小鼠卵母细胞孤雌激活及孤雌胚体外培养研究

本研究比较了几种化学激活剂对小鼠卵母细胞孤雌激活的影响以及激活后在不同培养培养液中的发育情况,并探讨了一种有效地孤雌激活和体外培养方法。分析结果表明:乙醇 6-DMAP(2.5mmol/L,4h)和SrCl2 6-DMAP(2.5mmol/L,4h)组的卵裂率和囊胚率都显著高于其他各组,说明他们能够较好的激活小鼠卵母细胞,而且,乙醇组合优于SrCl2组合,单个化学激活剂不能完全激活小鼠卵母细胞;激活的昆白小鼠MⅡ卵母细胞在含15?S的CZB培养液中可较好地克服2-cell阻滞并形成桑囊胚,在培养48h添加1.1mg/mL葡萄糖对胚胎的继续发育有利。     

不同方法对滩羊卵母细胞孤雌激活及重构胚发育影响的研究

旨在探索宁夏滩羊卵母细胞孤雌激活及胚胎培养条件,并建立转Cherry基因滩羊体细胞重构胚的融合与体外培养体系。试验分析了3种激活方法即电激活、Ionomycin结合6-DMAP与电激活结合6-DMAP对成熟的滩羊卵母细胞激活的影响,以及3种胚胎培养液mSOFaa、M16和KSOM的培养筛选,并优化了滩羊体细胞重构胚的融合与体外培养条件。结果表明:在卵母细胞孤雌激活中,最适电压为1 600V/cm,获得卵裂率和囊胚率分别为58.5%和19.5%;5μmol离子霉素结合2 mmol 6-DMAP能有效地激活成熟的滩羊卵母细胞,卵裂率为81.0%,囊胚率为26.2%;并发现mSOFaa胚胎培养液的卵裂率和囊胚显著优于M16和KSOM培养液;在转基因重构胚融合中发现在电压1 800V/cm、脉冲时间10μs、脉冲次数2次和间隔时间为1s的条件下,卵裂率和囊胚率为40.0%、21.4%。本试验建立的滩羊孤雌激活和转基因重构胚融合与体外培养体系为宁夏滩羊的分子育种奠定了基础。     

6-DMAP对延边黄牛末期卵母细胞核移植的影响

为探讨6-二甲基氨基嘌呤（6-DMAP）在延边黄牛末期去核的体细胞克隆中的作用,本试验主要研究了单独使用离子霉素处理与离子霉素联合添加6-DMAP处理对体外成熟的老化延边黄牛卵母细胞的激活率的不同影响;以及不同时期添加6-DMAP对重构胚后期发育能力的影响。试验结果显示,体外成熟的老化卵母细胞,使用离子霉素单独处理后91%被激活,而在离子霉素联合添加6-DMAP处理后却没有细胞被激活,也就是没有第二极体的排出。另外,融合后使用6-DMAP处理,所得重构胚的囊胚发育率最低,而激活后的卵母细胞立即用6-DMAP处理,所得重构胚的发育能力与无6-DMAP处理的相近。综上所述,离子霉素联合添加6-DMAP可抑制老化的延边黄牛卵母细胞的激活,阻止第二极体的排出,但对重构胚的后期发育没有影响。而融合使用添加6-DMAP的培养液,抑制了重构胚的后期发育。     

电脉冲及6-DMAP对小鼠卵母细胞孤雌激活的研究

实验研究了不同电脉冲条件、6-DMAP作用不同时间以及二者联合使用时对注射hCG后18～19h采集的小鼠卵母细胞孤雌激活及发育的效果。结果表明：4、鼠卵母细胞电激活后，放入2mmol／L6-DMAP的CZB中作用6h，其激活率和囊胚率都显著高于单独使用一种激活方法。其中场强2．0kv／cm，脉宽80μS，3次脉冲结合6-DMAP组的激活率和囊胚发育率最高，与其他两组差异显著。     

大鼠卵母细胞孤雌激活方法的研究

本试验以Wistar大鼠为动物模型,研究了单独应用乙醇、电刺激、 6-DMAP以及乙醇/6 DMAP、 电刺激/6-DMAP 对大鼠卵母细胞激活的影响。结果表明,单独应用电刺激、乙醇、6-DMAP均能激活大鼠卵母细胞,但激活率较低,卵母细胞的发育不能超过2-细胞阶段。单独应用电刺激时,相同的场强条件下,电刺激2次的激活效果明显优于电刺激1次的激活效果,并且电刺激场强在100 V/mm时卵母细胞的激活率最高。乙醇或电刺激联合6-DMAP激活大鼠卵母细胞,激活率和后期囊胚发育率都明显优于单独使用1种激活方法激活的效果。     

