Isolation, Identification Of Mouse Primordial Germ Cells And Epigenetic Regulation In The Migration

Primordial germ cells (PGCs), as precursors of sperm and oocytes, are important for biological survival and reproduction. Mammals PGCs, originated from embryonic epiblast, migrate from the yolksac at embryonic 7.5 days to the mesenteric through hindgut endoderm. During the migration process, PGCs continuously proliferate and eventually arrive at genital ridge at E13.5, where PGCs differentiate into spermatocyte and oocytes. In the migration process of PGCs, cell fate determination, cells-cells interaction, cell migration, epigenetic reprogramming and other important cellular activities are involved, therefore the isolation and purification of PGCs is crucial to study the proliferation and migration.This research were foucsed on the isolation, identification of mouse PGCs and epigenetic regulation of gene expression in the migration by flow cytometry.1. The isolation and identification of mouse PGCsFirstly, the location of SSEA-1 and OCT4 in mouse embryos at E10.5 and E13.5 was determined by immunohistochemical observations, the results showed that SSEA-1 and OCT-4 were located in embryos dorsal mesentery at E10.5 and SSEA-1 in embryos genital ridge at E13.5; Then, the last 1/3 part of E10.5 and the genital ridge of E13.5 were taken and treated by 0.05% trypsin digestion into single cell suspension. After marked by SSEA-1 and sorted through flow cytometry, the enriched cells were identified by Alkaline phosphatase (AKP) and SSEA-1 staining, and the sorted cells were detected to the expression of PGCs marker genes (Fkbp6, Mov1011,4930432K21Riken, Spo11), pluripotent genes (Nanog, Oct4) and germ cell specific genes (Mvh, DazP) through the real-time PCR. The sorting rate of SSEA-1 positive cells in E10.5 and E13.5 were 3±1.41% and 10.12±2.56% respectively. The AKP positive rate of these sorted cells was around 90%, and SSEA-1 staining were all positive. Comparing expression levels of PGCs marker genes between SSEA-1 positive and negative cells revealed significantly higher expression in positive cells of Fkbp6, MovlOLl,4930432K21Riken, Spo 11(P<0.001). The expression levels of pluripotency genes Nanog, Oct4 and germ cells specific genes Mvh, Dazl in the sorted cells at E13.5 were significantly higher than E10.5 (P<0.001). These results indicated that the sorted cells had characteristics of mouse PGCs.2. Epigenetic regulation of gene expression in PGCs migrationTo further explore the molecular regulation of PGCs migration, the gene expression and subcellular localizastion of MMPs and TIMPs were investigated in E10.5 and E13.5 PGCs. Real-time PCR results showed that compared the E13.5 PGCs, the expression of Mmp2 and Mmp9 were high in E10.5 PGCs, and the expression of MTl-Mmp was significantly higher in E10.5 PGCs(P<0.001); the expression of Timp1, Timp2 and Timp3 were higher in E13.5 PGCs than in E10.5 PGCs(P<0.001). By SSEA-1 and MT1-MMP double staing FACS and immunofluorescence, MT1-MMP was detected in E13.5 PGCs, and not in E10.5 PGCs. Bisulfites PCR sequencing were used to detect the DNA methylation of above gene expression, DNA methylation level in the promoter region of Mmp2 gene did not change between PGCs in E10.5 and in E13.5, while in E13.5 PGCs DNA methylation level of promoter region of Timpl obviously increased by 19%(P<0.05), Timp3 clearly reduced by 16%(P<0.05), which suggested that the change of DNA methylation level in promoter region associated with the different expression of Timpl and Timp3 of PGCs in different periods.In summary, sorted cells by SSEA-1 had PGCs characteristics with expression of PGCs marker, pluripotent and germ cells specific genes. MMP/TIMP were dynamic regulated at E10.5 and E13.5 PGCs, high expression of MT1-MMP might promote PGCs migration to the genital ridge. In addition, the expression of MMP/TIMP is related to its DNA methylation in genetic regulatory region, low methylation of Timp3 in E13.5 PGCs caused increased its expression.