小鼠不同卵龄卵母胞细纺锤体位置及对去核效率的影响

探讨小鼠不同卵龄卵母细胞纺锤体位置与去核效率之间的关系。用Spindle-View观察小鼠不同卵龄卵母细胞纺锤体位置,然后对其进行去核操作。观察发现卵龄10,12h的纺锤体处于I,II,III区的比率分别为95%,3%,2%;33%,33%,23%。两组进行盲吸去核,去核率分别为90%和22%。在Spindle-View下去核卵龄10h的卵母细胞去核率为95%,其中有56%(n=59)的卵母细胞去不到1/6的卵胞质就可去核,而卵龄12h的卵母细胞去核率为87%,其中有76%(n=85)的卵母细胞吸去1/3的卵胞质才可去核。小鼠卵母细胞不同卵龄对纺锤体位置和核移植程序中去核效率有显著影响。     

细胞松弛素B对猪卵母细胞去核的影响

适宜的细胞松弛素B(CB)浓度可提高卵母细胞的成功去核率。将有明显第一极体排出的卵母细胞放入含有3.5×10-3、5×10-3、6.5×10-3、7.5×10-3mg.ml-1CB的NCSU37成熟培养液中孵育30min,采用盲吸法去核,将去核后的卵母细胞置入含5×10-3g.ml-1的Hoechst33342培养液中染色15min,用荧光显微镜检验去核率。结果表明:CB处理组去核率显著高于对照组(P<0.05)。不同浓度梯度处理组中,CB浓度过低(3.5×10-3mg.ml-1)或过高(7.5×10-3mg.ml-1)都使去核成功率(54.44%,51.46%)下降,溃破率(28.21%,34.11%)增高,与5×10-3、6.5×10-3mg.ml-1组的去核成功率(75.83%,73.83%)、溃破率(9.86%,11.62%)差异显著(P<0.05),且5×10-3mg.ml-1组的去核成功率高于6.5×10-3mg.ml-1组,差异不显著(P>0.05)。证明5×10-3mg.ml-1浓度的CB有利于第一极体和核质的吸出,同时可降低细胞膜撕裂,胞质破溃流出,获得较高的去核效率。     

改进的末期去核方法解决延边黄牛卵母细胞第一极体残留问题

传统的末期去核方法仅仅去掉了第二极体及其附近少量的细胞质,第一极体仍然残留在卵母细胞中.本研究中,将末期去核方法进行了改进,即在去除第二极体及其附近少量胞质的同时去除第一极体.采用改进的末期去核法和传统的末期去核方法对延边黄牛卵母细胞进行去核操作,比较两种方法的去核率以及重组胚后期发育的整体效率.改进的末期去核法在去核率[(90.51±3.05)%]以及囊胚形成率[(8.78±2.38)%]上与传统的方法[分别为(87.00±1.82)%、(6.56±2.95)%]相似;传统的末期去核法在去核操作上耗时较长[(44.13±2.60)s],但在注核操作上用时略短于改进的末期去核法[(30.33±1.17)s];改进的末期去核方法所构建的重组胚异常发育率比传统的末期去核法低[(18.02±2.83)%],但差异不显著.改进的末期去核方法,去除第一、第二极体及其附近少量细胞质,可达到较高的去核率,并且支持发育到囊胚,同时解决了第一极体残留的问题.     

小鼠卵母细胞去核时间对卵丘细胞核移植的影响

对超数排卵的小鼠分别在注射HCG后14~16,16~18,18~20和20~22h采小鼠卵母细胞用于核移植,观察小鼠卵母细胞的去核时间在体细胞核移植中的作用。结果显示,在注射hCG后14~16,16~18,18~20和20~22h,小鼠卵母细胞去核效率分别为80.09%(338/422),73.12%(283/387),55.02%(230/418)和48.74%(193/396);重构胚激活率分别为66.20%(190/287),75.43%(175/232),84.78%(156/184)和85.14%(126/148);重构胚的囊胚发育率分别为13.94%(40/287),7.33%(17/232),3.80%(7/184)和1.35%(2/148)。试验证实小鼠卵母细胞在注射hCG后14~16h进行去核较为适宜。     