水牛卵母细胞孤雌激活方法的优化

为了研究离子霉素(Ion)在不同环境下对水牛孤雌激活效果的影响及水牛卵母细胞在6-二甲基氨基嘌呤(6-DMAP)液中以聚集激活或分离激活方式对激活效果的影响。试验对比了水牛卵母细胞在25℃室温环境和38.5℃CO2培养箱环境下的Ion激活效果,还比较了水牛卵母细胞在6-DMAP液中进行聚集激活和分离激活的孤雌激活效果。结果表明:试验组8-细胞胚胎率(54.11%)和囊胚率(31.51%)均高于对照组的8-细胞胚胎率(51.68%)和囊胚率(27.52%),但无统计学差异;在6-DMAP液中分离激活组(1组)的囊胚率明显优于聚集激活组(2组)的囊胚率。     

电脉冲及6-DMAP对小鼠卵母细胞孤雌激活的研究

实验研究了不同电脉冲条件、6-DMAP作用不同时间以及二者联合使用时对注射hCG后18～19h采集的小鼠卵母细胞孤雌激活及发育的效果。结果表明:小鼠卵母细胞电激活后,放入2mmol/L6-DMAP的CZB中作用6h,其激活率和囊胚率都显著高于单独使用一种激活方法。其中场强2.0kv/cm,脉宽80μs,3次脉冲结合6-DMAP组的激活率和囊胚发育率最高,与其他两组差异显著。     

山羊卵母细胞孤雌激活及孤雌胚的体外培养

为了比较不同的激活方法对卵母细胞孤雌激活的影响,以及培养液(CR1aa)中添加不同类型的血清对孤雌激活胚体外发育的影响。采用Eth 6-DMAP、A23187 6-DMAP和Ion 6-DMAP三种方法激活山羊卵母细胞,Eth 6-DMAP组孤雌激活胚的卵裂率和囊胚率(35.2%和4.8%)显著低于A23187 6-DMAP组(68.9%和24.1%)和Ion 6-DMAP组(88.1%和48.0%),表明以Ion 6-DMAP激活最为理想。在培养液(CR1aa)与颗粒细胞共同培养条件下,分别添加100 mL/L的发情牛血清(OCS)、胎牛血清(FBS)和新生牛血清(NCS)。添加OCS和FBS后,孤雌胚囊胚率分别为48.1%和45.0%,显著高于添加NCS组(26.0%)。表明OCS、FBS和NCS能有效的提高孤雌胚的体外发育能力,尤其是对提高孤雌胚的囊胚发育能力更佳。     

不同激活条件及半胱氨酸处理对猪ICSI胚胎发育影响研究

本试验探讨了不同辅助激活方法(Calciumionophore A23187激活、Calciumionophore A23187+6-DMAP联合激活和电激活)、不同精子预处理方法(液氮冻融处理和0.1%Triton X-100处理)和在添加半胱氨酸的胚胎培养液中培养不同时间(0 h、4 h、12 h和168 h)对猪卵母细胞内单精子注射(ICSI)胚胎体外发育的影响。结果显示:与无辅助激活相比,A23187+6-DMAP联合激活和电激活均能显著提高ICSI卵母细胞的激活率、卵裂率和囊胚率(P0.05),A23187+6-DMAP联合激活能显著提高ICSI卵母细胞的受精率(P0.05)。液氮冻融精子组ICSI卵母细胞的雄原核形成率显著高于活精子组(P0.05)。在添加半胱氨酸的胚胎培养液中培养4 h的ICSI卵母细胞受精率、雄原核形成率和囊胚率显著高于0 h组(P0.05)。以上结果表明,猪卵母细胞在ICSI后需要辅助激活来启动胚胎顺利发育,A23187+6-DMAP激活效果较好。液氮冻融精子可以促进ICSI后雄原核的形成。半胱氨酸处理4 h对猪ICSI卵母细胞受精和发育均有促进作用。     

