Cumulus Granulosa Cells Promote Oocyte Maturation In Vitro By Crotonylation

【Objective】 To explore the differences in overall crotonylation modification and regulatory enzymes of cumulus granulosa cells in different stages of oocyte in vitro maturation,and then to explore the influence of crotonylation modification on oocyte in vitro maturation,so as to provide research basis for improving oocyte culture system in vitro.【Methods】1.Comparing crotonylation modification enzymes(EP300,CBP,SIRT1,SIRT2,SIRT3,HDAC3 and CDYL)of cumulus granulosa cells in different stages by GEO database.2.Through the collection of clinical samples,dot blotting was used to detect the overall difference in crotonylation modification of cumulus granulosa cells in the oocytes at different stages in vitro maturation;Q-PCR was used to detect the differences of crotonylation modification enzymes(EP300,CBP,SIRT1,SIRT2,SIRT3,HDAC3,CDYL)of cumulus granulosa cells at different stages;and the correlation with each other;3.The primary cultured cumulus granulosa cells were treated with sodium crotonate,and the effects of sodium crotonate on the level of crotonylation and the EP300 and CBP of crotonyltransferase were detected by dot blotting、PCR and WB.CCK8 was used to detect the effect of crotonylation on the proliferation of cumulus granulosa cells and 293 T cell lines.WB assay were used to detect the effect of crotonylation on apoptosis of granulosa cells and 293 T cell lines.PCR was used to detect the expression of extended genes of cumulus(HAS2,PGR,PTGS2,PTX3,TNFAIP6).4.Comparing the maturation rate of oocyte between experimental group and control group by IVM model of mice.【Results】 1.The statistical analysis results of GEO database showed that the expression of crotonylated hydratase(CDYL)in MII phase was significantly lower than that in GV phase,indicating that there were differences in the expression of cumulus granulosa cells at different stages of oocyte.2.Cumulus granulosa cells at different stages of oocyte were collected.The results of dot blotting showed that the overall level of crotonylation was gradually increased in mature cumulus granulosa cells.The expression of EP300 was positively correlated with the expansion of the cumulus and the level of crotonylation.3.The primary cultured cumulus granulosa cells were treated with sodium crotonate.The results of dot blotting and PCR showed that sodium crotonate could promote the increase of overall crotonylation level and the up-regulation of EP300 expression in cumulus granulosa cells.WB results showed that crotonylation could promote the proliferation of cumulus granulosa cells and inhibit their apoptosis,similar to the effect on 293 T cells.Moreover,it can up-regulate the expression of follicular extension gene,accelerate the cumulus granulosa cell expansion,and resume the meiotic process of oocytes.4.The maturation rate of oocytes in the crotonylation group was significantly higher than that in the control group in mouse IVM model,suggesting that crotonylation can promote oocyte maturation in vitro.【Conclusions】 In the process of oocyte in vitro maturation,cumulus granulosa cells corresponding to oocytes at different stages enhance the level of crotonylation modification by upregulation of crotonyltransferase EP300,accelerate the expansion of cumulus,and promote oocyte maturation in vitro.