猪卵母细胞第1极体与核相对位置变化对去核率的影响

为探讨猪受体卵母细胞体外成熟过程中第1极体(pbⅠ)与细胞核相对位置的动态变化规律及其对去核效率的影响,准确判定受体卵母细胞去核"窗口期"提供试验依据。将体外采集的GV(Germinal vesicle)期卵母细胞随机分为3组,分别成熟培养42～44 h、44～46 h、46～48 h,并采用Hoechst33342荧光染色法对成熟过程中pbⅠ与细胞核相对位置进行检测判定。结果显示,随着体外成熟时间的延长,卵母细胞的成熟率呈逐渐增加的趋势,但pbⅠ与细胞核的位置发生偏离的比率逐渐增加,而去核率则表现为逐渐降低的趋势,pbⅠ偏离率与去核率之间呈现一定程度的负相关;体外成熟44～46 h,既能获得较高的卵母细胞成熟率(80.2%),又可以保证理想的去核率(81.2%)。说明延长体外成熟时间所造成的去核率显著降低与pbⅠ发生偏离有关,体外成熟44～46 h是该试验条件下猪受体卵母细胞去核的最佳"窗口期"。     

小鼠卵母细胞去核方法的初步研究

[目的]寻求既简单又有效的提高小鼠卵母细胞去核率的方法。[方法]分别采用3种不同的方法(盲吸法、点击法、负压-点压法),研究对MII期小鼠卵母细胞去核率的影响。[结果]结果表明,采用负压-点压法去核,其卵母细胞的去核率可达62.8%,与其他两种方法所获得的去核率差异显著(P<0.05)。[结论]负压-点压法是一种简单、有效的小鼠卵母细胞去核方法。     

减少细胞质对激活小鼠卵母细胞体外发育的影响

 以溶液的表面张力为主要动力 ,辅以显微操作法去除一定量的卵母细胞胞质。测量了去除的细胞质体的直径 ,第一组为 38.6 4 μm ,第二组为 6 1.5 2 μm。小鼠卵母细胞的平均直径为 80 μm ,则第一组细胞质体占卵母细胞体积的 11.3% ,第二组细胞质体占卵母细胞体积的 4 5 .5 %。去除细胞质后两组卵母细胞均被氯化锶激活 ,其培养后卵裂率没有显著差异 (80 .0 %vs 77.9% ) ,两者都显著低于未经去质处理的激活卵母细胞和受精卵的卵裂率。两者的囊胚发育率虽没有显著差异 (37.3%vs 36 .8% ) ,但均显著低于未经去质处理的激活卵母细胞和受精卵的囊胚发育率 (4 8.3%和 73.3% )。去除 11.3%和 4 5 .5 %细胞质的卵母细胞孤雌激活胚、未经去质处理的激活卵母细胞和受精卵 ,体外培养的囊胚细胞数 (17.2 8± 7.16 ,11.5 0± 6 .83,2 1.86± 7.5 2 ,4 4 .6 7± 6 .86 ) ,各组间差异显著。     

猪体细胞克隆研究初探

本研究对猪卵母细胞的体外成熟和猪颗粒细胞核移植进行了初步探索试验一证明,体外成熟48h,来自3-6mm大小卵泡的卵母细胞成熟质量较好,当卵泡较大或较小时,成熟质量较差;实验二表明,当卵母细胞成熟液添加7%浓度的发情母牛血清时,成熟效果较好;实验三证明,在猪的克隆过程中,去核时当吸掉1/4卵母细胞胞质时重构胚的分裂率是12.8%,显著高于吸掉1/3卵母细胞胞质时2.5%的分裂率.     

牛卵母细胞的去核与激活

比较了Ionomycin(离子霉素)和乙醇及其与其他物质的组合激活牛卵母细胞的效果,并研究了去核时间、细胞松弛素B、末期去核等对去核效率的影响。结果表明,配合使用6-二甲氨基嘌呤(6-DMAP)或放线菌酮(CHX)可显著提高Ionomycin或乙醇激活牛卵母细胞的效率,Ionomycin+6-DMAP激活的牛孤雌胚在8/16细胞之后的发育率明显高于乙醇+6-DMAP(或CHX)。牛卵母细胞的去核在成熟培养20h或之前进行效果较好,此时有较高比率的卵母细胞中第一极体与染色体位置靠近。牛卵母细胞在不含细胞松弛素B的操作液中可进行去核,但所得核移植胚胎的卵裂率明显下降,而对8/16细胞胚之后的发育没有明显影响。牛卵母细胞的末期去核率显著高于中期,二者核移植胚胎的早期发育率基本相同。     

