| The production of mature eggs and the smooth progress of embryonic development are essential prerequisites for the generation of normal individuals.Therefore,the study of the molecular mechanism regulating oocyte meiosis and preimplantation embryo development is of great significance for improving the production efficiency of cloned animals,promoting the establishment of animal models of human diseases and promoting the progress of assisted reproduction technology.As one of the major epigenetic modifications,histone acetylation is regulated by histone acetylases(HATs)and histone deacetylases(HDACs),the latter are further divided into four families in mammals.Among the classic histone deacetylases,The effects of class I and II family members on mouse oocyte maturation and preimplantation embryo development have been reported,while the role of HDAC11,the only member of class IV family,in porcine oocyte maturation and preimplantation embryo development remains unknown.In this study,we added specific inhibitor Elevenostat(JB-3-22)of HDAC11 to investigate the effects of HDAC11 on oocyte maturation and preimplantation embryo development in pigs.It mainly includes the changes of nuclear maturation and cytoplasmic maturation of oocytes after HDAC11 inhibition,the changes of histone acetylation modification level,expression of pluripotency related genes,zygotic activation genes and transcriptomics,etc.One of the possible mechanisms that lead to the above effects is the change of H4K16 ac level.Therefore,we subsequently up-regulated and down-regulated H4K16 ac levels by microcytoplasmic injection of H4K16 acetylated and non-acetylated mutants(H4K16Q and H4K16R)m RNA for further verification.Furthermore,we also down-regulated the expression of ESCO2 in pig preimplantation embryos by injecting si RNA,so as to observe its effect on the development of pig preimplantation embryos during IVF and its relationship with H4K16 ac.The main results are as follows:1.In this study,the effect of HDAC11 specific inhibitor Elevenostat(JB-3-22)on nuclear maturation and cytoplasmic maturation of porcine oocytes was observed by adding HDAC11 specific inhibitor into in vitro maturation liquid.We found that inhibition of HDAC11 had no significant effect on cytoplasmic maturation indicators such as mitochondrial function,cortical particle migration,and reactive oxygen species(ROS)levels.However,oocyte maturation can be hindered by significantly increased levels of ?-Tubulin acetylation and H4K16 acetylation,abnormal spindle assembly and chromosome arrangement during nuclear maturation.We also performed RNA-seq assay,which showed that HDAC11 inhibition also led to transcriptome changes in oocytes.After treatment with JB-3-22,157 genes were up-regulated and 106 genes were down-regulated,among which the up-regulated genes were mainly related to m RNA splicing and cyclin binding,and the down-regulated genes were mainly related to the nucleotide transmembrane transport and so on.2.In this study,JB-3-22 was added into the embrynic culture medium to analyze the changes in the development efficiency and histone acetylation modification level of IVF preimplantation embryo development in pigs by HDAC11.The results showed that inhibition of HDAC11 significantly reduced the blastocyst rate of preimplantation embryos(control group: 20.71 ± 0.91%,treatment group: 0.0 ± 0.0%),high acetylation of histone H4(H4K16ac and H4K12ac),and decreased expression of pluripotency related genes(NANOG,OCT4 and SOX2),among which the increased level of H4K16 ac may be one of the main reasons.Therefore,we then used cytoplasmic injection of H4K16 acetylated mutant H4K16 Q m RNA to rapidly increase the level of H4K16 ac in oocytes and also observed the same phenotype above.It reveals that the increase of H4K16 ac level is the key cause of preimplantation embryonic development arrest for pigs.3.In order to investigate the effect of reduced H4K16 ac level on preimplantation embryo development of pigs,we down-regulated H4K16 ac level by cytoplasmic injection of H4K16 non-acetylated mutant H4K16 R m RNA,and then observed its effect on preimpantation embryo development of pig.The results showed that injection of H4K16 R significantly reduced the blastocyst rate of preimplantation embryos(control group: 18.48 ± 2.267%,treatment group: 0.0 ± 0.0%),with the decrease of H4K5 ac and H3K4me3 levels,the increase of ?-H2A·X level and the down-regulation of zygotic activating genes(ZSCAN4,TDG,MYC,etc.).RNA-seq results showed that H4K16 R m RNA injection affected the genome-wide transcription of 4 cells,down-regulating the expression of 1637 genes and up-regulating the expression of 727 genes.The expression of zygotic activation-related genes was significantly down-regulated.Our study reveals that down-regulation of H4K16 ac inhibits the development of preimpantation embryos in pigs,possibly by down-regulating the expression of zygotic activating genes and reducing the repair capacity of DNA double-strand breaks.We also down-regulated the expression of ESCO2 in preimplantation embryos by cytoplasmic injection of si RNA,and found that down-regulated ESCO2 could lead to development arrest of preimplantation embryos,accompanied by decreased H4K16 ac level.Therefore,our study also confirmed that ESCO2 was also involved in regulating H4K16 ac levels during preimplantation embryo development in pig. |
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