不同化学激活方法和电激活参数对猪体外成熟卵母细胞早期孤雌发育的影响

利用屠宰场猪卵巢卵母细胞,在体外成熟培养44～48 h后,对核成熟卵母细胞进行激活.试验1为不同化学激活方法10% 乙醇 10 mg/L 放线菌酮组、2.5 mmol/L 氯化锶 10 mg/L放线菌酮组、5 μmol/L离子霉素 2.5 mmol/L 6-二甲氨基嘌呤(6-DMAP)组、200 μmol/L 硫柳汞 8 mmol/L二硫苏糖醇组和对照组(不用任何激活剂).试验2为用不同电场强度和脉冲时程进行电激活之后放入胚胎培养液中进行体外培养7 d.结果显示,(1)4种不同化学激活方法处理卵子的1原核形成率、2原核形成率和原核形成率都显著比对照组高(P＜0.05),其中离子霉素 6-DMAP组的2原核形成率和总原核形成率最高,分别为(23.1±3.5)%和(65.2±3.5)%,显著高于其他处理组(P＜0.05);(2)4种不同化学激活方法处理组的囊胚率都比对照组高(P＜0.01),其中离子霉素 6-DMAP组的卵裂率及囊胚率最高,分别为(46.6±18.5)% 和(5.6±4.2)%;(3)本试验所用的4种不同化学激活方法对猪4-细胞孤雌胚SDS-PAGE电泳的蛋白质表达图谱没有显著影响;(4)电场强度为1.7 kV/cm、脉冲时程为50、70 μs时猪体外成熟卵母细胞激活效果最好,卵裂率、囊胚率、囊胚细胞数分别为(77.4±9.7)%、(12.4±3.7)%、(17.6±5.9)%和(75.1±10.6)%、(12.3±2.6)%、(19.1±8.1).以上结果说明本试验所用的4种化学激活方法均能有效激活猪体外成熟卵母细胞,其中离子霉素 6-DMAP的激活效果最理想;在本实验室条件下,采用1.7 kV/cm的电场强度、50～70 μs的脉冲时程均能有效地激活猪体外成熟卵母细胞;本试验所用的4种不同化学激活方法对猪4-细胞孤雌胚SDS-PAGE电泳的蛋白质表达图谱没有显著影响.     

绵羊卵母细胞的孤雌激活

本文探讨了不同激活方法对绵羊卵母细胞的孤雌激活和其后的发育。结果表明 ,电激活可以激活绵羊卵母细胞孤雌发育到囊胚 ;Ca2 + 载体A2 3187和CHX组合 ,Ionomycin和 6 DMAP组合可以激活绵羊卵母细胞 ,其卵裂率与电激活相比差异显著。 7%乙醇激活绵羊卵母细胞 7min效果较好。不同场强、不同脉冲次数对绵羊卵母细胞激活都有影响 ,以 1.2kV/cm ,间隔 30 μs和 3次脉冲效果较好。而电激活与化学激活联合可以更好的激活绵羊卵母细胞     

Determination of optimal conditions for parthenogenetic activation and subsequent development of rat oocytes in vitro

The present study was undertaken to determine optimal conditions for parthenogenetic activation and subsequent development of rat oocytes. Oocytes from immature Wistar-Imamichi (WI) and Sprague Dawley (SD) rats were activated by electrical stimulation in combination with 6-dimethylaminopurine (6-DMAP) to assess whether different rat strains display different responses to activation treatment. Since the cleavage rates of activated oocytes were significantly higher in WI than SD strain rats, WI rats were used for the subsequent experiments to determine the effects of post-hCG time, culture duration, different activation protocols (electrical stimulation with 6-DMAP or ionomycin with 6-DMAP) and osmolarity of the activation medium on the activation and subsequent development of WI rat oocytes. For oocytes activated by electrical stimulation combined with 6-DMAP, the percentages of oocytes that were activated and that developed to blastocysts were higher when oocytes were collected at 18-20 h than at any other time points after hCG injection (16, 22-24 h). Culturing for 2-6 h before activation treatment markedly decreased the percentage of activated oocytes that developed to beyond the four-cell stage. There were no differences in the percentages of oocytes with pronuclear formation and subsequent development to the two-cell and blastocyst stages between oocytes that were activated by electrical stimulation or ionomycin, both followed by 6-DMAP treatment. Activation of oocytes by ionomycin and 6-DMAP, both in low osmolarity media (246 mOsM), markedly increased the cleavage rates and percentages of high quality blastocysts (71%). The optimal conditions determined in the present study with simplified activation protocols and high efficiency of activation and subsequent development of WI rat oocytes will be helpful for further research involving nuclear transfer in the rat.     