猪卵母细胞孤雌激活方法及影响因素的研究

 研究了离子霉素、电刺激、二硫苏糖醇、6-DMAP和硫柳汞等单独或结合使用及极体形态对猪卵母细胞的激活率、原核类型及激活卵卵裂的影响。结果表明:(1) 单独用离子霉素(88.9%)或电脉冲(80.0%)处理卵母细胞的激活率与二者同6-DMAP结合处理的激活率(84.3%和79.1%)差异不显著,但6-DMAP处理明显(P<0.05)提高电激活卵的1PN比率,而对离子霉素激活卵作用不明显。(2) DTT后处理显著提高硫柳汞处理对卵母细胞的激活率(由4.6%提高到82.6%)。(3) 极体皱缩(85.4%)和极体     

Effects of Ooplasmic Transfer on Rabbit Oocyte Fertilization and Early Embryonic Development

In order to evaluate the effects of ooplasm on oocyte fertilization and early embryonic development and to study the mitochondrial DNA (mtDNA) heterogeneity of early embryos, microinjection was first performed to transfer a small amount (5 to 7%) of donor ooplasm into recipient oocytes, then the eggs were fertilized with rabbit sperm through intracytoplasmic sperm injection (ICSI). In group 1 (homogeneous ooplasmic transfer), both the donor and recipient rabbit oocytes were at metaphase Ⅱ (MⅡ). In group 2 (heterogeneous ooplasmic transfer), the donor was mouse MⅡ oocyte and the recipient was rabbit MⅡ oocyte. In the control group, only ICS! was done on rabbit oocyte without ooplasmic transfer.No significant difference (P＞0.05) was observed in blastocyst development rates between group 1 (13.0%, 3/23) and the control group (16.7%, 4/24), but significant difference (P＜0.05) was examined in blastocyst development rate between group 2 (0, 0/27) and the control group. Blastomeres cleaved unequally and embryonic fragments increased after ooplasmic transfer and ICSI. In early embryos, in group 2, donor mouse mtDNA was detected in 2-cell embryos (3/3), 4-cell embryos(3/4), 8-cell embryos (4/4), and morulae (2/2). The mtDNA fingerprinting analysis showed that mouse mtDNA detected in heterogeneous embryos of different developmental stages had exactly the same sequence as that of the donor mouse mtDNA, thus indicating that homogenous ooplasmic transfer had no significant influence on rabbit oocyte fertilization and early embryonic development, and that heterogeneous ooplasmic transfer did cause notable reduction in blastocyst development rate. Heterogeneous mtDNA sequence in early embryos did not mutate. Compared with the control group,the embryonic quality declined after ooplasmic transfer operation in the present experiment.     

印迹基因Igf2r的甲基化进程与兔卵母细胞直径关系的研究

本研究以3～4月龄兔为研究对象,收集卵母细胞,按直径将其划分为75～95μm、95～105μm、105～115μm3个组,通过亚硫酸盐测序法检测了印迹基因Igf2r的甲基化程度,并运用RT-PCR对Dnmt1、Dnmt3a、Dnmt3b、Dnmt3l、Lsh的表达量进行检测。结果表明,在3组卵母细胞内,Igf2r的甲基化比率依次为15.7%、60%、91.5%,甲基化程度随卵母细胞直径的增加而不断提高。     

绵羊卵巢卵母细胞体外成熟培养的研究

研究了绵羊卵巢卵母细胞体外成熟培养,并就影响体外成熟的因素进行了探讨。试验表明:添加10%和15%的胎牛血清(FCS)绵羊卵巢卵母细胞的体外成熟率(81.25%和83.75%)明显高于不添加FCS的对照组的卵母细胞成熟率(65.00%);在基础培养基中10%的FCS存在的条件下,添加一定质量分数的促卵泡素(FSH)和促黄体素(LH)时,卵母细胞的体外成熟率明显高于不添加FSH和LH的对照组的绵羊卵母细胞的体外成熟率;培养24 h和26h卵母细胞体外成熟率(75.00%和80.00%)明显高于培养20 h的卵母细胞体外成熟率(63.30%);从3~6 mm卵泡中抽取的卵母细胞体外成熟率(81.50%)明显高于从1~3 mm卵泡中抽取的卵母细胞的体外成熟率(53.30%)。     

