Mechanism Of Neddylation On The Function Regulation Of Follicular Granulosa Cells In Sheep

The normal development of ovarian follicles is key to the reproductive process in which granulosa cells(GCs)play an important role.The ubiquitin-like protein(UBL)neural precursor cell expressed developmentally downregulated protein 8(NEDD8)is related to Neddylation,a process in which substrates conjugate.similar to ubiquitylation,Neddylation involves three enzymatic complexes: NEDD8 activating enzyme E1(NAE1),conjugating enzyme E2(UBE2M/UBE2F),and E3 ubiquitin ligases,participating in the regulation of protein stability and function.Neddylation is an important protein post-translational modification,which plays a crucial regulatory role in oocyte maturation,embryonic development and blastocyst hatching.However,there are few studies concerning the mechanism of action associated with Neddylation on sheep follicular GCs.Therefore,RNA-seq,proteomics,transmission electron microscopy(TEM),RTCA,flow cytometry and other technical methods were used to explore the effects of Neddylation on the proliferation,apoptosis and steroid hormone synthesis in sheep follicular GCs.The main research contents and results are as follows:1.Expression and localization of NEDD8 in sheep follicular GCsThe current study demonstrated that NEDD8 was strongly expressed in sheep ovarian follicular granulosa cells by immunohistochemistry and immunofluorescence techniques.In the process of culturing sheep follicular GCs,GCs were transfected with Neddylation specific inhibitor MLN4924 in vitro,MTT results showed that MLN4924 progressively attenuated the proliferation of GCs at concentrations of 0.5,1,5,and 10 μM(P < 0.01).x CELLigence RTCA S16 instrument was used to monitor the cell index in real time,after adding MLN4924 for 48 h,the cell index of the 1 μM group was significantly lower than that of the control group(P < 0.05),whereas the 5 and 10 μM groups displayed more significantly cytotoxic effects against sheep follicular GCs than in the control group(P < 0.01).Western blotting results showed a significant reduction in neddylated cullins(NEDD8-cullins)and Cullin 1 was observed at 1 μM with increasing concentrations of MLN4924(P < 0.05).Three NEDD8 small interfering RNA(NEDD8-si RNAs)were synthesized,and the results of q RT-PCR and western blotting showed that the expression of NEDD8-Cullins was significantly lower in NEDD8-si RNA1 after transfection at 48 h(P < 0.01).2.High throughput transcriptome and proteome sequencing analysis in sheep follicular GCsIn this study,high-throughput sequencing technology was used to analyze the transcriptome and proteome of sheep follicular GCs transfected with MLN4924(1 μM)and si-NEDD8,its main aim was to screen the key signal pathways,differentially expressed genes and proteins involved in the effects of Neddylation on the functions of sheep follicular GCs.Transcriptome sequencing GO and KEGG enrichment analysis revealed that differentially expressed genes were mainly involved in biological processes such as ovarian steroid biosynthesis,cell senescence and apoptosis.Proteomic KEGG enrichment analysis of different expressed proteins found that fatty acid and amino acid metabolism,tricarboxylic acid cycle,Wnt,PI3K/Akt/FOXO,steroid hormone synthesis,and cell cycle pathways were significantly enriched.Transcriptome and proteome association analysis further indicated that fatty acid and amino acid metabolism,tricarboxylic acid cycle,steroid hormone synthesis,cell cycle signaling pathways were involved in the regulation of sheep follicular GCs functions.Transcriptional and proteomic analysis laid the foundation for the subsequent study of the effects of Neddylation on the key signaling pathways and functions of sheep GCs.3.Effects of Neddylation on Akt/FOXO,JAK2/STAT3,TGF-β/Smad and Wnt pathway in sheep follicular GCsThe effects of Neddylation inactivation on the related genes of key signaling pathway were evaluated.Neddylation inactivation by MLN4924 increased the levels of phospho-Akt,JAK2,phospho-STAT3,and FOXO1(P < 0.05),and decreased the levels of phospho-FOXO3 a and STAT3(P < 0.05),but Neddylation inactivation by si-NEDD8 decreased the levels of JAK2,phospho-STAT3(P < 0.01,P < 0.05).Further,Neddylation inactivation could affect the TGF-β/Smad signaling pathway by increasing the levels of Smad1,Smad4,Smad7,Smad9 and TGF-β1 m RNA(P < 0.01,P < 0.05)and Smad7 protein(P < 0.01).After the inactivation of Neddylation,the β-catenin protein level was significantly down-regulated(P < 0.01),and the protein level of Wnt5 A was significantly up-regulated in Wnt signaling pathway(P < 0.05).4.Effects of Neddylation on functions in sheep follicular GCsBased on transcriptome and proteome KEGG analysis and the above key signaling pathways were involved in the regulation of steroid hormone synthesis,cell cycle and other cell functions,the further verifications found that Neddylation inactivation by MLN4924 and si-NEDD8 could significantly inhibit the synthesis of E2 and P4 in sheep follicular GCs(P < 0.01,P < 0.05),meanwhile the expression levels of steroids synthesis key genes and proteins were also significantly down regulated.Neddylation inactivation could attenuate the proliferation of sheep follicular GCs significantly and led cell cycle being arrested at G2/ M phase.The cell cycle related genes cyclin B1 and cdc-2 were significantly down regulated(P < 0.01),but both the m RNA and protein expression levels of Wee1 were significantly upregulated(P < 0.05).The flow cytometry and transmission electron microscopy results elucidated that Neddylation inactivation induced GCs cell apoptosis,the PARP,c-Myc,and NOXA increased significantly(P < 0.01,P < 0.05),whereas Mcl-1 decreased significantly(P < 0.05).In addition,MLN4924 could alter the mitochondrial morphology of GCs,increase cellular glucose utilization and lactate production,increase reactive oxygen species(ROS)generation,and promote sheep follicular GCs glycolysis,thus causing changes in mitochondrial functions.In summary,this study identified the signaling pathways and regulatory effects of Neddylation on sheep follicular GCs by high-throughput sequencing,and further verified that the effects of Neddylation on related genes expressions in PI3K/Akt/FOXO,JAK2/STAT3,TGF-β/Smad and Wnt key signaling pathways,as well as the regulation of cell functions including cell proliferation,cell cycle,apoptosis,steroid hormone synthesis,mitochondria function.Collectively,this study provides a molecular biological foundation for elucidating the mechanism of Neddylation in sheep follicular development.