The Reasearch Of Isolation And Establishment Of Mouse Oogensis Stem Cells And Their Differention Into Oocytes In Vitro

OSCs were enriched from the 7 days post parturition(7dpp)and 4 weeks old(4W)prepuberty mouse ovaries,respectively,with the method of differential attachment,and cultured in vitro.And the cells were named as 7dpp-putative OSCs and 4W-putative OSCs according to the mice's week age.After passages,the cells with a homogenious population were subjected to alkaline phosphates staining,marker gene expression analyses,immunofluorescence staining,transcriptomic analyses and ovarian transplantation examinations.The results showed that the cells isolated from 7dpp and 4W ovaries were ramarkablly different in morphology.The 7dpp-putative OSCs were replicated them self by “budding” with the formation of a string like colony.After cell passage,7dpp-putative OSCs proliferated and gathered into clusters.4W-putative OSCs were formed into a three-dimensional clone at the beginning of self replication.After passages,4W-putative OSCs gradually became flat morphologically more like epithelia.However,both types of cells expressed Oct4,Ddx4,Ifitm3 and Cdh1 genes,but with different expression levels.The 7dpp-putative OSCs expressd higher levels of Oct4 and Ddx4 and lower levels of Ifitm3 and Cdh1 than that of 4W-putative OSCs.In addition,7dpp-putative OSCs expressed Blimp1,as PGCs specific transcriptional factor,meanwhile,Blimp1 could not be detected in 4w-putative OSCs.The further analyses of transcriptome revealed that the gene expression patterns of 7dpp-putative OSCs and 4W-putative OSCs were significantly different from each other.The genes expressed in 7dpp-putative OSCs were enriched in the biological processes of germ cells and stem cells,whereas the up-regulated genes in 4W-putative OSCs were enriched in the biological processes of epithelial cell migration and proliferation.Importantly,the 7dpp-putative OSCs formed oocytes in the recipient's ovary in vivo.Taken all together,we concluded that OSCs exist in the 7 days post-natal mouse ovary,but not in the 4 week old mouse ovary.Next we extended our interesting to reconstitute the follicles by co-culturing the OSCs and ovarian somatic cells in vitro.In this section,the same methods were used to isolate 7dpp-GFP-OSCs from the GFP transgenic mice with the ovarian somatic cells isolated from wild-type mice(GFP negative)mice.Through the methodology of morphological observation,in vitro maturation of oocytes,intracytoplasmic sperm injection(ICSI),embryo transfer and Sycp3 immunofluorescence staining,the abilities of co-culture system to from reconstituted follicles were identified.Furthermore,the abilities of 7dpp-GFP-OSCs to differentiate into fertile oocytes through meiosis in vitro were analyzed.After follicle reconstitutaion,the co-culture results showed that OSCs could form follicles with post-natal mouse ovarian somatic cells and complete the entire oogeneis in vitro,including the entry into meiosis and formation of GV stage oocytes.Remarkely,more than 65.00% of GV oocytes could release the first polar body,finishing the maturation in vitro,which closed the maturation rate of the eggs generated in vivo.Moreover,the MII oocytes could be fertilized with intracytoplasmic sperm injection(ICSI)and the healthy offspring were borned by the recipients after transplantation of the embryos.Finally,the gene expression patterns of mouse embryonic stem cells(ESCs),primordial germ cells(PGCs)and 7dpp-OSCs were further characterized by RNA-seq and fluorescence quantitative PCR.The results showed that the specific gene expression levels related with pluripotency in 7dpp-OSCs was similar to that of PGCs,which were both significantly lower than that of ESCs.The specific gene expression levels of germ cells in 7dpp-OSCs were similar to that of PGCs,which were significantly higher than that of ESCs.The gene expression levels related with meiosis in 7dpp-OSCs were significantly lower than that of PGCs.In conclusion,this study has provided concreted evidence that OSCs indeed exist in the post-natal mouse ovaries,but not in the ovaries of 4w old mouse.This is the first time to demonstrate that the OSCs are capable of reconstitution of follicles with ovarian somatic cells in vitro and finishing the entire process of oogenesis to form the functional oocytes.Additionally,the study has answered why follicles pool cannot be regenerated in adult ovary.There can be no doubt that this study has provided a powerful cell model system to study oogenesis and formation follicles in vitro,and set a new milestone for investagtion of OSCs.