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目的探讨激素刺激时间、体外培养时间对小鼠卵母细胞核成熟及不同激活方案对卵母细胞孤雌激活、胚胎发育能力的影响。方法孕马血清促性腺激素(PMSG)超排处理,在体外培养的不同时间点检测卵母细胞核成熟率(每个处理至少重复3次,3只/重复,以下实验相同)。分别采用乙醇结合6-二甲氨基嘌呤(6-DMAP)法和SrCl2法激活卵母细胞,胚胎培养液选用CZB[胎牛血清(FBS)或牛血清清蛋白(BSA)]两种,确定最佳激活方案。对不同时间点成熟的卵母细胞进行激活,确定最佳激活卵龄。研究缩短PMSG刺激时间对卵母细胞发育的影响。结果将PMSG刺激时间从46h缩短至24h,卵母细胞获得最高核成熟率(97.6%vs 91.9%)的培养时间由14h延长至16h;缩短PMSG刺激时间,核成熟率不受影响,但能显著降低激活率(91.2%vs 37.1%)和囊胚率(20.9%vs0.0%)。两种方法体内成熟卵母细胞激活率均高于90%,但囊胚率差异显著(P<0.05)。卵母细胞体外培养至24～26h时,激活率(89.5%)和囊胚率(21.9%)均达到最高点。结论建立了一种小鼠卵母细胞体外成熟培养系统,即PMSG超排处理46h、卵母细胞培养24h,CZB(10mmol/L SrCl2)激活2.5h后采用CZB(0.5%BSA)进行胚胎培养。
Objective To investigate the effects of hormone stimulation time and in vitro culture time on the maturation of mouse oocytes and the different activation programs on oocyte parthenogenetic and embryo development. Methods The pregnant rats were treated with superoxide dismutase (PMSG), and the maturation rates of oocytes were measured at different time points (at least 3 times per treatment, 3 / repeat, the same in the following experiments). The oocytes were activated by ethanol combined with 6-DMAP and SrCl2, respectively. Two kinds of embryonic culture medium, CZB (FBS) or bovine serum albumin (BSA) Good activation program. Oocytes were activated at different time points to determine the best activated egg age. Study to reduce the impact of PMSG stimulation on oocyte development. Results The time of PMSG stimulation was shortened from 46h to 24h, and the incubation time of oocytes with the highest nuclear maturation rate (97.6% vs 91.9%) was prolonged from 14h to 16h. Shortening of PMSG stimulation time, nuclear maturation rate was not affected but significant Reduce the activation rate (91.2% vs 37.1%) and blastocyst rate (20.9% vs0.0%). The activation rates of mature oocytes in both methods were higher than 90%, but the blastocyst rate was significantly different (P <0.05). Oocytes cultured in vitro to 24 ~ 26h, the activation rate (89.5%) and blastocyst rate (21.9%) reached the highest point. Conclusion A mouse oocyte maturation culture system was established in vitro. PMSG was treated with superovulation for 46h. The oocytes were cultured for 24 hours. CZB (0.5% BSA) was used to culture embryos after activation of 10mmol / L SrCl2 for 2.5h.