冷冻保存能降低绵羊卵母细胞受精能力  

Parthenogenetic activation induced by vitrification solution can decrease sperm penetration rate of in vitro matured ovine oocytes

作　　者：田树军[1,2,3] 孙树春[1] 闫长亮[2] 朱士恩[2] 杨慧欣[1] 

机构地区：[1]河北农业大学动物科技学院,河北保定071001 [2]中国农业大学动物科技学院,北京100094 [3]河北省牛羊胚胎工程技术研究中心,河北保定071001

出　　处：《中国兽医学报》2008年第10期1233-1238,共6页Chinese Journal of Veterinary Science

摘　　要：本实验研究了体外成熟绵羊卵母细胞在含抗冻保护剂DMSO和EG的冷冻液中暴露和OPS法玻璃化冷冻保存后对体外受精卵裂率、精子入卵率、皮质颗粒分布、酶溶解透明带时间及雌原核形成的影响。将体外成熟24h的绵羊卵母细胞分为3组:(1)对照组,卵母细胞不进行处理;(2)毒性组,卵母细胞在冷冻液中进行暴露但不投入液氮中冷冻;(3)冷冻组,卵母细胞利用OPS法进行玻璃化冷冻。处理组卵母细胞在浓度递减的蔗糖溶液中脱除抗冻保护剂。结果,卵母细胞体外受精的卵裂率和单精子入卵率,毒性组(62.3%和29.3%)和冷冻组(67.6%和28.2%)显著低于对照组(78.4%和45.0%)(P<0.05),毒性组和冷冻组间无显著差异(P>0.05)。为了研究冷冻保存导致卵母细胞受精能力降低的机制,处理组及对照组一部份卵母细胞分别于处理后0h(IVM24h)、2h(IVM26h)和体外受精后12h(IVF12h)测定皮质颗粒分布和用0.1%链霉蛋白酶溶解透明带时间,另一部份卵母细胞则在不含有精子的受精液中孵育12h后测定雌原核形成率。结果,在IVM24h和IVM26h,皮质颗粒呈完全释放的比例,毒性组(41.2%和40.8%)和冷冻组(41.7%和51.8%)显著性高于对照组(7.1%和18.4%)(P<0.05);IVM26h酶溶解透明带时间,毒性组(435.6±16.6)s和冷冻组(422.3±14.6)s显著长于对照组(381.6±15.3)s(P<0.05);雌原核形成率,毒性组(58.7%)和冷冻组(63.9%)组显著高于对照组(8.2%)(P<0.05)。上述结果表明,含DMSO和EG的冷冻液对体外成熟绵羊卵母细胞具有孤雌激活作用,引起皮质颗粒的提前释放,导致透明带变硬,降低卵母细胞的受精能力。The present research was designed to investigate the effect of cryoprotectant agents （DMSO and EG）, exposure or vitrification on the cleave rates in vitro fertility, the sperm penetration, cortical granules （CG） migration, dissolution time of zona pellucida （ZP） and female pronucleus formation of in vitro matured ovine oocytes. After in vitro maturation for 24 h, the oocytes were randomly allocated into three groups： untreated （control） ;exposed to vitrification solution （VS） without being plunged into liquid nitrogen （toxicity）;vitrified by open-pulled straw method （vitrification）. The treated oocytes were diluted in sequential sucrose solutions. The results showed that the cleave rates in vitro fertility and the rates of monospermy in toxicity group （62. 3% and 29. 3%） and vitrification group （ 67.6 % and 28.2 % ） decreased as compared with the control group （ 78.4 % and 45.0 % ） （ P〈0.05）, and they were similar between the treated groups （P〉0.05）. To find the reason that the VS containing DMSO and EG decreased the monospermy rates,a part of oocytes were used to test the distribution of CG and the resistance of ZP to 0. 1% pronase immediately （IVM 24 h） ,after another 2 h of incubation （IVM 26 h） ,and after 12 h of in vitro fertilization （IVF 12 h） respectively. Another part of oocytes were used to examine the female pronucleus formation rates after 12 h of culture in fertilization medium with the absence of sperm. The results showed that the rates of CG complete release in the oocytes （IVM 24 h and 26 h） of toxicity group （41.2% and 40.8%） and vitrification group （41.7% and 51.8%） were higher than that of control group （7. 1% and 18.4%）（P〈0.05）. The ZP digestion time in the oocytes （IVM 26 h） of the toxicity group （435.6±16.6） s and vitrification group （422.3±14.6） s was longer than that of control group （381.6±15.3） s （P〈0.05）. The rates of female pronueleus formation in toxicity group （58. 7%）

关 键 词：绵羊 卵母细胞 DMSO EG 玻璃化冷冻 孤雌激活 

分 类 号：S826.35[农业科学—畜牧学]