Effect Of DNMT1 On The Functions Of Ovarian Granulosa Cells In Pigs

The follicles are the basic structural and functional units of porcine ovaries,and their main physiological functions are ovulation and secretion of sex hormones that required by the body.In the process of follicle growth and development,excessive apoptosis of granulosa cells in follicles will lead to the phenomenon of atresia in follicles,and then affect the reproductive production capacity of pigs.DNA methylation is that the free CH₃is transferred to specific bases of DNA molecules under the catalysis of DNA methyltransferases（DNMTs）,and then regulates gene transcription and expression.The DNMT1 is a main role for DNA methylation formation in mammals.So we aimed to study the effect of DNMT1 on granulosa cell function in sows.Firstly,we used DNMTs inhibitor5-Aza-CdR to treat porcine ovarian granulosa cells（pGCs）.Secondly,we used experimental techniques such as qPCR,EdU,Annexin V-FITC,BSP to investigate the effects on pGCs proliferation,apoptosis,cell cycle,E2 synthesis ability after inhibiting DNMT1 gene expression.Finally,we used qPCR and BSP to investigate the key gene of the related signaling pathways mRNA expression and DNA methylation level after inhibiting DNMT1gene expression.The main results of this study are as follows:（1）The inhibition of DNMT1 expression up-regulates the expression level of Caspase-3、Caspase-8、BAX、BAK、BIM genes mRNA and down-regulates the expression level of MCL-1 gene mRNA,and then promote apotheosis of pGCs.（2）The inhibition of DNMT1 expression down-regulates the expression levels of PCNA、CDK1、CDK2、CCNA1、CCND1 genes mRNA,and then inhibits proliferation progression and cell cycle of pGCs.（3）The inhibition of DNMT1 expression down-regulates the expression level of CYP19A1、HSD17B1 genes mRNA,and then inhibits the E2 synthesis and secretion of pGCs.（4）The inhibition of DNMT1 expression reduces the DNA methylation level of apotheosis signaling pathway genes Caspase-3、BAK promoters,proliferation and cell cycle signaling pathway genes PCNA、CCNA1 promoters.Based on the above results,the main conclusion of this study is that the inhibition of transcription expression of DNMT1 may down-regulate the DNA methylation level of Caspase-3 and BAK genes promoters and up-regulate the mRNA expression of Caspase-3and BAK genes,thus promote apoptosis of pGCs.The inhibition of transcription expression of DNMT1 may down-regulate DNA methylation level and down-regulate the mRNA expression of PCNA、CDK1、CDK2、CCNA1、CCND1 genes and down-regulate the DNA methylation level of PCNA and CCNA1 genes promoters,and then inhibit the process of cell proliferation and cell cycle in pGCs.The inhibition of transcription expression of DNMT1 may down-regulate the mRNA expression of CYP19A1 and HSD17B1 genes,and then inhibit the synthesis and secretion of E2 in pGCs.This results can play a theoretical reference for deeply exploring DNMT1 regulats the mechanism of ovarian granulosa cell function and follicular development.