Isolation And Characterization Of Female Germline Stem Cells In Mice

It is regarded that the number of fermale germ cells in mammalian ovary has been determined at birth or soon after birth,the germ cell pool is thought to be extuasted due to the inability of fermale germ cell renewal,but this traditional idea has been challenged recently.Here I report a new method to isolate female germline stem cells(FGSC)from neonatal ovary.Through multi-round attachment in gelatin-coated wells,ovary germ cells were enriched after attachment.During short-term culture,some female germ cells exhibited dividing activity in FGSC culture condition,indicating that fermale germ cells still have self-renewal ability after birth.Moreover,maximum activity of cell dividing in the female germ cells can be obtained from ovary of newborn mice,especially postnatal 1-3 days.The diameters of germ stem cells were among 15-25 ?m,and actively dividing cells finally formed germ cell cyst-like structure,in which cells contacted through cytoplasm bridge,while nucleus were sepearated.To investigate which cytokine is indispensible during cell dividing,growth factors deperived condition was used in cell culture.I observed that cells in GDNF deperived condition still exhibited diving activity.In addition,when the estrogenic hormone E2 was added in GDNF-contained culture conditon,cell proliferation was significantly depressed.These results suggested that GDNF and E2 could act coordinately to induce germ cell differentiation,and the differentiated germ cells were unable to be maintained in the culture and led to cell number decline.To testify the hypothesis I conducted Dual-Luciferase Reporter Assay to analyse the influence of E2 hormone on Gfral promoter acitivity.E2 hormone can increase Gfra1 transcriptional activity,thus led to an increasment of GDNF receptor protain GFRA1,This observation indicated that the increasement of GDNF-GFRA1-RET signaling could trigger the germ cell differentiation.When compared with muli-round attachement islated germline stem cells,the VASA+ female germline stem cells isolated through MACS method exhibited different characteristics such as cell diameter,stem related genes expression and differentiation ability,which indicated that MACS isolated germline stem cells resembled PGC,while muli-round attachement islated stem cells were more similar to oogonial cells.In conclusion,multi-round attachment method is sufficient for fermle germ cells isolation,numbers of fermale germline stem cells in ICR strain mice decrease with aging,and E2 and GDNF can influence the fermale germ cell differtiantion.These results enrich our understanding of mice fermale germ cell features.