| Polyamines(putrescine,spermidine,and spermine)are essential components of living cells,and it is involved widely in cell proliferation,differentiation,apoptosis,and regulation of cancer,aging,and reproductive function.Ornithine decarboxylase(ODC)is a key rate-limiting enzyme in the process of polyamine biosynthesis,which catalyzes ornithine decarboxylation to form putrescine and plays a key role in the process of polyamine biosynthesis.However,there are no reports on the regulation of follicular development by ODC,and the mechanism is still unclear.In this study,we constructed ODC gene overexpression vector and synthesized si RNA interfering with ODC gene,and transfected them into primary follicular granulosa cells of Sichuan white goose.The effects of overexpression and interference of ODC gene on polyamine metabolism,hormone level,proliferation and apoptosis of follicular granulosa cells.The main results are as follows:(1)The vector for overexpressing the ODC gene in geese was successfully constructed,and si RNA sequences interfering the ODC gene expression were designed and synthesized.After the interference of ODC gene expression for 24 h,48 h and 72 h,ODC gene expression in granulosa cells decreased significantly,and ODC protein level increased significantly at 24 h and 48 h(P < 0.05).After overexpression of the ODC gene for 24 h,48 h and 72 h,ODC gene expression and protein level in goose ovarian granulosa cells increased significantly(P < 0.05).After interfering with the expression of ODC gene,the granulosa cell activity was significantly lower than that of the control group(P < 0.05).However,when the ODC gene was overexpressed,the goose follicular granule cell activity was significantly higher than that of the control group(P < 0.05).(2)The expression of OAZ1,OAZ2,AZIN,SPMS,SMO,APAO,and SSAT genes increased significantly after interfering ODC gene for 24 h,and the expression of SPDS and SAMDC were significantly decreased(P < 0.05).There was no significant difference in SPMS and SAMDC expression at 48 h(P > 0.05);At 72 h,the expression levels of AZIN1,SMO,APAO,and SAMDC were significantly increased(P < 0.05).When the ODC gene was overexpressed for 24 h,the expression levels of OAZ1,SPDS,AZIN1,SMO,and SSAT were significantly increased,while the expression of SAMDC was significantly decreased(P < 0.05).At 48 h,the expression of SPMS was significantly increased,and the expression of OAZ2 was significantly decreased(P < 0.05).At 72 h,the expression levels of OAZ1,AZIN1,SPMS,and SSAT were significantly increased,and the expression of APAO was significantly decreased(P < 0.05).(3)The content of putrescine in granulosa cells that interfered with ODC expression was significantly decreased(P < 0.05);the concentration of putrescine in granulosa cells overexpressing ODC was significantly increased(P < 0.05).(4)When the expression of ODC gene was disturbed for 48 h,the E2 content in the medium was significantly lower than that in the control group(P < 0.05).At 24 h,the expression of PR was significantly lower than that in the control group(P < 0.05).When the ODC was overexpressed for 24 h,the P4 content in the medium was significantly lower than that in the control group(P < 0.05).At 48 h,the P4 content was significantly higher than that in the control group(P < 0.05).ER and PR expression were significantly higher than the control group(P < 0.05).The expression of PR was continuously increased at 72 h(P < 0.05).(5)The expression levels of SMAD1,PCNA,CCND1,and CASPASE 9 were significantly decreased after interfering the ODC gene granulosa cells at 24 h,and the expression of BAX,CASPASE 3 and CASPASE 8 increased significantly(P < 0.05).At 48 h,the expression of BAX,CASPASE 8 and CASPASE 9 were significantly increased,and the expression of CCND1 gene was significantly decreased(P < 0.05).After 72 h,the expression of CCND1,PCNA and SMAD1 were significantly decreased,while the expression of BAX and CASPASE 3 were significantly increased(P < 0.05).After overexpression of ODC gene,the expression levels of SMAD1 and CCND1 were significantly increased at 24 h,and BAX was significantly decreased(P < 0.05).At 48 h,the expression of SMAD1,CCND1 and BCL-2 were significantly increased,while the PCNA and CASPASE 8 were significantly decreased(P < 0.05).At 72 h,the expression levels of SMAD1 and CCND1 were significantly increased,and the CASPASE 8was significantly decreased(P < 0.05).In conclusion,ODC not only regulates ovarian function by mediating P4 secretion and rec eptor gene expression in follicular granulosa cells,but also regulates proliferation and apoptosi s of granulosa cells by mediating SMAD1,CCND1,BCL-2 and BAX gene expression. |
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