Investigation On The Derivation Of Embryonic Stem Cell Lines Using Artificial-developed MII Oocytes Of Mouse

Objective: To investigate the possibility of the derivation of mouse embryonic stem cells (ESC)from the artificial-developed MII oocytes for establishing a eligible model to solve the lack of human MII oocytes which restrict the therapeutic cloning.Methods: 0.5 day newborn B6D2F1 mouse ovaries were allotransplanted into the renal capsule of ovariectomized recipient B6D2F1 mice. After 14 days, preantral follicles were isolated from the mice ovarran graft by mechanical, long-term enzymatical, short-term enzymatical or mech-enzym-combined method respectively. Preantral follicles recovered were cultured in vitro for 12 days under different conditions and the rates of follicle survival, ovulation, maturation of oocytes in vitro and fertilization rates of the MII oocytes were examined. In experiment 1, the cumulus-oocyte complexes (COCs) were harvested at different time course after adding hCG ( human chorionic gonadotropin) in culture medium and IVF was successively undertaken using the harvested COCs. The maturation and fertilization rates of the COCs harvested at different time course of hCG treatment and the developmental capacity of embryos were evaluated. In experiment 2,follicles were cultured in media supplemented with 5% fetal calf serum (FCS) in group 1, 1% FCS in group 2. After 12 days, the maturation rates of oocytes, fertilization rates of the MII oocytes and the developmental capacity of embryos were evaluated. The blastocysts derived from the artificial-developed MII oocytes obtained from above procedure were transferred to a fibroblast feeder and cultured with conventional technic to breed embryonic stem cells.Results: 200 mouse ovaries were transplanted into the renal capsule of 8-10 weeks old B6D2F1 female ovariectomized recipient. After 2 weeks, 197 grafts were recovered with the recovering rate 98.5%. 97.13±5.30 follicles were isolated from the recovered graft tissues using mech-enzym-combined method. The survival rate in culture and ovulation rate of follicles were higher in mechanical method than in enzymatical method. Ovulation rate was higher in short-term than in long-term enzymatical method. The number of isolated and ovulated follicles per ovary, the rates of maturation of oocytes were significantly higher in mech-enzym-combined method than in other methods, but the feitilization rates were not different in each methods . In Experiment 1, the rate of maturation of oocytes in 26h hCG treatment was higher than any other hCG time course. In Experiment 2, the fertilization rate of 5% FCS group was higher than 1%FCS group. Blastocysts derived from the aritificial-developed MII oocytes growed well and formed columnar inner cell mass (ICM) on the feeder. After micromanipulated isolating and trypsinizing, cells from ICM amplificated rapidly on feeder and formed the typical ES colonies. The ES colonies were then passed for two generations (P1 and P2).Conclusion: The mech-enzym-combined method is best method evaluated. The best time course of hCG treatment is 26 hours for ovulation, maturation and fertility. 5%FCS is favourable for follicle development and the fertilizing potency of oocytes. The primordial follicles can be triggered to full maturation by our artificial-developed system, and ESC line can be established from this artificial-developed MII oocytes of mouse.