Isolation, Establishment And Functional Study Of The Mouse Female Germline Stem Cell Line

In traditional view of reproductive biology the germ cells in mammal ovaries are considered lack the capacity of mitosis after birth, fertility lost when germ cells in the pool exhaust with the growth of age. However, Johnson and colleagues in 2004 found the discordance in follicle loss versus atresia: the incidence of atretic follicles was higher than the decline of the follicles, by counting the number of non-atretic and atretic follicles in mouse ovaries at different ages, which indicated the capacity of proliferation in mouse ovary. In search of the evidence of germ cell proliferation in neonatal and adult mouse ovaries, our group characterized the proliferating germ cells in vivo, predicted the existence of transmembrane domain of MVH as a sorting marker, and isolated these putative germ line stem cells by double enzymes digestion and subsequent MVH-magnetic beads cell sorting. Cultured with similar condition of spermatogonial stem cells, female germ line stem cell (FGSC) lines were established and have been maintained for more than 60 passages. Subsequently, we characterized these cells on mRNA, protein, epigenetic levels by RT-PCR, fluorescent immunocytochemistry, Cobra assays, and verified their biological function by ovary transplantation assays, finally identified them as germ line stem cells. Furthermore, we enhanced the sorting efficiency by optimizing the purification steps and using different markers. The improvement of purification protocol facilitates the establishment of FGSCs line. The discovery of FGSCs and the establishment of FGSC line modified the traditional doctrine of reproductive biology and opened up a novel field, and brought light for the cure of female infertility and gynecological diseases in clinic as well. Moreover, we developed a new tool for transgenic engineering. Therefore, our discovery has great significance on reproductive biology theory and potential for research and clinic use.