黄淮白山羊孤雌胚胎的体外培养

采用离子霉素和6-DMAP对黄淮白山羊体外成熟卵母细胞进行联合激活,分别在不同的培养体系进行体外培养,观察孤雌胚胎的发育情况。培养体系分别为:无共培养条件下,M199(10?S)、CR1aa和HTF P1;颗粒细胞共培养条件下,CR1aa、HTF P1和SOFaa。结果发现,无共培养条件下,CR1aa和HTF P1组孤雌胚胎的卵裂率显著高于M199组(P<0.05),但3组均未有囊胚;共培养条件下,HTF P1组的卵裂率显著高于CR1aa和SOFaa组(P<0.05),而CR1aa组的囊胚率却显著高于其他2组(P<0.05)。综合试验结果说明,CR1aa联合颗粒细胞共培养能够获得较好的培养效果,适用于山羊孤雌胚胎的体外培养。     

Survivability and developmental competences of domestic cat (Felis catus) oocytes after Cryotech method vitrification

The aim of this study was to evaluate the applicability of the Cryotech technique for the vitrification of domestic cat (Felis catus) oocytes, as a model for other feline species threatened with extinction. This technique, in which oocytes are stored in a minimal volume of medium, is already widely used in human assisted reproductive technology. In the first part of this study, a viability test (EtBr/FDA) was used to evaluate the toxicity of the vitrification media (solutions). After IVM, oocytes were placed in vitrification and warming solutions according to the manufacturer's procedure, with or without exposure to liquid nitrogen. The solutions and the vitrification procedure each caused a reduction in oocyte viability, with survival rates of 71.4% in oocytes exposed to the Cryotech media (without cooling in liquid nitrogen), and 62% in oocytes that were vitrified. In the second part of the experiment, parthenogenetic activation was used to evaluate the developmental potential of oocytes previously vitrified using the Cryotech method. After warming, the oocytes were activated using a combination of 0.7 µM ionomycin in TCM 199 medium (5 min) followed by 2 mM 6-DMAP in TCM 199 supplemented with 10% FBS (3 hr), then cultured and evaluated every 24 hr for parthenogenetic cleavage. In the experimental group, 23/50 (46%) cleaved embryos were obtained. Domestic cat oocytes, vitrified by the Cryotech method, are characterized by high survival rates. However, it is necessary to improve the technique to increase the developmental competence of embryos obtained from vitrified oocytes.     

Activation of Pig Oocytes using Calcium Ionophore: Effect of the Protein Kinase Inhibitor 6-dimethyl aminopurine

The aim of our study was to investigate the parthenogenetic activation of in vitro matured pig oocytes after their combined treatment with calcium ionophore A 23187 and the inhibitor of protein kinases, 6-dimethylaminopurine (6-DMAP) and to study the further embryonic development of oocytes activated using this treatment. The oocytes were exposed to ionophore (10, 25 or 50  μ M ) for 0.5, 1, 3, 5 or 7 min and then cultured with 6-DMAP (0 or 2  μ M ) The highest activation rate (up to 88% of the activated eggs reached the pronuclear stage) was observed after combined treatment of the oocytes with 50  μ M ionophore and 6-DMAP. The highest rate of embryonic development was observed after treatment with 25  μ M ionophore without 6-DMAP, when up to 51% of the eggs developed beyond two-cell stage, 2% of the eggs developed up to the stage of morula and up to 3% of the eggs reached the stage of blastocyst. When 50  μ M ionophore was used, the embryonic development of the activated eggs was arrested before the morula and blastocyst stage. After treatment of the activated eggs with 6-DMAP, we did not observe any development beyond the stage of 16 blastomeres. We can conclude that combined treatment with calcium ionophore A 23187 and 6-DMAP increases the activation rate in pig oocytes matured in vitro , but this combined treatment exerts a detrimental effect on further embryonic development of the activated eggs.     

