猪卵泡卵母细胞体外成熟与冷冻保存

研究了激素、猪卵泡液、不同类型血清对猪卵泡卵母细胞体外成熟的影响 ;比较了卵泡直径小于 2 mm、2～ 5 mm和大于 5 mm的卵母细胞体外成熟能力的差异 ,并对不同发育阶段猪卵母细胞的冷冻保存进行了研究。结果表明 :猪卵母细胞体外培养 4 8h时 ,培养的前 2 4 h培养液中加入激素 ,后 2 4 h去掉激素 ,卵母细胞的 A级成熟率 (5 1.73% )和总成熟率 (83.2 5 % )最高 ,极显著高于前 2 4 h不加激素 ,后 2 4 h添加激素培养的成熟率 (P<0 .0 1) ;也显著高于不含激素的培养液连续培养 4 8h的成熟率 (P<0 .0 5 ) ;但与添加激素连续培养 4 8h的成熟率差异不显著 (P>0 .0 5 )。在体外成熟培养液中 ,添加 10 % (体积分数 ) ECS的成熟率 (72 .86 % )显著高于添加 10 % (体积分数 ) NCS的成熟率(6 2 .2 1% ) (P<0 .0 5 ) ,而添加 10 % p FF则抑制卵母细胞的体外成熟。随着卵泡直径的增大 ,卵母细胞体外成熟能力逐渐增强。采用程序冷冻保存方法 ,成熟卵母细胞的成活率 (35 .5 9% )显著高于培养前 (2 4 .6 4 % )和培养 2 4 h(2 3.36 % ) (P<0 .0 5 )。培养 2 4 h(77.2 2 % )和培养成熟 (72 .81% )的卵母细胞解冻后的形态完整率均显著高于培养前(5 3.2 4 % ) (P<0 .0 5 )     

Effect of Human Menopausal Gonadotropin,17β-estradiol and Epidermal Growth Factor on the Quality of in vitro Maturation Bovine Oocytes

For optimizing in vitro maturation system of bovine oocytes,we firstly examined the influence of four different hormonal regimes(FSH+LH,HMG,FSH+LH+E₂ and HMG+E₂) on oocyte maturation rates.Then we studied the effects of epidermal growth factor (EGF) in the above defined medium on bovine oocyte maturation,in vitro development and quality of parthenogenetic embryos.The cell apoptotic index of parthenogenetic blastocysts was detected by TUNEL.No significant difference was observed in maturation rates in four groups supplemented with different hormones.However,human menopausal gonadotropin (HMG) provided steady maturation results in replicates.Maturation of oocytes was promoted by supplementation with 17β-estradiol (E₂).Combination of HMG and E₂ gave rise to steady and efficient mature results.The presence of EGF at 30 ng/mL concentration significantly increased maturation rate and blastocyst rate and reduced apoptotic cells in parthenogenetic blastocysts.Therefore,the optimal oocyte maturation solution could be supplemented with 0.075 IU/mL HMG,1 μg/mL E₂ and 30 ng/mL EGF.     

白藜芦醇和褪黑素对绵羊胎儿成纤维细胞转染及卵母细胞体外成熟的影响

试验旨在利用白藜芦醇(Re)和褪黑素(MT)提高绵羊胎儿成纤维细胞电穿孔转染(电转)效率、优化卵母细胞体外成熟体系,以提高转基因动物生产效率。将绵羊胎儿成纤维细胞分为6组:对照组、添加白藜芦醇高、中、低(R^-5、R^-6、R^-7)3种浓度组及白藜芦醇和褪黑素联合添加组(R^-5M^-5、R^-6M^-7),通过电转效率和细胞凋亡情况确定电转体系优化方案;通过对细胞周期、线粒体膜电位及活性氧(ROS)的检测研究其作用机制。将绵羊卵母细胞分为对照组及白藜芦醇和褪黑素(R^-5M^-5、R^-6M^-7)联合添加组,通过绵羊卵母细胞体外成熟后卵丘细胞扩展、第一极体排出及体外受精胚胎的卵裂率检测其对卵母细胞体外成熟的作用。研究结果显示,分离的绵羊胎儿成纤维细胞P3代增殖旺盛,细胞生长曲线呈典型的S型;与对照组相比,所有试验组胎儿成纤维细胞的电转效率均显著提高(P<0.05),R^-5M^-5组电转效率最高;R^-6M^-7组显著降低细胞凋亡率(P<0.05),R^-5、R^-6、R^-5M^-5和R^-6M^-7组均显著促进细胞增殖(P<0.05);R^-6、R^-7、R^-5M^-5和R^-6M^-7组G1期细胞增加,S期细胞相对减少,但差异均不显著(P>0.05);R^-6、R^-7、R^-5M^-5和R^-6M^-7组成纤维细胞内ROS水平均显著降低(P<0.05),R^-6、R^-5M^-5和R^-6M^-7组线粒体膜电位均显著增加(P<0.05);R^-6M^-7组显著促进绵羊卵丘细胞扩展、提高卵母细胞第一极体排出率和体外受精胚胎发育能力(P<0.05)。综上,白藜芦醇和褪黑素可以优化绵羊胎儿成纤维细胞电转体系,提高卵母细胞体外成熟效率,为白藜芦醇和褪黑素在转基因动物生产中的应用提供参考依据。     

