摘要
目的观察不同激活方法对小鼠卵母细胞激活的影响，以优化核移植的小鼠卵母细胞激活方案。方法昆明种小鼠经腹腔注射7．5～10U孕马血清促性腺激素（PMSG），48h后腹腔注射同样剂量的人绒毛膜促性腺激素（hCG）。注射hCG后14-16h收集卵母细胞，18h后用单个激活剂激活卵母细胞。单个激活剂包括钙离子载体（A23187，5μmol／L，5min）、乙醇（Eth，7％，7min）、氯化锶（SrCl2，10mmol／L，20min）。结果单个激活剂激活小鼠卵母细胞时，卵裂率同其他组（48％～70％）相比，SrCl2组最高（72％）。囊胚发育率（9％～20％）均高于对照组（1％，P〈0．05），且SrCl2组囊胚率（20％）高于A23187组（9％）和乙醇组（12％，P〈0．05）。单个激活剂同蛋白激酶抑制剂6-二甲氨基嘌呤（6-DMAP）联合使用能使卵母细胞获得完全的激活。乙醇＋6-DMAP组（95％和35％）和SrCh-q-6-DMAP组（92％和31％）卵裂率和囊胚形成率明显高于其他各组（49％～53％和3％～17％，P〈0．05），两组间没有差异。A23187＋6-DMAP组的囊胚率（17％）与对照组（3％）有显著性差异（P〈0．05）。本文还比较了6-DMAP在不同浓度（0．2mmol／L和2．5mmol／L）以及不同激活时间（1．5h和3．5h）与单个激活剂（乙醇或SrCl2）组合对小鼠卵母细胞激活的影响。在高浓度（2．5mmol／L）短时间（1．5h）的6-DMAP组合组囊胚率（34％～36％对3～23％，P〈0．05）和卵裂率较高。结论乙醇和SrCl2与6-DMAP组合，均能较好地激活小鼠卵母细胞，组合中乙醇组合优于SrCl2。此外，6-DMAP的浓度和激活时间也是影响激活效率的重要因素。单个化学激活剂不能完全激活小鼠卵母细胞。
Objective Oocyte activation is one of the essential elements determining the success of nuclear transfer and the subsequent development of cloned embryos both in vitro and in vivo. Experiments were conducted to optimize the protocol for oocyte activation in a regular nuclear transfer study by examining parthenogenesis of mouse oocytes activated by different stimuli. Methods Oocytes derived in vivo were collected at 14h-16h from KM mice after ovulation treatment, and activated ＋18h post-ovulation treatment by single activation agents including calcium ionophore （A23187, 5μmol/L, 5min）, ethanol （Eth, 7%, 7min） and strontium （SrCl2, 10mmol/L, 20min）. Results Cleavage rates were highest in the strontium treated group （72%） compared to other groups （48%-70%）. Blastocyst rates were higher in the single activation agent groups （9%-20%） than that in the control group （1%）（P〈0. 05）. And blastocyst rate was higher in strontium treated group （72%） than that in both ionophore-treated group （9%） and ethanol-treated group （12%）（P〈0. 05）. Combined single agent treatment （calcium stimulators） with protein kinase inhibitor, 6-DMAP was used to achieve a full oocyte activation. Both deavager and blastoeyst formation rates were significantly higher （P〈0. 05） in EthW6-DMAP （95% and 35%） and strontiumW6-DMAP （92 and 31%） treatment groups than that of other groups （49%-53% and 3%-17%, respectively, P〈0. 05）. There was no difference in two groups. Blastocyst formation rate was significantly higher （P〈0. 05） in A23187＋6-DMAP group （17%） than that in control group （3%）, Low （0. 2mmol/L） and high （2. 5mmol/L） concentrations of 6-DMAP treatments with different treatment duration （1.5h and 3.5h） in the combined groups were also compared. Blastocyst formation and cleavage rates were greater in the high concentration with less treatment duration groups （34%-36% vs 3%-23%, P〈0. 05）. Conclusion All the Eth or the strontium ＋6-DMAP combined treatment could better activate mouse oocytes. The Eth ＋6-DMAP combined treatment was better than the strontium ＋6-DMAP combined treatment, and single activation agent could not fully activate mature mouse oocytes.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2007年第1期16-19,共4页
Medical Journal of Chinese People's Liberation Army
基金
国家"十五"863"组织工程"重大专项(2003AA205004)
国家"十五"863航天领域课题(2002AA744064)
