential

Studies have shown that the main causes of low development efficiency of IVF embryos and somatic cell nuclear transfer embryos include excessive DNA damage and epigenetic reprogramming abnormalities.Transcription factors play an important role in regulating gene expression and embryonic development.ELF4,a member of the ETS family of transcription factors,plays an important role in cell cycle regulation,cell differentiation and DNA damage repair.However,the role of ELF4 in porcine oocyte maturation and preimplantation embryo development has not been reported.Therefore,microinjection of interfering RNA was used to knock down ELF4 to study its effects on porcine oocyte maturation,pre-implantation development of IVF embryos and preimplantation development of somatic cell nuclear transfer embryos.The results are as follows:1.Effect of ELF4 on porcine oocyte maturationIn this study,porcine oocytes were selected as the research object,and RNA interference technology(siRNA)was used to knock down ELF4 to detect the effect of low level ELF4 on porcine oocyte maturation.The results showed that the level of ELF4 in MI phase of porcine oocyte was normally higher than that in MII phase.After siRNA interference,oocyte maturation was significantly decreased in the si-ELF4 group compared with the control group(70.11% control vs.67.09% si-scramble vs.51.87%si-ELF4;p < 0.01).After RNA knockdown of ELF4,mitochondrial membrane potential and reactive oxygen species levels were not significantly different between the three groups,but CCNB1 transcription level in si-ELF4 group was lower than that in control group.The above results indicated that ELF4 significantly affected the maturation of porcine oocytes.Low level of ELF4 can reduce CCNB1 and affect oocyte maturation.2.Effect of ELF4 on development of porcine in vitro fertilization(IVF)embryosIn this experiment,porcine IVF embryos were used as research objects,and the expression of ELF4 was down-regulated by microinjection of siRNA to study its effect on the developmental ability of pig IVF embryos.The results showed that compared with the control group,after ELF4 gene knockdown,the blastocyst rate was significantly decreased(20.70% control vs.17.49% si-scramble vs.2.40% si-ELF4;p< 0.001)and blastocyst mass decreased significantly,DNA damage degree of 4-cell embryos in porcine IVF increased significantly,H3K9me3 and DNA methylation levels increased abnormally,and the expression of ZGA-related genes decreased significantly in si-ELF4 group.The above experimental results indicated that knocking down ELF4 damaged the genomic stability of IVF embryos,resulting in abnormal H3K9me3 and DNA methylation,which was not conducive to ZGA and embryonic development of porcine IVF embryos.3.Effect of ELF4 on preimplantation development of porcine cell nuclear transfer(SCNT)embryosBy comparing the expression changes of ELF4 in IVF and SCNT embryos at various stages of preimplantation development,we found that the expression level of ELF4 in SCNT embryos was significantly higher than that in IVF embryos.Therefore,we hypothesized that the abnormally elevated ELF4 transcript level in SCNT embryos may be responsible for the retardation of SCNT embryo development.To test this conjecture,we down-regulated the expression of ELF4 by cytosolic injection of siRNA,and investigated the possible effect of ELF4 on promoting the developmental potential of porcine SCNT embryos.The results showed that,compared with the control group,after ELF4 knockdown,the SCNT embryo 4-cell rate was significantly increased in the si-ELF4 group(27.21% control vs.24.91% si-scramble vs.36.19% si-ELF4;P < 0.01),the number of blastocyst cells increased significantly(47.00 control vs.46.33 siscramble vs.60.67 si-ELF4;p < 0.001),the DNA damage degree,DNA methylation level and H3K4me3 modification level of SCNT embryo 4-cells in the si-ELF4 group were significantly reduced,while the H3K4me3 modification level was significantly increased.It can be concluded from the above experimental results that ELF4 knockdown plays an important role in maintaining the stability of SCNT embryo genome and normal DNA methylation and H3K4me3 modification level,which is helpful to regulate the normal development of porcine SCNT embryo.