Contribution Of Golgi Apparatus In The Mouse Oocytes And Mouse Nuclear Transfer Embryos

This study was designed to investigate the effect of golgi apparatus on mouse oocytes and mouse nuclear transfer embryos developing in vitro, In the research, we got immature oocytes through gonadotrophin treatment and established mouse reconstructed embryos following nuclear transfer, which between mouse fibroblasts and mouse enucleated oocytes, then we observed the dynamic changes of golgi in the oocytes and embryos through laser scanning microscopy with the use of membrane trafficking inhibitor BFA(Brefeldin A) and golgi specific maker(β-COP), meanwhile, we study some relating factors involve in mouse oocytes and mouse nuclear transfer embryos developing in vitro .The main contents and results were as following:1. mouse oocytes maturating in vitro1) The addition of FSH(follicle-stimulating hormone) or insulin in DMEM medium is superior than the addition of BSA(Bovine serum albumin) for the nude oocytes in vitro maturation, in vitro fertilization and cleavage(P<0.05).2) Among the 4 concentrations of BFA: 5μM 0.5μM 50nM 5nM,the second concentration is most significance concerning its effects on the mouse oocytes in vitro maturation.3) the nude oocytes are parthogenetically activated in the presence of 10% ethanol and/or 6-DMAP(6-dimethylaminopurin), and when the two chemicals are used in combination the rate of activation is the highest among the four groups.4) Put the GV oocytes in M2 medium containing BFA(5ng/mL) and dbcAMP(100ug/mL), 1 hour later employing immunocytochemixtry with β-COP, no distinct structure was labeled by the golgi maker, while after washed out the drug and allowed the oocytes a 1-h recovery period in fresh dbcAMP(100ug/mL) containing M2 medium, golgi markers was again visible.5) GV oocytes were cultured in M2 medium containing BFA(5μg/mL) and dbcAMP(100|Ag/mL) for 1 hour, then washed the BFA and allow the oocytes to recover in fresh M2 medium for another hour, subsequently estimated their rates of first polar body extrusion, rates of in vitro fertilization and rates of parthogenetically activation, the results are lack of statistical significance(P>0.05), furthermore, we got the similar results using the mature oocytes under the same conditions.2. The separation and culture of mouse fibroblast cell1) We obtained the fibroblast cell from mouse subcutaneous tissue by issue culture method and enzyme digestion, the purity and proliferation rates of the cells are good, after 15 passage of the primary mouse fibroblast cells, the cell still have high activity.2) Among the three culture medium: M16. DMEM/F12 and CZBM(CZB modified), the former two are superior than the last one for primary mousefibroblast cell culture(P<0.05).3) Cultured fibroblasts cells of mouse were preserved at liquid nitrogen with cryogen of 10%DMSO in DMEM medium. The seeding efficiencies were not significant change when cells were thawed after lmth, 3mth, 6mth, 12mth(P>0.05).3. Establishment of reconstructed embryos following nuclear transfer1) The mouse fibroblast cells were serum starved for 5 days or treated with Nocodazole(10μg/ml) for 24h, then calculated the percentage of the treated cells and the control cells in G₀, G₁, G₂ and M phases of the cell circle by using flow cytometry, respectively. Mouse fibroblast cells treated with serum starvation for 5 days contained higher percentages of G0+G1; whereas the cells treated with Nocodazole contained higher percentages of G2+M; The results of two synchronization treated groups and control group are notable (PO.05).2) The receptor oocytes were divided into two groups, the first group were oocytes maturated in vitro, and the second group were oocytes maturated in vivo. After nuclear transfer, the survival rates of the reconstructed embryos came from different groups of receptors were 19.2% and 32.3%, respectively.3) The donor of mouse fibroblast cell were treated with serum starvation for 5d, and done with the 10ug/ml Nocodazole for 24h, were used to recombine nuclear transfer embryos. After the recombined embryos were activated by 6-DMAP+CCB+ethanol, their rates of cleavage were 18.24% 10.27% 9.15%(control), respectively.4) The receptor oocytes were divided into A, B, C three groups, group A and B were experimental groups compared group C. In group A, oocytes were cultured in TCM199 medium containing BFA(5ug/mL) for lh; in group B,...