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小鼠卵母细胞体外成熟培养系统的建立与优化
Establishment and optimization of the culture system of mouse oocyte in vitro maturation
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% vs 0.0%）。 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 study the effects of the duration of pregnant mare serum gonadotropin(PMSG) priming and in vitro culture (IVC) on nuclear maturation, and the effects of the different activation schemes on the abilities of parthenogenetic activation
and development. Methods To detect the rate of nuclear maturation at different time points of IVC by using PMSG priming(each treatment had at least 3 replicates, 3 mice per replicate).In order to determine the optimum activation scheme, two schemes
including: (1) ethanol combining with 6-dimethylaminopurine(6-DMAP) and (2) SrCl2 were used, CZB［fetal bovine serum(FBS) and CZB［bovine serum albumin(BSA)］ were used for embryo culture. In order to determine the optimum activation age, the oocytes matured
at the different time points were activated. To investigate the effect of PMSG on the ability of oocyte development by shortening the duration of PMSG priming. Results The time of IVC when oocytes reached the highest rate of nuclear maturation (97.6% vs 91.9%)
was prolonged from 14 hours to 16 hours if shortening the duration of PMSG priming from 46 hours to 24 hours. Shortening the duration of PMSG priming did not affect the rate of nuclear maturation, however, the rates of activation（91.2% vs 37.1%）and
blastocyst（20.9% vs 0.0%）were significantly reduced. Two schemes used in the present study were able to induce the activation rate of oocytes to more than 90% in the in vitro maturation system, however, the rates of blastocyst were significantly different
(P<0.05). The rates of activation (89.5%) and blastocyst (21.9%) reached the highest points from 24 hours to 26 hours during IVC. Conclusion A relatively ideal IVC system has been established in the present study. PMSG stimulation duration is 46 hours,
oocytes cultured for 24 hours, activated with CZB (10mmol/L SrCl2) and embryos cultured in CZB (0.5% BSA).
卵母细胞 / 孤雌激活 / 体外培养 / 胚胎培养 / 小鼠
Oocyte / Parthenogenetic activation / In vitro culture / Embryo culture / Mouse
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