The Involvement Of NR4A1 In The Mechanisms Of Porcine Ovarian Granulosa Cells Differentiation And Granulosa-Lutein Cells Regression

Porcine corpus luteum(CL)develops from ovarian follicles.Follicular granulosa cells(GCs)could differentiate into granulosa-lutein cells(GLCs),and the latter secrete progesterone(P4)which is essential for porcine estrus and pregnancy.NR4A1,a rapid response gene and transcription factor,can be rapidly up-regulated by multi stimuli and involved in life activities.NR4A1 has been proved to be expressed in porcine luteal steroidogenic cells,and PGF2a induced luteal NR4A1 expression in rats,suggesting NR4A1 might be associated with the formation and regression of porcine CL.In this study,based on differentiated GCs(GLCs)model,techniques such as RNAi,RIA,CCK-8,RT-qPCR,WB were applied to investigate the effect of NR4A1 in GCs differentiation and GLCs regression,which will provide a theoretical basis for future study on the formation and regression of CL.The main contents are as follows:1.Establishment of porcine differentiated GCs model in vitroTo establish a model for in vitro differentiation of porcine ovarian GCs,porcine follicular(5～8 mm)GCs were isolated and pre-cultured for 6 d(70%～80%aggregation),then the Forskolin(FSK,10 nmol·L-1)was added into the medium.48 h later,cell morphology,proliferation,and gene expressions of luteal markers were evaluated;the P4 and steroidogenic proteins levels were measured during 0～96 h.Results showed that FSK changed cell morphology,enlarged cell bulk,decreased cell proliferation activity(P<0.01).RT-qPCR results showed FSK decreased gene expression of GCs marker(FSHR)and increased gene expression of luteal markers(PTGFR,LHCGR)(P<0.01).RIA and WB results showed FSK increased P4 synthesis and steroidogenic proteins(StAR,CYP11A1,HSD3B)expression levels(P<0.01).All these results indicate FSK could change GCs morphology,inhibit cell proliferation,enhance P4 anabolism,and promote GCs to differentiate into luteal cells.2.Study on the involvement of NR4A1 in the differentiation of porcine GCsTo investigate the involvement of NR4A1 in GCs differentiation,pre-cultured porcine GCs(6 d)were divided into the control group,FSK group,siNR4Al group,and FSK+siNR4A1 group.After 48 h of treatment,the changes of GCs proliferation,lipid droplets(LDs)and P4 synthesis were detected.CCK-8 results showed GCs proliferation activity decreased in FSK group(P<0.05),but not in FSK+siNR4A1 group.ORO staining results showed FSK promoted LDs accumulation;whereas compared with FSK group,LDs content were significantly decreased in FSK+siNR4A1 group(P<0.05).RIA and WB results showed FSK up-regulated P4 and P4 synthesis-related proteins levels,decreased E2 and CYP19A1 protein levels(P<0.05),which was reversed by siNR4A1.RT-qPCR results showed FSK decreased Cyclin B1/D1 and CDK1/2 mRNA expression,increased P21/P27 mRNA expression(P<0.05),but adverse results were obtained when NR4A1 expression were inhibited.In summary,NR4A1 is involved in GCs differentiation,and inhibition of NR4A1 expression slowed GCs differentiation process,which provide a basis for further study of the molecular mechanism of porcine ovarian luteal formation.3.Study on the involvement of NR4A1 in PGF2α-regulated apoptosis and progesterone synthesis of porcine GLCsTo investigate the involvement of NR4A1 in PGF2α-regulated porcine luteal cells regression,follicular GCs were isolated and pre-cultured for 6 d,then the GLCs model was established by FSK.After that,GLCs were divided into the control group,PGF2α group,siNR4A1 group,and PGF2α+siNR4A1 group.After 0～24 h of treatment,the changes of P4 synthesis,apoptosis-related proteins and signaling pathway proteins expressions were detected.CCK-8 results showed PGF2α reduced GLCs viability(P<0.05),which was reversed by siNR4A1.RIA results showed P4 level was higher in PGF2a+siNR4A1 group than that in PGF2α group(P<0.01).WB results showed P4-related proteins expression in PGF2α+siNR4A1 group were higher than those in PGF2α group(P<0.05);ATF3,phospho-MAPKs,NF-κB and apoptosis-related proteins expression were lower in the PGF2α+siNR4A1 group than those in the PGF2α group(P<0.05).Thus,NR4A1 might promote PGF2α-regulated apoptosis and P4 decline through MAPKs/ATF3 and NF-κB pathways in GLCs,which provide a basis for further study of the molecular mechanisms of porcine luteal regression.In summary,NR4A1 is involved in the regulation of porcine GCs differentiation and GLCs regression through cell proliferation,apoptosis,steroidogenesis pathways,which provide a theoretical basis for further understanding of the mechanism of porcine luteal formation and regression.