| Different from mammals,chicken Primordial germ cells(PGCs)can enter the vascular network after origin and migrate to the gonadal germ ridge along with the blood circulatory system.Based on the unique migration characteristics of chicken PGCs,which can be further developed into sperm and ovum,chicken PGCs can be genetically modified and xenografted to produce transgenic animals,and even the protection of germplasm resources of endangered birds and the precise breeding of important genetic traits can be realized through this method.Although the lack of access to PGCs in early studies limited its specific application,with the development of sequencing technology,a large number of key genes and signaling pathways affecting the development of PGCs have been mined,thus optimizing the in vitro culture and induction pathways of chicken PGCs and alleviating the dilemma of insufficient PGCs data to some extent.However,new problems are:in the course of specific studies,it is found that there are significant differences in the migration ability of chicken PGCs of different genders in the process of allotransplantation,and there are also significant differences in the germ-line ability of PGCs of different development times in the specific process.In order to further clarify the specific differences of PGCs at different developmental times and between different genders,this study intended to screen the differentially expressed genes of PGCs at the same developmental time points of the same gender(3.5 days,4.5 days and 5.5 days)by transcriptome sequencing technology,and identify the specific differences by bioinformatics analysis.The results of transcriptome sequencing were verified by biological identification of male and female PGCs cultured at different time points,providing technical guidance for further application of PGCs.The results are as follows:(1)Isolation,culture and identification of female and male PGCs in chickens at different timesIn order to obtain PGCs in male and female gonads at different developmental stages for transcriptomic sequencing,C-KIT antibody was used in this study to mark the suspensions of male and female gonads at different developmental points,and the proportion of PGCs in cell suspensions was analyzed by flow cytometry.The results showed that:During development,the proportion of PGCs in male and female gonads was less than 3%,but significantly increased(P<0.01).There was no significant difference in the proportion of PGCs in male and female gonads at the same developmental point(P>0.05).After 10 days of cultured PGCs,the cells showed smooth and clear circular structure with high cell purity.Further flow cytometry showed that the purity of cultured PGCs ranged from 25%to 60%.The identification results of PGCs showed that both male and female PGCs were PAS positive,CVH positive and SSEA-1 positive at different developmental time points.The results of RT-qPCR showed that CVH and PouV were expressed in both male and female PGCs cells of different embryonic ages,which were associated with PGCs reproduction.All these results indicated that male and female PGCs with high purity in different developmental processes were successfully isolated and obtained,which could be used for subsequent RNA extraction and transcriptome sequencing.(2)Differences between male and female PGCs at the same developmental stage were compared based on transcriptome sequencingTo compare the specific differences between male and female PGCs,we extracted total RNA from female and male PGCs cultured for 3.5 days,4.5 days and 5.5 days for transcriptional sequencing and screening of differentially expressed genes.PCA analysis and sample correlation analysis showed that male and female PGCs of the same embryo age were similar but still different.A total of 206 differentially expressed genes(109 up-regulated and 97 down-regulated)were detected between Female3.5 vs Male3.5 PGCs.GO analysis indicated that differentially expressed genes in cell migration(chemotaxis,cell chemotaxis),cell proliferation(cell proliferation,regulation of cell proliferation),cell differentiation(positive)regulation of myoblast differentiation,keratinocyte differentiation)and cell reproductive development(female pregnancy,sperm principal piece,male genitalia development)and other related GO items were significantly enriched.Further analysis showed that the expression of WNT3A,SIX3 and LOC107049174(proliferation-related genes)was higher in females than in males.The expressions of ACKR4 and CXCR4(migration-related genes)were higher in males than in females.The expressions of FHL1 and BRINP3(differentiation related genes)were higher in males than in females.The expression of CORIN,DNAH8 and LOC100859467(genes related to germ cell development)was higher in females than in males.The results of KEGG analysis were consistent with those of GO