| 摘要: |
| 【目的】研究周期依赖性蛋白激酶抑制剂(Roscovitine，ROS)对山羊卵母细胞体外成熟及孤雌胚发育的影响。【方法】将山羊卵母细胞置于含不同浓度(0(对照)，20，40，80，120，160，200 μmol/L)ROS的成熟液中培养24 h统计第一极体排出率，初步筛选山羊卵母细胞成熟培养液中添加ROS的有效浓度；将山羊卵母细胞在含不同浓度(0(对照)，40，80，120，160，200 μmol/L)ROS的抑制培养液中培养8或16 h，再转入常规培养液中培养至24 h，统计第一极体排出率，确定ROS适宜的浓度和作用时间。将卵母细胞放入添加40 μmol/L ROS的培养液微滴中进行抑制培养，抑制培养8 h后分别再常规培养0，2，4，6，8，10，12，14，16 h，统计各处理第一极体排出率。将山羊卵母细胞在最佳ROS处理方案下抑制培养后进行孤雌激活，观察孤雌胚的发育情况。【结果】成熟培养液中加入40 μmol/L ROS抑制培养山羊卵母细胞8 h后再转入常规培养液中培养至24 h，是比较理想的ROS处理方案。山羊卵母细胞成熟培养的16 h以前，全程抑制和抑制8 h再常规培养2个试验组第一极体排出率与常规对照组无显著差异；分别培养18，20，22 h时，2个试验组第一极体排出率均极显著低于对照组；24 h时，抑制8 h再常规培养组第一极体排出率(58.76%)与对照组(63.13%)接近，无显著差异。试验组成熟卵母细胞孤雌激活胚的囊胚率(46.75%)显著高于对照组(36.04%，P＜0.05)。【结论】山羊COCs成熟培养液中加入40 μmol/L ROS培养8 h再转入常规培养液培养至24 h，是山羊卵母细胞核质同步成熟较适宜的方案，此方案可以有效抑制18～22 h核成熟卵母细胞的减数分裂恢复，推迟4～6 h成熟，达到培养24 h核质同步成熟的目的，提高了体外培养卵母细胞的质量。 |
| 关键词: Roscovitine 卵母细胞 体外成熟 山羊 |
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| 基金项目:转基因生物新品种培育重大专项(2009ZX08008-010B)；家畜疫病病原生物学国家重点实验室开放基金项目(SKLVEB2010KFKT005) |
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| Effects of roscovitine on maturation and parthenogenetic embryo development capacity of goat oocytes in vitro |
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| Abstract: |
| 【Objective】The study was to investigate the effects of roscovitine(Ros),a potent,selective inhibitor of cyclin-dependent kinases on maturation and parthenogenetic embryo development in vitro of goat oocytes.【Method】Cumulus oocyte complexes (COCs) were incubated in the maturation medium containing different concentrations of ROS (0(control),20,40,80,120,160 and 200 μmol/L) for 24 h,the percentage of polar body 1 extrusion was counted,then the efficient concentration of ROS was preliminary selected;COCs were incubated in the medium containing 0(control),40,80,120,160 and 200 μmol/L of ROS for 8 h or 16 h then transferred into the normal maturation medium and cultured to 24 h to determine the feasible working concentration and time of ROS based on the percentage of polar body 1 extrusion;The goat oocytes were incubated in the medium containing 40 μmol/L of ROS for 8 h,then transferred into normal maturation medium to incubate for 0,2,4,6,8,10,12,14 and 16 h respectively.The percentage of polar body 1 extrusion was calculated.Under the optimal protocol,the developmental capacity of embryo following parthenogenetic activation from the matured oocytes was evaluated in vitro.【Result】Oocytes of goats were incubated in normal maturation medium containing 40 μmol/L of ROS for 8 h,then transferred into normal maturation medium and cultured to 24 h.This was the optimal protocol of the working concentration and inhibition time of ROS for inhibition on meiotic resumption of goat oocytes.Oocytes were cultured for less than 16 h.There was no statistic significant difference between the experimental groups (including 24 h full-range inhibition,and inhibition for 8 h then following the normal maturation incubation) and the control groups.The percentage of the polar body 1 extrusion of the 2 experimental groups was significantly lower than the control groups at 18,20 and 22 h;However,the percentage of the polar body 1 extrusion was 58.76% which was very close to the rate of control group (63.13%),and there was no statistic difference.Subsequently,the blastocyst rate (46.75%) had significant difference with the control group (36.04%) of the parthenogenetic embryo from the activated goat oocytes of experimental groups（P＜0.05）.【Conclusion】It was the optimization protocol of nuclear and cytoplastic synchronized maturation of goat oocytes that oocytes were cultured in the normal maturation medium containing 40 μmol/L of ROS for 8 h,then cultured in normal maturation medium to 24 h;It effectively inhibited nuclear maturation of oocytes after cultured for 18 h to 22 h,delayed the meiotic resumption about 4 h to 6 h for achieving the purpose of 24 h nuclear and cytoplastic synchronized maturation,thus improved the quality of oocytes in vitro. |
| Key words: Roscovitine oocytes maturation in vitro goat |
