Effects And Mechanism Of Histone Epigenetic Modifications Regulated By HDAC6 And HDAC11 On Mouse Oocyte Maturation

Abnormal oocytes maturation is a main cause of spontaneous abortions,birth defects and developmental disabilities in humans.Find and explore key factors and related pathways that regulate oocyte maturation is of great significance to human assisted reproduction and embryonic development.Several members of the HDACs family participate in oocyte maturation via regulating histone modifications.Among them,HDAC6 is one of the key factors involved in oocyte maturation.However,the mechanism how HDAC6 regulate oocyte maturation has not been fully elucidated.HDAC11 could regulate mitosis process in mammalian cells,but little is known about the role of HDAC11 in meiosis and the underlying mechanism.In this study,by adding the specific inhibitors of HDAC6 and HDAC11,we analyzed their effects on oocyte maturation,meiotic apparatus assembly,mRNA expression and histone modification,which could provide a theoretical basis for uncovering the important role of HDAC6 and HDAC11 in oocyte maturation.The main research contents and results are as follows: 1.Inhibition of HDAC6 affects mouse oocyte maturation and mRNA expressionWe first analyzed the expression pattern of HDAC6 in oocytes by detecting the expression and promoter DNA methylation status of HDAC6 in oocytes.Next,the effects of HDAC6 inhibition on oocyte nuclear maturation and mRNA expression were analysed by adding HDAC6 inhibitor TubasinA(TubA).The results showed that: 1)HDAC6 showed highly expression in oocytes,corresponding with its low promoter DNA methylation level;2)Compared with the control group(71.7 ± 1.3%),the oocyte maturation rate in 0.1 μg/mL and 10 μg/mL TubA(47.3 ± 0.8%;3.17 ± 2.0%)treatment groups were significantly decreased.With the significantly increased abnormalities in spindle/chromosome arrangement and K-MT attachment.Furthermore,TubA treatment also activated SAC expression without affecting its function;3)Single-cell mRNA-seq results showed that TubA treatment resulted in 121 down-regulated and 212 up-regulated genes in oocytes.Among them,cell cycle-related genes CCNB1,CDK2,SMAD3,YWHAZ and DNA methylation-related genes DNMT1 and DNMT3 B all showed a significant decrease after TubA treatment.In summary,the expression of HDAC6 in oocytes was regulated by its promoter methylation level.Inhibition of HDAC6 disrupted the assembly of meiosis appratus and affect mRNA expression.2.Inhibition of HDAC6 affect histone epigenetic modification during mouse oocytes maturationTo further analyze the mechanism how HDAC6 inhibition affects oocyte maturation,we tested the effects of HDAC6 inhibition on α-tubulin acetylation and histone modifications(acetylation,phosphorylation,and methylation),we further analyzed the possible interaction between histone acetylation and phosphorylation.The results showed that: 1)Compared with control group,HDAC6 inhibition by TubA and Tubacin both resulted in increased acetylation level of α-tubulin and H4K16;2)Elevated H4K16 acetylation induced by HDAC6 inhibition caused decreased phosphorylation level of H3T3 and H3S10.Conversely,inhibiting phosphorylation level of H3T3 by Haspin inhibitor 5-ITu showed elevated H4K16 acetylation level in mouse oocytes;3)Inhibition of HDAC6 leaded to a significant increase in H4K5,H4K12 and H3K4 acetylation and H3K9me3 levels,but had no significant effect on H3K4me3 levels.In summary,TubA treatment affected α-tubulin acetylation and histone acetylation,phosphorylation,and methylation modifications.These epigenetic changes may be the potential molecular mechanisms how HDAC6 affects meiosis apparatus assembly and mRNA expression.These results also indicated that mutual exclusive interaction exists between H4K16 acetylation and H3 phosphorylation in oocytes 3.HDAC11 promotes meiotic apparatus assembly during mouse oocyte maturation via decreasing H4K16 and α-tubulin acetylationWe first examined the expression of HDAC11 in oocytes.At the same time,the role of HDAC11 played on oocyte maturation and histone modification were studied by adding HDAC11 specific inhibitor(JB3-22).The results showed that: 1)HDAC11 expression could be detected in oocytes.HDAC11 expression in GVBD oocytes was similar to that in GV oocytes,but its expression in MI and MII oocytes was significantly lower than that in GV oocytes(P < 0.01);2)HDAC11 inhibition significantly affected GVBD and maturation rate of oocytes.With the increased abnormalities in spindle arrangement/chromosome assembly and K-MT attachment in JB3-22 treated oocytes.At the same time,JB3-22 treatment also affected the activation and function of SAC in oocytes;3)Immunofluorescence staining and WB detection revealed that inhibition of HDAC11 significantly increased the acetylation levels of α-tubulin and H4K16.Above all,HDAC11 is an important factor in oocyte maturation.It plays important roles in accurate chromosome/spindle alignment,K-MT attachment,and SAC function.This effect may be achieved by deacetylating α-tubulin and H4K16.