Efficient strontium-induced activation of mouse oocytes in standard culture media by chelating calcium

Without using sperm, artificial oocyte activation is essential for current assisted reproductive technologies, particularly somatic cell nuclear transfer and round spermatid injection. Strontium has been widely used as an activator of oocytes especially in the mouse, by which efficient oocyte activation requires Ca(2+)-free medium. In this study, we examined whether Sr(2+) can efficiently activate oocytes in Ca(2+)-containing culture media when calcium is chelated. Ethylene glycol-bis (beta-aminoethyl ether) -N, N, N', N'-tetraacetic acid (EGTA) was added to three standard culture media (CZB, M16 and KSOM) for mouse embryos because it preferentially binds Ca(2+) rather than Sr(2+). We found that treatment with 5 mM Sr(2+) and 2 mM EGTA left fewer than 1% of oocytes at the MII stage, which is comparable to that of Ca(2+)-free medium. As a result, addition of 2 mM EGTA along with 5 mM Sr(2+) in either CZB, M16 or KSOM made more than 80% of available activated oocytes, which was comparable to or better than 72% in a Ca(2+)-free Sr(2+) medium, since EGTA-Sr(2+) activation led to significantly less oocyte degeneration than Ca(2+)-free Sr(2+) activation. Furthermore, we demonstrated that this activation method can support the birth of cloned embryos. Thus, addition of EGTA to typical Ca(2+)-containing culture media can easily produce activation media that does not interfere with embryonic development.     

Studies on Survival of Horse Oocytes After Rapid-i Method Vitrification

The aim of the present study was to determine the vitality and developmental competence of equine oocytes after in vitro maturation (IVM) and vitrified by Rapid-i method. In experiment 1, oocytes after IVM were vitrified using media: EquiPro VitKit (group 1) or medium containing 18% Ficoll, 40% ethylene glycol, and 0.3 M sucrose (group 2). For evaluation of toxicity effect, oocytes were exposed to media without a plug to liquid nitrogen. To evaluate viability, oocytes were stained with fluorescein diacetate and ethidium bromide. In experiment 2, oocytes after IVM and vitrification were activated by 7.5 μM ionomicin in TCM 199 (5 minutes) combined with 2 mM 6-DMAP in TCM 199 with 10% fetal bovine serum (4.5 hours). Survival rate was: 63% in group 1 (n = 54), 55% in group 2 (n = 69), and 73.2% (n = 56) in the control group. After parthenogenetic activation, 10.2% (n = 49) of 2–4 blastomeres were observed. This percentage was lower than in the nonvitrified group: 38.5% (n = 53).     

小鼠卵细胞体外成熟培养及孤雌激活

[目的]利用小鼠卵母细胞体外成熟培养及3种激活方法（乙醇激活、氯化锶激活、乙醇-氯化锶激活）对小鼠卵母细胞进行了孤雌激活研究。[方法]小鼠经注射孕马血清促性腺激素（PMSG）48h后,摘取卵巢获得3级未成熟卵母细胞．在成熟培养液（M199100mL＋丙酮酸钠2．2mg＋抗100IU／mL＋LH2IU／mL＋FCS10％）中对小鼠未成熟卵细胞进行体外成熟培养,获得的成熟卵母细胞分别在乙醇、氯化锶、乙醇一氯化锶不同激活剂中激活,[结果]A级卵母细胞成熟率为79％．B级卵母细胞成熟率为70％,C级卵母细胞成熟率为55％。A级卵母细胞使用氯化锶激活率可达80．0％,为最佳方法。[结论]A级卵母细胞成熟培养效果最好。孤雌激活氯化锶激活效果高于乙醇．．     

不同激活方法对小鼠卵母细胞孤雌发育及二倍体形成的影响

卵母细胞的孤雌激活是研究哺乳动物受精机制和发育机理的有效方法,也是细胞核移植、显微注射受精技术、孤雌胚胎干细胞等研究内容中的重要环节。本试验分别用乙醇、SrCl2、钙离子载体A23187对小鼠卵母细胞进行单独孤雌激活,并分别与6-DMAP联合运用,对小鼠卵母细胞进行联合孤雌激活。结果显示：（1）不同激活剂单独或联合激活,对卵母细胞的激活率有显著影响（P〈0.05）,SrCl2组的激活率最高（92%～94%）;（2）相同激活剂对卵母细胞孤雌囊胚的发育率在单独激活组（SrCl2组,18%）和联合激活组（SrCl2＋6-DMA组,53%）中存在显著差异（P〈0.05）;（3）相同激活剂对卵母细胞二倍体率在单独激活组（钙离子载体A23187组,21%）和联合激活组（钙离子载体A23187＋6-DMA组,77%）中均存在显著差异（P〈0.05）。结果表明：（1）SrCl2可以对小鼠卵母细胞进行有效的孤雌激活;（2）联合激活法可以显著提高孤雌卵母细胞的囊胚发育率;3、6-DMAP可以抑制第二极体排出,显著提高孤雌激活卵母细胞的二倍体率。