Effects And Mechanism Of Histone Deacetylase SIRT6 On Porcine Oocyte Maturation

Oocyte in vitro maturation as a key step of embryo engineering has a great application value in animal breeding,genetic resource conservation and biological researches.The low efficiency and poor quality of oocyte IVM is due to an inadequate understanding underlying oocyte maturation.SIRT6 is an NAD+ dependent histone deacetylase and is involved in the regulation of oocyte meiotic maturation in mouse oocytes.However,the role of SIRT6 in porcine oocyte maturation is yet to be known.The purpose of this study was to investigate the effects of maternal and cumulus cell-derived SIRT6 on porcine oocyte maturation.These studies will clarify the regulatory role of SIRT6 in porcine oocyte maturation and provide a basis for better understanding the biological function and mechanism of SIRT6 in porcine oocyte maturation.Fluorescence quantitative PCR and immunofluorescence staining analyses revealed that SIRT6 m RNA and protein were expressed in oocytes and cumulus cells during porcine oocyte maturation.Treatment with OSS₁28167（SIRT6 inhibitor,400 μM）showed that cumulus expansion（0.048±0.0073 mm2 Vs.0.026±0.0020 mm2）,germinal vesicle breakdown（GVBD）（24h: 94.41±3.41 % Vs.23.73±1.55 %,28h:100.00±0 % Vs.25.01±4.44 %,32h: 100.00±0 % Vs.40.35±6.72 %）,first polar body（PB1）extrusion rate（80.60±1.28 % Vs.13.95±1.41 %）of cumulus-oocyte complexes（COCs）and development efficiency of parthenogenetically activated embryos（cleavage rate: 85.52±4.41 % Vs.43.69±9.97 %,blastocyst rate: 28.70±2.07 % Vs.0±0 %）were significantly reduced in the treatment group compared with the control group.In contrast,cumulus cell-removed oocytes were exposed to the OSS₁28167.We found that PB1 emission rate of oocytes was significantly decreased compared with the control group.However,inhibition of SIRT6 derived from cumulus cells and oocytes disrupted spindle and chromosome formation.In addition,SIRT6 inhibition in cumulus cells significantly reduced the expression of genes associated with cumulus expansion and gap junction formation,and decreased phosphorylated CDK1 levels in oocytes.Finally,inhibition of adenylate cyclase activity partially restored the GVBD（23.97±7.57 % Vs.57.28±12.06 %）and PB1 extrusion rates（12.77±2.56 % Vs.35.99±4.06 %）of SIRT6 inhibitor-treated COCs.In conclusion,these results demonstrated that SIRT6 was involved in regulating porcine oocyte maturation.SIRT6 expression in cumulus cells regulated the expansion of cumulus cells and the GVBD of oocyte,while SIRT6 expression in oocytes was involved in regulating the discharge of the first polar body.SIRT6 expression in both cumulus cells and oocytes was involved in regulating the formation of spindles and chromosomes during egg maturation.SIRT6-mediated adenylate cyclase activity regulated meiosis initiation and maturation of porcine oocytes.