| In order to understand the difference of cumulus cell transcriptome before and after porcine oocyte maturation,on the basis of comparing the genes related to lipid metabolism between normal ovary and cyst ovary,cumulus cells around oocytes in germinal vesicle stage(GV stage)and second meiotic metaphase(MII)were collected,and the differences of m RNA and micro RNA transcripts in the two stages were compared by RNA sequencing,bioinformatics analysis and in vitro verification.The roles of bone morphogenetic protein 2(BMP2),ssc-mi R-101 and ssc-mi R-155-5p in cumulus expansion were also studied.The results are as follows:1.In order to study the differences of the genes related to lipid metabolism between normal ovary and cyst ovary,ovarian tissues,granulosa cells and oocytes were collected respectively,and the expressions of genes related to fat metabolism were compared by real-time fluorescence quantitative PCR.The results showed that the expression level of FABP3 and CPT1 in cyst ovarian tissue was significantly higher than that in normal ovary tissue,the expression level of FABP3 in granulosa cells of cyst ovary group was significantly higher than that of normal ovary group,and the expression of CPT1 and FABP4 in oocytes of cyst ovary was significantly higher than that of GV and MII oocytes of normal ovary.2.The coding transcripts of cumulus cells around GV phase and M Ⅱ oocytes were sequenced by RNA-Seq,and the candidate genes were verified,and different concentrations of BMP2 were added to the maturation medium to detect the effect of oocyte maturation.The results showed that there were 3 789 differentially expressed genes in cumulus cells around GV oocytes compared to MII oocytes,including 2 085up-regulated genes and 1 704 down-regulated genes,and the candidate differentially expressed genes HAS,PTX3,TNFAIP6,CPT1,CD36,BMP2 and FOXO1 were verified by q PCR,and the results were basically consistent with those of RNA-Seq.The addition of BMP2 with 50 ng/ml to the maturation medium significantly promoted the maturation of oocytes and significantly increased the transcription level of maturation promoting factor MPF in oocytes.However,under the condition of lack of follicular fluid,50 ng/m L BMP2 could promote oocyte maturation and MPF transcription in oocytes at 24 hours after in vitro maturation,indicating that there may be some antagonism between BMP2 and follicular fluid.3.Micro RNA sequencing of cumulus cells around GV phase and M Ⅱ oocytes was carried out by RNA-Seq.After verifying some candidate mi RNA,ssc-mir-101 and ssc-mir-155-5p were selected as research objects,target genes were predicted by target gene prediction website and verified by q RT-PCR,Western blot and luciferase reporter assay,and the role of these two micro RNAs in oocyte maturation was studied.The results showed that compared with the oocytes around MII oocytes,there were 40 differentially expressed micro RNA genes in cumulus cells around GV oocytes,of which 13 genes were up-regulated and 27 down-regulated,and the candidate differentially expressed ssc-mi R-101,ssc-mi R-130b-5p,ssc-mi R-140-5p,ssc-mi R-155-5p,ssc-mi R-let-7f were verified by q PCR,and the results were basically consistent with those of RNA-Seq.The mi RNA target gene prediction website shows that HAS2 and PTGS2 genes might be the target genes of mi R-101,while SOCS1 might be the target genes of ssc-mi R-155-5p.The accuracy of the prediction was verified by luciferase reporter assay.Transfection of mi R-101 and mir-155 mimics in cumulus cells in vitro significantly inhibited the expression of predictive target genes,while the inhibitors promoted the expression of predictive target genes.The oocyte-cumulus cell complex was transfected with ssc-mi R-101 and ssc-mi R-155-5p mimics and inhibitors mediated by liposome.It was found that transfection of mimics could inhibit the expanding of cumulus cells.. |
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