Mechanism Study Of Chicken PGCs Formation Regulated By The Coordination Of Oxidative Phosphorylation And Methionine Metabolism.

Primordial germ cells(PGCs)are the only gamete cells,which play an important role in the pRotection and breeding of livestock and poultry genetic resources.Poultry PGCs can migrate to the gonads and settle in the blood,which makes the migration path of PGCs an effective way to realize the recovery of poultry species and also provides a good cell material for the conservation of poultry resources.However,the amount of PGCs isolated in vivo is small and difficult to be cultured for a long time.The application and popularization of PGCs in poultry resource conservation and livestock production are severely restricted.Therefore,the quantitative source of PGCs is the fundamental problem that limits the application of PGCs.The transcriptomic,metabolome and DNA methylation sequencing of ESCs and PGCs were conducted in the previous stage,and the combined analysis results showed that the level of oxidative phosphorylation was significantly increased in PGCs,and the metabolites methionine and Wnt9A genes were also highly expressed,suggesting that oxidative phosphorylation,methionine and Wnt9A might be involved in the regulation of PGCs formation.In this study,the role of oxidative phosphorylation,methionine and Wnt9A in PGCs formation was systematically studied from the point of view of cell energy metabolism,metabolites and key genes.On this basis,WB,Chip-qPCR and dual luciferase reporting system were used to analyze the regulatory mechanism of methionine-mediated H3K4me2 modification involved in regulating the expression of target gene Wnt9A and ultimately affecting the formation of PGCs.Specific experimental results are as follows:(1)Exploring the function of cell energy Metabolism in PGCs formation based on Transcriptomics In order to clarify the change mode of cell energy metabolism during the differentiation of ESCs into PGCs,the transcriptome data of chicken ESCs and PGCs were analyzed in this study,and the results showed that:The key genes regulating oxidative phosphorylation pathway(Idh,Ndufa10,Cox4il)are significantly up-regulated in PGCs,while the key genes regulating glycolysis pathway(Pkm,Ldh,Glut)are significantly down-regulated,and the qRT-PCR detection results are also consistent with the sequencing analysis results.These results suggest that the cell energy metabolism pattern may change from glycolysis to oxidative phosphorylation during the differentiation of chicken ESCs to PGCs.At the same time,the mitochondrial membrane potential level in PGCs was significantly higher than that in ESCs,while the lactic acid accumulation level was significantly lower than that in ESCs(P<0.01),which further confirmed that the cell energy metabolism mode changed from glycolysis to oxidative phosphorylation during the differentiation of ESCs into PGCs.In addition,this study also added 2-DG(glycolysis inhibitor,promoting oxidative phosphorylation level)and Rotenone(Rote,oxidative phosphorylation inhibitor,promoting glycolysis level)on the basis of the previously established differentiation system of ESCs induced by BMP4 to PGCs.It was found that the addition of 2-DG significantly increased the DDX4-positive cell rate at 6d induction(P<0.01),indicating that the level of oxidative phosphorylation can promote the formation of PGCs.(2)Screening key metabolites involved in PGCs formation and verifying their functions based on metabolomics.In the process of ESCs formation to PGCs,the metabolic mode of cells changes from glycolysis to oxidative phosphorylation,and which metabolites are involved in this process.Therefore,based on the previous metabolome sequencing results,The methionine involved in the regulation of this process was screened by KEGG analysis and related literature reports.The differentiation of ESCs into PGCs was induced by BMP4 induction system,and different concentrations of methionine were added to determine the function of methionine in the formation of PGCs.Through cell morphology observation,qRTPCR detection of PGCs marker genes,indirect immunofluorescence detection and flow cytometry,it was found that adding 0.4mM methionine could promote the induction and differentiation of ESCs into PGCs.(3)Explore the regulatory relationship between oxidative phosphorylation and methionine in the formation of PGCs.In order to explore the relationship between oxidative phosphorylation and metabolite methionine in promoting the formation of PGCs,BMP4 induction system was used to add 0.4mM methionine,respectively,and promote or inhibit oxidative phosphorylation.During induction,morphological observation of cells,expression of PGCs specific marker genes detected by qRT-PCR,indirect immunofluorescence and flow cytometry were used to explore the regulatory relationship between PGCS and PGCS.(4)Screening and Functional verification of target gene Wnt9A,which is involved in the regulation of PGCs formation by methionine Since oxidative phosphorylation depends on methionine to promote PGCs formation during the differentiation of ESCs into PGCs,do the two function through the regulation of key genes involved in PGCs formation?Therefore,based on the results of metabolomics and transcriptomic sequencing,Wnt9A,a gene involved in germ cell genesis and stem cell differentiation,was screened out and highly expressed in PGCs.The interference vector and overexpression vector of Wnt9A were constructed.Fluorescence observation and qRT-PCR detection results showed that shRNA2-Wnt9A had the best interference effect.The interference vector and overexpression vector of Wnt9A were transfected into ESCs by BMP4 induction system.The specific marker genes of PGCs were detected by cell morphology,qRT-PCR,indirect immunofluorescence and flow cytometry.It was found that Wnt9A could promote the differentiation of ESCs into PGCs.(5)Analysis of the mechanism of methionine’s involvement in regulating the expression of Wnt9A and promoting the formation of PGCs In order to explore how the metabolite methionine participates in the regulation and expression of Wnt9A to promote the formation of PGCs,different concentrations of methionine were added into the medium of PGCs.After 48h of culture,cells were collected to extract pRoteins.The histone H3K4me2 antibody was used to detect WB,and different concentrations of methionine could change the expression level of H3K4me2 in cells.In order to further clarify the mode of methionine regulation of Wnt9A,the double luciferase reporter vector of Wnt9A promoter region was constructed in this study.Cells transfected with the double luciferase reporter vector of Wnt9A promoter region were continued to be cultured in different concentrations of methionine medium for 48h,and the cells were collected for double luciferase activity detection.Methionine significantly up-regulated the transcription level of Wnt9A(P<0.05).Chip-qPCR showed that H3K4me2 could bind to the Wnt9A promoter region.The concentration of H3K4me2 in Wnt9A promoter region decreased after transfection with shMLL2,but increased after transfection with shMLL2.Further double luciferase assay showed that shMLL2 significantly down-regulated Wnt9A initiation activity(P<0.05).shLSD1 significantly increased the activation activity of Wnt9A(P<0.05).These results indicate that methionine can affect the expression of Wnt9A and promote the differentiation of ESCs into PGCs by regulating the H3K4me2 modification level of Wnt9A promoter.