Effect of Psammaplin A on in vitro Development of Bovine Aging Oocytes

To investigate the effect of histone deacetylation inhibitor Psammaplin A (PsA) on the development of bovine aging oocytes in vitro,oocytes were randomly divided into control group,aging group and 50 mmol/L PsA treated aging group (PsA group).Immunofluorescence staining and JC-1 were used to detect the blastocyst rate of bovine oocytes after parthenogenetic activation,the number of cells in blastocysts,apoptosis,reactive oxygen species (ROS),glutathione (GSH) and mitochondrial membrane potential intensity of embryos.The results showed that the blastocyst rate of the aging group was significantly lower than that of PsA and control groups (P<0.05).The blastocyst rate of PsA group was not significantly different from that of control group (P>0.05).The number of cells in the blastocysts of control group and PsA group were significantly higher than that of aging group (P<0.05).The number of cells in the blastocysts of PsA group was not significantly different from that of control group (P>0.05).The apoptosis rate in aging group was significantly higher than that of control and PsA groups (P<0.05),the apoptosis rate of PsA group was significantly higher than that of control group (P<0.05).The GSH level of MⅡ oocytes in aging group was significantly lower than that of control and PsA groups (P<0.05).There was no significant difference in GSH level between control and PsA groups (P>0.05).The ROS level of the embryos in aging group was significantly higher than that of control and PsA groups (P<0.05).The ROS level in PsA group was significantly higher than that of control group (P<0.05).The mitochondrial membrane potential of early embryos of aging group 4-8 cells was significantly lower than that of control and PsA groups (P<0.05).The mitochondrial membrane potential intensity of control group was significantly higher than that of PsA group (P<0.05).In summary,PsA could effectively delay the aging of bovine oocytes and improve the quality of oocytes.     

GMP玻璃化冷冻未成熟牛卵母细胞核成熟及冷冻损伤试验

本试验通过荧光染色的方法建立了未成熟牛卵母细胞在体外培养过程中第1次减数分裂的各个阶段的参考判定图谱;根据这个标准来观察毛细玻璃管(GMP)玻璃化冷冻对卵母细胞核成熟和冷冻损伤的影响。结果表明,从屠宰场废弃的卵巢表面卵泡内抽取的COCs,70%处于生发泡期,12.5%生发泡开始破裂,7.5%已开始浓缩,这说明从屠宰场获得的COCs有较高的异质性;卵母细胞在成熟培养22h时收获排出第一极体的卵母细胞,可得到丰度较高的极体-胞质染色体对称、紧密相邻的成熟卵母细胞;GMP玻璃化冷冻损伤主要有2种表现形式,首先,直接影响膜结构的完整性,包括细胞膜和核膜,这可从退化的细胞比例看出(8～24h,有21.9%～27.2%的细胞处于该阶段),其次,影响CONDENSED向MⅠ期的过渡,这可从处于CONDENSED卵母细胞的比例看出(8～24h,有24.1%～34.3%的细胞处于该阶段)。     

水牛卵泡颗粒细胞对卵母细胞体外成熟的影响

为了系统研究颗粒细胞对水牛卵母细胞体外成熟的影响,使用颗粒细胞条件液处理或单层颗粒细胞和卵母细胞共培养的方法,探讨颗粒细胞共培养对水牛卵母细胞体外成熟和早期胚胎发育的影响。结果显示,添加颗粒细胞传代接种第2天收集的20%颗粒细胞条件液到水牛卵母细胞成熟液中能显著提高水牛卵母细胞体外成熟率和囊胚发育率(P<0.05);然而单层颗粒细胞却显著抑制水牛卵母细胞体外成熟和早期胚胎发育(P<0.05)。结果表明,与单层颗粒细胞共培养相比,与颗粒细胞条件液共培养更有利于水牛卵母细胞体外成熟和早期胚胎发育。本研究为水牛卵母细胞体外成熟培养体系的完善提供一定理论依据。     

山羊卵泡卵母细胞的采集及体外成熟效果研究

为了获取高质量的卵泡卵母细胞，提高山羊体外胚胎生产的效率，采用切剖法与抽吸法采集卵泡卵母细胞，并对获得的可用卵母细胞进行了体外成熟培养。结果表明，切剖法能够获得较多的可用卵母细胞，可以明显增加用于体外成熟的卵母细胞数；卵母细胞的体外成熟率与卵泡直径密切相关，大卵泡与中卵泡的卵母细胞成熟率明显高于(P<0.05)小卵泡；山羊卵泡卵母细胞的体外成熟培养液中FCS的添加比例以10% ~ 15%为益。在一定范围内，提高FCS的浓度，并不能提高卵母细胞的成熟率。     

水牛卵母细胞孤雌激活及孤雌胚与体外受精胚发育比较

比较了水牛卵母细胞体外成熟时间对孤雌激活胚发育能力的影响,以及不同化学激活方法对水牛卵母细胞孤雌激活效果的影响,并在相同条件下,对孤雌激活胚与体外受精胚的发育能力进行了比较.结果表明,水牛卵母细胞体外成熟27 h或30 h的囊胚发育率(19.0%、17.7%)明显高于体外成熟21 h或24h的囊胚发育率(12.3%、13.8%);Ion联合6-DMAP激活水牛卵母细胞的效果优于其他几组激活方法;在相同条件下,孤雌激活胚与体外受精胚的发育能力存在着差异,其中卵裂率差异不显著,但孤雌激活胚的囊胚发育率显著高于体外受精胚.     

猪卵母细胞去核方法的改进

对猪体细胞克隆技术中关键步骤之一的去核方法作了较深入的研究。结果表明，由本实验室改进的Willadsen去核法——二步挤压去核法，无论在去核操作成功率（73．9％）上还是在去核效率（81．2％）上都明显好于McGrath-Solter去核法（42．5％、67．4％，P〈0．05）；同时，本试验还发现，卵母细胞成熟培养36h后去核组去核效率（89．1％）显著地高于成熟培养44h后去核组（55．8％，P〈0．05），而核移植重构胚的发育率2个组间无显著差异。