| Object:1.Establish a tissue engineering technology model to harvest developmentallycompetent MⅡ oocytes from primordial follicles.2. To generate embryonic stemcell using artificial-developed MⅡ oocytes derived from primordial follicles.Method:1.21-day-old femmal mice and recipient mice which had transplanted newborn ovaries for21days were primed with10IU Pregnant Mare SerumGonadotropin(PMSG)，and the different COCs were isolated respectively44h later forIVM. The process was performed under three systems, which used fetal bovine serum,bovine serum albumin, serum replacement as alternatives, then compared thematuration rate and blastocyst rate of oocytes cultured in various systems.2. Thefresh ovaries from the newborn mice were immediately transplanted to kidney capsuleof recipient mice to initiate oocyte growth. Grafts were recovered at23days afterreceived10IU PMSG for44h. Subsequently, the COCs were cultured for16h in vitroto generate mature oocytes. Embryonic stem cell–like colonies were established bysubculturing the inner cell mass cells from in vitro fertilization blastocysts.Result(s):1. Three different IVM systems have no significance in oocyte maturation,fertilization and post-blastocyst rate difference (P>0.05).2. Two colony-formingcell line was established by IVM-MⅡoocytes obtained from COCs of grafted ovaries.One established stem cells have successfully subcultured>6times. Colony-formingcells at the3th subpassage stained positive for alkaline phosphatase(AP), anti-SSEA-1.The colony-forming cells were transformed into a teratoma consisting of three germlayer-derived cells after being transplanted into kidney capsule of male mice.Conclusion(s):1. BSA and KSR proved to be an effective substitute for FBS. It isnoteworthy that KSR has better comprehensive advantages on the maturation of cumulus cells and the development of oocytes.2. To our knowledge, this is the firsttime that ESC have been produced by oocytes developed from non-recruitmentprimordial follicles of newborn mice. |
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