The Role Of Pcls In Germ Cell Development

Germ cells originated in the proximal ectoderm of 6.5 dpc embryos in mice,are called primordial germ cells(PGCs),and are induced by Wnt signals and BMP signals.PGCs began to migrate along the mesenteric to the genital ridge at 9.5 dpc,and are accompanied by proliferation during the migration process.At 12.5 dpc all PGCs enter into the genital ridge,13.5 dpc they stop mitosis,and then enter the process of sex differentiation.During the formation,proliferation,and migration of PGCs,epigenetic modifications such as DNA methylation,H3K9me2,and H3K27me3 undergo tremendous changes.It is reported that the increase of H3K27me3 in PGCs is related to PRC2.The formation and migration of PGCs determine the individual's reproductive ability.Genetic diseases may cause the reduction of primordial germ cells and cause infertility in adult.The abnormal migration of PGCs leads to extragonadal tumors.Mammalian Pcll,Pcl2,and Pcl3 are three genes that are homologous to Drosophila Pcl.They recruit PRC2 to the chromatin to methylate lysine 27 of H3 and regulate gene expression.At present,the epigenetic regulation of Pcls on chromatin has already relatively clear.In addition to functioning as a subunit of PRC2,Pcls itself can also function as a cytokine.According to reports,Pcls also play an important role in the occurrence and development of cancer.However,the role of Pcls in germ development cells is less studied.This study showed that Pcl2 and Pcl3 were significantly high expressed in PGCs,and Pcll-3 were highly expressed in early gonads.Pcll-3 were expressed in the testis and were highly expressed in spermatogonia and spermatocytes at specific stages of seminiferous tubules.The knockout of Pcl3 promoted the proliferation of PGCs,but led to an increase in the number of PGCs in the hindgut of 10.5 dpc mice,and a large number of PGCs were ectopic at 12.5 dpc.This article revealed the expression of Pcls in PGCs and adult testis,studied the role of Pcl3 in the proliferation and migration of PGCs,and deepened the understanding of the function of Pcls.Object:To study the role of Pcls in the proliferation and migration of mice primordial germ cells.Methods:1.Observe the distribution of Pcls in 9.5 dpc embryos through in situ hybridization experiments.2.Analyze and map the transcriptome data of GEO database to clarify the expression of each subunit of PRC2 in PGCs.3.Analyze and map the transcriptome data of GEO database to clarify the expression changes of Pcls in 9.5-15.5 dpc mice PGCs.4.Through gene expression differential analysis and gene enrichment analysis of multiple Pcls-related databases,look for Pcls-regulated factors related to the development of PGCs.5.Detect Pcls expression from 12.5 dpc mice primitive gonad to adult mice gonad by semi-quantitative experiment.6.The effects of Pcl3 knockout on the proliferation of PGCs was detected by ALP staining and EdU staining on 10.5 dpc mice.7.TAM induced Pcl3 knockout in mice,and the effects of Pcl3 knockout on PGCs migration was detected by sectioning and immunofluorescence staining of 10.5 dpc mice.8.The gonads and surrounding tissues of 12.5 dpc mice were sectioned and immunofluorescent stained to observe the effects of Pcl3 knockout on the number and migration of PGCs.9.Detect the expression of Pcls in adult male genital organs by Western Blot experiment and immunohistochemistry experiment.Rresult:1.In situ hybridization experiments show that Pcls were highly expressed in the dorsal neural tube,Pcl3 was significantly expressed in PGCs.2.13.5 dpc PRC2 subunit expression volcano graph results showed that Pcl2 and Pcl3 were significantly higher in PGCs compared with somatic cells,while the expression of Pcll in PGCs was not significantly different from somatic cells.3.The mapping analysis of the data in the GEO database showed that Pcl2 and Pcl3 were highly expressed in mouse PGCs from 9.5 dpc to 15.5 dpc,while the expression of Pcl1 was lower;with the development of mice,the expression level of Pcl3 decreased,the expression level of Pcl1 gradually increased,and the expression level of Pcl2 was basically the same.4.By drawing the Venn diagram,we found 876 genes that would be significantly affected by Pcl3 knockout.We found that these genes are enriched in the cell cycle,nuclear transport and p53 signaling pathways by performing gene enrichment analysis on 876 genes.5.By extracting total RNA from the gonads of fetal and adult mice and conducting semi-quantitative analysis,and performing gray-scale analysis of the results,it was found that Pcl2 was highly expressed in the gonads of fetal and adult mice,Pcl1 and Pcl3 were highly expressed in the early gonads.Compared with Pcl1 and Pcl2,Pcl3 was less expressed in adult gonads and germ cells.6.The Pcl3 knockout of embryos were induced by TAM,and the whole embryos were stained with ALP.It was found that the number of PGCs in knockout mice increased compared with that of control.The EdU stain was used to detect cell proliferation,and it was found that compared with the control group,the percentage of proliferating cells in PGCs increased significantly after Pcl3 knocked out.7.TAM induced Pcl3 knockout in embryos,frozen section and SSEA-1 immunofluorescence staining were performed on the collected embryos,and the position of PGCs was counted.It was found that there was no significant difference in the percentage of PGCs in the hindgut and mesentery,but the total number of PGCs in the hindgut increased significantly compared with the control group.8.The ALP staining of the gonads of 12.5 dpc Pcl3 knockout embryos showed that the number of PGCs increased;through the 12.5 dpc gonads and surrounding tissue sections,SSEA-1 immunofluorescence staining we found that compared with the control group,a lot of PGCs still in the mesentery in 12.5 dpc knockout embryos.9.Through the Western Blot experiment on the protein extracted from the testis and epididymis of 3M adult mice,we found that Pcls were expressed in the testis,but the expression in the epididymis is very low.By conducting immunohistochemical experiments on testicular tissue,we found that Pcls were highly expressed in spermatocytes at specific stages of seminiferous tubules.Conclusions:1.Pcl2 and Pcl3 are highly expressed in PGCs,and the expression of Pcls are correlated during the development of PGCs.2.Pcl3 knockout promotes the proliferation of PGCs,the number of PGCs in mice increases,and the number of PGCs retained in the hindgut increases.3.The number of PGCs that migrate into the 12.5 dpc Pcl3 knockout mice gonads increase,and the number of PGCs still in the mesentery increase compared to the control group.