Study On The Regulation Of Orexin A On The Expression Of Progesterone Secretion-Related Genes In Luteinized Granulosa Cells Of Sheep

As an important neuropeptide hormone,Orexin A affects reproductive function by binding G protein coupled receptor.In order to study the effect of Orexin A on the gene expression of sheep luteinized granulosa cells and its signal pathway,reference laboratory established sheep ovarian granular cell culture system for cell culture,joint added follicle-stimulating hormone(FSH),luteinizing hormone(LH),estradiol(E2)luteinizing particles cells,identified by ELISA.The RNA of luteinized granulosa cell group and the luteinized granulosa cell group with Orexin A was extracted and sequenced by Illumina sequencing technology.The transcriptome data were analyzed by bioinformatics.Based on sequencing data quality control,differentially expressed genes were screened,annotated and enriched.The quantitative real-time PCR and Western blot were used to verify the genes with significant differences.PRRT2 screened out may be involved in cGMP/PKG,MAPK.Wnt and other signal pathways to affect the progesterone secretion of luteinized granulosa cells,so further verification of PRRT2.Different concentrations(0.01 ?mol/L,0.1 ?mol/L,1 ?mol/L)of PRRT2 inhibitor(HY-10230)were added to luteinized granulosa cells and those with added Orexin A.The secretion of progesterone in the supernatant was detected by ELISA,and the content of PRRT2 and StAR in luteinized granulosa cells with added HY-10230 and Orexin A was detected by WB.The results showed that the progesterone secretion of the luteinized granulosa cells group was significantly higher than that of the non-luteinized granulosa cells group,indicating that the ovarian granulosa cells had been luteinized.A total of 123 differentially expressed genes were obtained by transcriptome sequencing,of which 54 were up-regulated and 69 down-regulated.Many highly expressed genes related to cell cycle regulation,reproduction and metabolism were found.Combined GO analysis and KEGG pathway analysis indicates that the differential genes were mainly involved in:metabolic pathways,oxidative phosphorylation,cancer signaling pathways,Ras signal pathway,MAPK signaling pathways,NF-kappa B signal pathway,transcriptional regulation,etc.;q-PCR was used to verify these significantly different genes that related to reproduction,metabolism,cell proliferation and apoptosis(PRRT2,ATP8,KIRREL,ID1,SOX4,TBX3),the expression levels of ATP8,PRRT2 and KIRREL increased significantly after adding Orexin A(P<0.05),while the expression levels of ID1,SOX4 and TBX3 decreased significantly after adding Orexin A(P<0.05),which was consistent with the sequencing results.PRRT2 was further verified with WB.The secretion of HY-10230 progesterone was significantly decreased(P<0.01),and the optimal inhibitory concentration was 1 ?mol/L.The expression levels of PRRT2 and StAR protein increased significantly after adding Orexin A(P<0.05),and the expression levels of PRRT2 and StAR decreased significantly after adding HY-10230(P<0.05).The WB results were consistent with the sequencing and q-PCR results,and showed that Orexin A can affect the secretion of progesterone by PRRT2.The experiment provided evidence to clarify the effect of Orexin A on reproductive regulation in sheep.