analysis.These results indicate that the ratio of female PGCs to male PGCs at 3.5d indicates that the reproductive and reproductive related abilities of female PGCs are higher than those of male PGCs,and the migration and differentiation abilities of male PGCs are higher than those of female PGCs.A total of 588 differentially expressed genes(303 up-regulated and 285 down-regulated)were detected in Female4.5 vs Male4.5 PGCs.GO analysis showed that differentially expressed genes were involved in cell migration(chemotaxis,cell chemotaxis),cell proliferation(keratinproliferation),and cell differentiation(cell differentiation)differentiation,transdifferentiation)and cell reproductive development(female pregnancy,sperm principal piece,flagellated sperm motilityf and other related GO items were significantly enriched.Further analysis showed that the expression of SDR16C6 and WNT16(proliferation-related genes)was higher in females than in males.The expressions of ACKR4,AvBD1,AvBD7 and CXCR4(migration-related genes)were higher in males than in females.DDX4,INSM1 and MYOG(differentiation related genes)were expressed more in males than in females.The expressions of SLC26A3 and PRLHRL(genes related to germ cell development)were higher in males than in females.The results of KEGG analysis were consistent with those of GO analysis.These results indicate that the reproductive capacity of female PGCs at 4.5 days is higher than that of male PGCs,and the migration,differentiation and reproductive regulation of male PGCs are higher than that of female PGCs.A total of 1760 differentially expressed genes(514 up-regulated and 1246 downregulated)were detected in Female5.5 vs Male5.5 PGCs.GO analysis results showed differentially expressed genes in cell migration(chemotaxis,cell chemotaxis),cell proliferation(chondrocyte proliferation,cardiac muscle cell)proliferation),cell differentiation(transdifferentiation,myoblast differentiation),and cell reproductive development(female pregnancy,male genitalia)development)and other related GO items were significantly enriched.Further analysis showed that the expression of SDC3 and TENM4(proliferationrelated genes)was higher in males than in females.CXCR4(migration-related gene)expression was higher in males than in females.The expressions of FGF6,WNT11 and INSM1(cell differentiation)were higher in males than in females.The expression of COR1 CRH(genes related to germ cell development)was higher in males than in females.The results of KEGG analysis were consistent with those of GO analysis.These results indicate that the reproductive,differentiation and reproductive related abilities of male PGCs at 4.5 days are higher than those of female PGCs,while the migration ability of male PGCs is lower than that of female PGCs.(3)Transcriptome sequencing was used to compare the differences between PGCs of the same sex at different developmental stagesTo compare the specific differences between male and female PGCs,we extracted total RNA from female and male PGCs cultured for 3.5 days,4.5 days and 5.5 days for transcriptional sequencing and screening of differentially expressed genes.PCA analysis and sample correlation analysis showed significant differences between PGCs samples of different embryo ages.A total of 2731 differentially expressed genes(1228 up-regulated and 1503 downregulated)were obtained from Female3.5 vs Female4.5 PGCs.According to GO analysis,proliferatio,regulation of cell proliferation,cell differentiation,and myoblast are proliferatio of cells The differentiation,osteoclast differentiation and cell reproductive development(male sex determination,sexual reproduction)and other related GO items were significantly enriched.Further analysis showed that the expression of ACE and BMP4(proliferation-related genes)was higher in 3.5 days than 4.5 days.The expressions of CXCR4,PDGFRA,S1PR1 and CCR2(migration-related genes)were higher in 3.5 days than 4.5 days.The expressions of ANKRD2 and MBNL1(differentiation related genes)were higher in 4.5 days than in 3.5 days.The expression of DMRT1(germ cell development related gene)was higher in 4.5 days than in 3.5 days.The results of KEGG analysis were consistent with those of GO analysis.These results indicate that the proliferation and migration of female PGCs at 3.5d is higher than that at 4.5d,and the differentiation and reproductive related regulation at 3.5d is lower than that at 4.5d.A total of 7109 differentially expressed genes(4133 up-regulated and 2976 downregulated)were obtained from Female4.5 vs Female5.5 PGCs.GO analysis indicated that differentially expressed genes function in cell migration(chemotaxis,cell chemotaxis),cell proliferation(cell proliferation,regulation of cell proliferation),cell differentiation(stem cell)differentiation,cell differentiation,germ cell development,male germ cell nucleus and other related GO items were