Study On Maturation In Vitro Of Murine Oocytes And Ovary Transplantation In Transgenic Mouse

With the increasing development of life science, laboratory animals played an increasingly obvious role in scientific research. Mouse was one of the most widely used kinds of laboratory animals. C57BL/6J strain mouse, a widely used inbred mouse, was common used in oncology, physiology, immunology and genetics due to its steady genetic background. Most transgenic mice are produced from C57BL/6J mice. It was important for how to carry out genetic resource protection of these genetic engineering mice and to establish an efficient conservation protocol of murine resources. In vitro fertilization combined with embryo cryopreservation was well functional in resource protection. However, in vitro maturation (IVM) of mouse oocytes was a basic link to produce vitro embryos. Allograft orthotopic ovary transplantation (OT) was an effective way to save the reproductive function of females and could get pregnant natural. Both IVM and OT, basic work for resource protection of mouse and having wide foreground in treating infertility, was of important significance. However, the in vitro fertilization (FVF) rate of in vitro maturated oocytes was still very low, and little research had been done on the allograft orthotopic ovary transplantation. These problems needed to be resolved.This study took C57BL/6J mice as research objects, the purpose of it was intended to increase the IVF rate of IVM oocytes and get offspring come from the donor ovaries, so as to establish a basis for the conservation of C57BL/6J mice and to provide reference for other relevant research.1. Research on in vitro Maturation of Mouse OocytesThe C57BL/6J mice were used in present experiment. To get test-tube mouse, various affecting factors were studied, including PMSG treatment, age of mouse, IVM media, FBS and mOTF supplement, duration of culture, kinds of oocytes, laser drilling in zona pellucida, sperm capacitating duration et al.The results were showed as following.(1) PMSG treatment could improve the IVF rate efficiently,73.10%vs68.73%, P<0.05.(2) Oocytes isolated from4w-9w mice were better both in quality and quantity, both in COC and DO. The old age group had high IVF rate, but the number of oocytes isolated from it was low, just14oocytes/mouse vs116oocytes/mouse in4w-9w mouse.(3)Of the three differetnt culture medium supplement with10%FBS, M16suited better for IVM of mouse oocytes than DMEM, MEM-α. IVF rate was51.56%vs45.58%vs38.10%.(4) Supplement with both10%FBS and10%mOTF could get higher IVF rate than the one supplent with only10%FBS. The IVF rate was80.07%vs51.56%, it showed significant difference, P<0.05.(5) IVM for18h could get higher IVF rate than any other groups.(6) COC had better maturation and higher IVF rate than DO,79.82%vs53.86%, significant different, P<0.05.(7) Choosing DO with pbl and laser drilling in zona pellucida could improve the IVF rate of in vitro maturated DO,69.91%vs58.75%, significant different, P<0.05.(8)Compared with traditional sperm capacitating duration, the4h group got higher IVF rate.83.87%vs49.51%, significant different, P<0.05.Concluded the above research results, an optimal program was made. Choose4-week old mouse, intraperitoneally inject PMSG,10IU/mouse, isolate immature oocytes48h later, culture in M16+10%FBS+10%mOTF medium for18h, IVF with4h capacitated sperm. The IVF rate showed no significant difference with in vivo maturated oocytes,85.14%vs90.19%, P>0.05.2-cell embryos were cultured in vitro and hatched blastocysts were got.2-cell embryos were transfer into the fallopian tube of pseudopregnancy mouse and test-tube mice were got.2. Research on Allograft Orthotopic Ovary TransplantationThis study took three strains of transgenic mice and its background mice as research objects. Transgenic mice Ptet-mPTA1and LAP/rtTA were made from inbred mouse C57BL/6J, HBV-x from ICR/JCL. The transgenic mice were ovary donors, the background mice were ovary acceptors. Natural mate the acceptors with males which were negative of target gene2months later after operation. Test the target gene in the offspring by extracting DNA, PCR and agarose gel electrophoresis. Positive ones were judged as from the donor ovary, negative ones the acceptors.The results showed as following.(1) In both Ptet-mPTA1and LAP/rtTA mice groups, respectively,4mice were operated, all survived, pregnant. Positive genes were detected in the offspring. In HBV-x group, positive target gene was not detected.(2) Dissect the negative acceptors, some of the transplant ovary didn’t grow, some died, some adhered to the around tissues, and some without vascularization.(3) Dissect the positive acceptors, the transplant ovaries were healing with the left part of the acceptor’s ovaries, with vascularization. Ovarian follicles, immature oocytes were observed under microscope. IVM-IVF the oocytes,2-cell embryos were got.