| Notch signaling pathway is widely distributed in metazoan animals and has attracted much attention in regulating cell proliferation in recent years.Notch2,a member of the Notch family,plays an important role in granulosa cell(GCs)proliferation and follicular development.However,the regulatory mechanism of Notch signaling pathway has been few reported on luteinized granulosa cells(LGCs)during ovulation and atresia of follicles,and the regulatory mechanism of Notch2 on bovine follicular development remains to be further studied.In addition,bone morphogenetic protein 4(BMP4)is one of the most important members of the BMP family and plays a key role in follicular development.Notch and BMP/Smad signaling pathway interact with each other in varieties of cellular processes.However,their cross-talk in reproduction have not been clarified completely.Firstly,the segmented CDS region of bovine Notch2 gene was cloned and analyzed with bioinformatics methods after spliced.The localization of Notch2 receptor and its ligand Jagged of Notch signaling pathway in bovine antral follicles and LGCs were detected respectly by immunohistochemistry and cellular immunofluorescence assays.Then the expression of genes related to cycle,apoptosis,steroidogenesis and antioxidant level were detected after LGCs silenced or overexpressed Notch2.The cell viability was detected by cck-8,and steroidogenesis was detected by ELISA.Additionally,the expression of key genes of BMP/Smad signaling pathway was detected after LGCs silenced or overexpressed Notch2.Meanwhile,the expression of Notch signaling key genes were detected after LGCs were treated with BMP4.The results are as following:(1)The length of bovine Notch2 gene complete CDS region is 7416 bp,encoding 2471 amino acids.Notch2 protein is an unstable hydrophilic transmembrane protein with a molecular weight of 265309.51 and an isoelectric point of 4.95.The α-helix accounted for 15.22%,extended strand accounted for 9.42%,β-turn accounted for 5.27% and random coil accounted for 70.09% in the protein secondary structure of Notch2.The cleavage site of signal peptide was found between the 25~26 amino acids and the signal peptide type was Sec/ SPI.There are 5 potential N-glycosylation sites and 203 potential phosphorylation sites.Bovine Notch2 protein includes 19 calcium-binding EGF-like domains,3 LNP domains,2 Notch proteins,1 ankyrin repeats,1 domain of unknown function,and 1 ankyrin-like protein.Homology alignment of genes showed that it has the closest relationship with water buffalo and farthest relationship with rodent.(2)After bovine follicular LGCs Notch2 silenced,the mRNA expression of cell cycle gene Cyclin D2 had no significant change(P>0.05),CDK4 decreased(P<0.05),cell cycle suppressor gene P21 increased(P<0.05),apoptosis gene Bcl-2/Bax decreased(P<0.05),apoptosis executive gene Casepase3 increased(P<0.05),and the cell viability significantly decreased(P<0.05).In addition,silencing Notch2 had no effect on the secretion of estradiol(E2)and m RNA expression of CYP19A1(P>0.05),but the secretion of progesterone(P4)and the m RNA expression of STAR,3β-HSD decreased(P<0.05).After bovine follicular LGCs Notch2 overexpressed,the m RNA expression of cell cycle gene Cyclin D1,Cyclin D2,CDK4 and PCNA increased(P<0.05),there was no significant difference in CDK1 m RNA expression(P>0.05),steroidogenesis gene CYP11A1,STAR and 3β-HSD increased(P<0.05),antioxidant gene SOD and CAT increased(P<0.05).(3)Key genes of BMP/Smad signaling pathway were detected after bovine follicular LGCs silenced or overexpressed Notch2.After Notch2 was silenced,the m RNA expression of BMP signaling downstream factors Smad1,Smad5,Smad8 and phosphatase PPM1 A were down-regulated(P<0.05),Smad4 decreased but there was no statistically difference(P>0.05),membrane receptor ALK3 had no difference(P>0.05).After Notch2 was overexpressed,the m RNA expression of BMP signaling membrane receptor ALK3,downstream factors Smad1,Smad4,Smad5,Smad8 and phosphatase PPM1 A all increased(P<0.05).Moreover,bovine follicles LGCs were treated with FSH and different concentrations of BMP4,and results showed that 2 ng/m L BMP4 down-regulated the m RNA expression of Smad1,but there was no significant difference(P>0.05).FSH promoted the inhibition of Smad1 by 2 ng/m L BMP4(P<0.05).Similarly,steroidogenesis gene STAR and CYP11A1 were down-regulated in LGCs treated with BMP4 and FSH(P<0.05).Adding FSH alone inhibited the m RNA expression of Notch intracellular domain 2(NICD2)(P<0.05),but activated the m RNA expression of Hes1(P<0.05).The addition of 1~2 ng/m L BMP4 inhibited the m RNA expression of Hes1 induced by FSH(P<0.05).In the presence or absence of FSH,5ng/m L BMP4 promoted the m RNA expression of NICD2 compared with 2 ng/m L BMP4(P<0.05),but there was no statistical difference in the promotion of target gene Hes1 m RNA(P>0.05).In conclusion,Notch2 promote proliferation,P4 secretion,antioxidant effect and inhibit apoptosis in bovine follicles LGCs,but it has no effect on E2 production.Notch2 activate BMP signaling,while BMP4 inhibit P4 secretion and Notch downstream signaling in the presence of FSH. |
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