significantly enriched.Further analysis showed that the expression of WNT3 and ACE(proliferation-related genes)was higher in 5.5 days than 4.5 days.The expressions of CXCR4 and PTAFR(migration-related genes)were higher in 4.5 days than in 5.5 days.The expressions of GPM6A,HOXA7 and ZNF750(differentiation related genes)were higher in 4.5 days than in 5.5 days.The expression of DZIP1,REC8 and SYCP1(genes related to germ cell development)in 5.5 days was higher than that in 4.5 days.The results of KEGG analysis were consistent with those of GO analysis.These results indicate that the migration and differentiation ability of female PGCs at 4.5d is higher than that at 5.5d,and the proliferation and reproductive related ability at 5.5d is higher than that at 4.5d.A total of 2780 differentially expressed genes(1273 up-regulated and 1507 downregulated)were obtained from Male3.5 vs Male4.5 PGCs.GO analysis indicated that differentially expressed genes function in cell migration(chemotaxis,cell chemotaxis),cell proliferation(cell proliferation,regulation of cell differentiation),and cell differentiation(regulation)of cell differentiation,cell morphogenesis involved in neuron differentiation)and germ cell development,male germ cell nucleus and other related GO items were significantly enriched.Further analysis showed that the expression of TGFBI,WNT2B and CGTL(proliferation-related genes)was higher in 3.5 days than in 4.5 days.The expressions of CMTM3,CXCR4,DEFB4A and RHOG(migration-related genes)were higher in 3.5 days than in 4.5 days.The expressions of SCG2 and LOC121106433(differentiation related genes)were higher in 3.5 days than 4.5 days.The expressions of BOLL,TDRD1 and REC8(genes related to germ cell development)in 4.5 days were higher than those in 3.5 days.The results of KEGG analysis were consistent with those of GO analysis.These results indicate that in male PGCs,the proliferation,migration,and differentiation abilities at 3.5 days are higher than those at 4.5 days,and the regulation of reproductive related abilities at 4.5 days are higher than those at 3.5 days.A total of 206 differentially expressed genes(109 up-regulated and 97 down-regulated)were obtained from Male4.5 vs Male5.5 PGCs.GO analysis results of differentially expressed genes in cell migration(chemotaxis,cell chemotaxis),cell proliferation(cell proliferation,stem cell)proliferation),transdifferentiation(transdifferentiation,cell differentiation),and reproductive cell development(sperm flagellum,maternal process involved in female pregnancy)and other related GO items were significantly higher.Further analysis showed that the expression of BCAT1(proliferation-related gene)was higher in 5.5 days than 4.5 days.The expressions of DEFB4A and HOXB9(migration-related genes)were higher in 4.5 days than in 5.5 days.The expressions of MYOG,SOX9 and ILDR2(differentiation related genes)were higher at 4.5 days than 5.5 days.The expression of NME5(gene related to germ cell development)was higher at 5.5 days than at 4.5 days.The results of KEGG analysis were consistent with those of GO analysis.These results indicate that migration,differentiation,and reproductive regulation in male PGCs at 4.5 days are higher than those at 5.5 days,and proliferation is lower than those at 5.5 days.(4)Comparison of female and male PGCs in chickenIn order to verify the accuracy of the sequencing results,we compared cell proliferation,migration and differentiation between male and female PGCs at different developmental times by RT-PCR,EdU detection and other methods.The results showed that the expression levels of C-kit and Dazl in male and female PGCs at 4.5 and 5.5 days were significantly higher than those in female and female PGCs at 3.5 days(P<0.05).The expression of CXCR4 in 3.5d PGCs was significantly higher than that in 4.5d and 5.5d PGCs(P<0.05).These results indicated that the migration ability of PGCs gradually decreased and the differentiation ability increased with the development of PGCS.EDU cell proliferation assay showed no significant difference between male and female PGCs at 3.5d(P>0.05),but female PGCs at 4.5d and 5.5d were significantly higher than male PGCs(P<0.05).With the development of female PGCs,there was no difference in the reproductive ability of male PGCs at 3.5 and 4.5 days(P>0.05),but the reproductive ability of male PGCs at 5.5 days was significantly higher than that at 3.5 and 4.5 days(P<0.05).These results are consistent with sequencing analysis.Based on the above results,it can be concluded that there are differences in the primordium germ cells isolated from the gonads of female and male chicken embryos,which are specifically manifested in cell differentiation,proliferation and migration,etc.Our results provide a theoretical basis for further research and application of PGCs. |
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