The Role Of DCN In Porcine Follicular Atresia And Granulosa Cell Apoptosis

There are a large number of follicles in the primitive follicular library of mammals,but only a few of follicles can eventually mature and ovulate,and most of the follicles in each stage of development will be atretic and eventually degenerate.Follicular atresia directly affects the development of mammalian reproductive potential.However,the molecular mechanism of follicular atresia is not fully understood.To parse the molecular mechanism of pig follicular atresia,using RNA-seq technology,this paper builds the pig health early ovarian follicles and atresia of follicles transcriptome expression spectrum,using PCR amplification of pig follicular atresia DCN related gene coding sequence,using bioinformatics method analysis of the characteristics and nature of the protein pig DCN gene sequences,use the technical analysis such as RNAi and express the DCN in pigs,the effect and mechanism of follicular granulosa cell apoptosis.The results of this study have some significance for the analysis of the potential molecular mechanism of porcine follicular atremia and granulosa cell apoptosis.The research results of this paper mainly include the following four aspects:(1)Transcriptomics analysis of healthy follicles and early atresia follicles in pigsA total of 1,157 differentially expressed genes were identified in healthy follicles and early atretic follicles by RNA-seq,among which 660 genes were significantly up-regulated and 497 genes were significantly down-regulated.Ten differentially expressed genes were selected for qPCR verification,and the results were highly consistent with those of RNA-seq.GO functional enrichment analysis found that differentially expressed genes were enriched in 212 cell components,biological processes and molecular functional items,among which Wnt signaling pathway,hypoxia response,organel components and calcium ion binding were related to ovarian function and follicular atremia.Enrichment analysis of KEGG pathway found that differentially expressed genes were significantly enriched in 25 signaling pathways,among which TGF signaling pathway and steroid synthesis pathway were related to ovarian function and follicular atresia.The analysis of the cross-regulation network of differentially expressed genes encoding proteins revealed 15 key genes,among which TGF-1,VEGFA and IGF-1 were candidate genes for mammalian fertility traits.The discovery of these key genes and important signaling pathways lays the foundation for the molecular mechanism of porcine follicular atresia.(2)Amplification and bioinformatics analysis of porcine follicular atresia candidate gene DCNDCN gene of TGF signaling pathway was selected among differentially expressed genes for further study.Correlation analysis showed that the mRNA level of DCN gene in porcine follicles was significantly positively correlated with the P4/E2 ratio,an important indicator of follicular atresia(P=0.0144,r=0.4053),indicating that DCN was a candidate gene for porcine follicular atresia.The DCN coding region of pig was obtained by PCR amplification and sequencing,with the full length of 1,083 bp and the coding protein containing 360 amino acids.Sequence alignment showed that the DCN coding region and amino acid sequence of the coding protein were homologous with those of other mammals.It was found that the relative molecular weight of pig DCN protein was 39.89 kDa and the theoretical isoelectric point was 8.84.It was a basic protein with stable physical and chemical properties.It was found that pig DCN protein contains 13 helices,9 folds and 22 random curls.Domain analysis showed that porcine DCN protein contained 1 glyosaminoglycan attachment site(GAS),2 cysteine residues clusters,1 leucine-rich repeat n-terminal domain(LRRNT)and multiple leucine-rich repeat sequences(LRR).The amplification,sequence characteristics and physicochemical properties of pig DCN gene provide scientific basis for studying its function and mechanism.(3)DCN is a pro-apoptotic gene of porcine follicular granulosa cellsIn order to analyze the role of DCN gene in porcine follicular atremia,the expression of endogenous DCN gene in porcine follicular granulocyte cultured in vitro was knocked down by small interfering RNA(siRNA).Flow cytometry(FACS)detection showed that the apoptosis rate of granulocyte cells was significantly reduced.Meanwhile,the eukaryotic expression vector of DCN gene was constructed in this paper and transfected into porcine follicular granulosa cells.FACS detection showed that the apoptosis rate of porcine follicular granulosa cells was significantly increased after DCN overexpression,indicating that DCN could promote porcine follicular granulosa cells apoptosis.In addition,qPCR showed that DCN overexpression could significantly up-regulate the expression of pro-apoptotic gene BAX in porcine follicular granulosa cells,while the expression of anti-apoptotic gene BcI-2 was not significantly changed.Western blotting found that expression levels of apoptosis marker cleaved caspase-3 were significantly up-regulated after DCN overexpression.These results confirmed that DCN gene is a pro-apoptotic gene in porcine follicular granulosa cells.(4)DCN is an inhibitor of the TGF signaling pathway in porcine follicular granulosa cellsPrevious studies have confirmed that DCN is an inhibitor of TGF-signaling pathway.In order to analyze the effect of DCN on TGF-signaling pathway in porcine follicular granulosa cells,Western blotting was used to analyze the protein levels of SMAD3(P-Smad3),the marker protein of TGF signaling pathway,in porcine follicular granulosa cells after DCN was knocked down and overexpressed.The results showed that the total SMAD3(T-SMad3)protein level in porcine follicular granulosa cells did not change significantly after DCN was knocked down,but the[P-Smad3 protein expression level increased significantly.In contrast,there was no significant change in T-Smad3 protein level in porcine follicular granulosa cells after DCN overexpression,while P-Smad3 protein expression level was significantly decreased,indicating that DCN as a negative regulator inhibited the activity of TGF signaling pathway in porcine follicular granulosa cells.In conclusion,transcriptomic expression profiles of healthy and early atresia follicles in pigs were constructed,and key genes and important signaling pathways of atresia in pigs were identified.The coding region of DCN was amplified,and it was found that DCN is the pro-apoptotic gene and TGF signaling pathway of porcine follicular granulocyte.The results provide a basis for analyzing the molecular mechanism of porcine follicular atresia and granulosa cell apoptosis and provide a potential target for improving sow fertility.