脉冲场强和波形对小鼠卵母细胞电活化效果的影响

实验以一定持续时间与不同场强搭配的一次电脉冲对注射ｈＣＧ后１７和１９ｈ的小鼠卵母细胞进行电刺激，研究脉冲场强和波形对印母细胞活化效果的影响．结果说明：（１）在一定持续时间下，只有当场强增至一定值时才获得较高活化率（ｈＣＧ后１７ｈ卵，４０％～５５％左右；ｈＣＧ后１ｇｈ卵，４２％～７０％左右）；（２）在某一持续时间，获得较高活化率的场强不止一个，而是有一定范围；低于这一范围的场强因刺激强度不够至使许多卵母细胞不能活化，高于这一范围的场强则因刺激强度过大造成卵大量死亡而使活化率下降；（３）随脉冲持续时间倍减，获较高活化率的场强约以０．２ｋＶ／ｃｍ的强度递增；（４）与注ｈＣＧ后１７ｈ卵相比，ｈＣＧ后１９ｈ卵获较高活化率的场强范围更宽；（５）在同等条件下，不规则波形脉冲的卵活化率、碎裂率和死亡率都明显高于方波脉冲．     

卵母细胞体外成熟时间对绵羊核移植效率的影响(英文)

[Objective] The study aimed to provide references for the time of oocyte maturation in vitro and enucleation in the course of sheep nuclear transfer(NT).[Method] Compared the effects of different maturation time of oocytes on enucleation efficiency and reconstructed embryo development by means of blind enucleation and fluorescence microscopy.[Result] Treatment of IVM(in vitro maturation)19-21 h was significantly higher than IVM 16-18 h treatment in oocyte maturation rate(P<0.05)and was significantly higher than IVM 22-24 h treatment in enucleation rate(P<0.05).Three treatments had no significant difference in cleavage rate and blastocyst rate(P>0.05),but IVM 19-21 h treatment was significantly higher than the other 2 treatments in average cell number of blastocysts(P<0.05).[Conclusion] The appropriate in vitro maturation time of oocytes was 19-21 h for sheep nuclear transfer,which could significantly improve the quality of blastocysts according to the cell number per blastocyst(P<0.05).     

Effects of in Vitro Maturation Time of Oocytes on Sheep Nuclear Transfer Efficiency

[Objective] The study aimed to provide references for the time of oocyte maturation in vitro and enucleation in the course of sheep nuclear transfer(NT).[Method] Compared the effects of different maturation time of oocytes on enucleation efficiency and reconstructed embryo development by means of blind enucleation and fluorescence microscopy.[Result] Treatment of IVM(in vitro maturation)19-21 h was significantly higher than IVM 16-18 h treatment in oocyte maturation rate(P<0.05)and was significantly higher than IVM 22-24 h treatment in enucleation rate(P<0.05).Three treatments had no significant difference in cleavage rate and blastocyst rate(P>0.05),but IVM 19-21 h treatment was significantly higher than the other 2 treatments in average cell number of blastocysts(P<0.05).[Conclusion] The appropriate in vitro maturation time of oocytes was 19-21 h for sheep nuclear transfer,which could significantly improve the quality of blastocysts according to the cell number per blastocyst(P<0.05).     

卵母细胞体外成熟时间对绵羊核移植效率的影响

[目的]为绵羊克隆试验中卵母细胞的体外成熟及去核时间提供参考。[方法]利用盲吸法结合荧光显微镜检查,对绵羊不同成熟时间卵母细胞去核的效率及其后续重构胚的发育进行对比。[结果]体外成熟培养19~21 h的绵羊卵母细胞在成熟率上显著高于体外成熟培养16~18 h(P(0.05),在去核成功率上显著高于体外成熟培养22~24 h(P(0.05);3个试验组在卵裂率、囊胚率上差异不显著(P(0.05),但是体外成熟培养19~21 h的试验组囊胚平均细胞数要显著高于其他2组(P(0.05)。[结论]体外成熟培养19~21 h的卵母细胞较适于作为受体细胞进行绵羊核移植,可以显著提高囊胚的质量。