不同化学激活剂对小鼠卵母细胞的激活效果

为探讨不同激活剂的卵母细胞孤雌激活效果及激活胚体外发育情况,用乙醇、CaA23187、SrCl2、6-DMAP及CB分别对小鼠卵母细胞进行了激活处理.结果显示,70 mL/L乙醇刺激小鼠卵母细胞时,以激活5～7 min的激活效果及孤雌胚发育较好,桑囊胚发育率可达29.11%;用SrCl2激活小鼠卵母细胞时,6～10 mmol/L为最佳处理浓度,3～6 h为最佳激活时间,桑囊胚发育率可达16.67%;以70 mL/L乙醇激活5 min,再用2 mmol/L 6-DMAP＋5 μg/mL CB激活3 h效果最佳,桑囊胚发育率高达35.77%.研究证实,小鼠卵母细胞经乙醇、6-DMAP和CB等复合激活后能较好地发育.     

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。     

Ca-A23187及6-DMAP对小鼠卵母细胞孤雌激活的研究

孤雌生殖(parthenogenesis)是指无雄性配子的任何作用,由雌性配子产生个体的繁殖。孤雌生殖技术通过人工刺激模拟受精过程,提高胞质内钙离子浓度,从而使卵母细胞在无需精子条件下被激活,是一项重要的生物技术。钙离子载体A23187作为常用的化学激活介质广泛应用于哺乳动物卵母细胞的激活,钙离子载体A23187通过Ca^2+的跨膜转运,能迅速诱导卵母细胞内Ca^2+浓度升高,激活哺乳动物的卵母细胞,而6-DMAP是蛋白质磷酸化抑制剂,能阻止蛋白质磷酸化,抑制MPF和CSF活性,同时抑制第二极体的释放,保证孤雌激活偶数目细胞的染色体为二倍体。本实验是来观察钙离子载体A23187(Ca-A23187)及6-甲基嘌呤(6-DMAP)对小鼠卵母细胞的孤雌激活作用。     

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。     

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。     

不同激活方法对猪体外成熟卵母细胞孤雌发育的影响

通过系统探讨不同孤雌激活方法对猪卵母细胞体外激活后发育效果的影响,比较了乙醇（Ethanol,EH）、离子霉素（Ionomycin,Ion：5μmol/L）、氯化锶（Strontium chloride hexahydrate,Sr^2＋：10 mmol/L）、6-二甲氨基嘌呤（6-dimethylaminopurine,6-DMAP：2 mmol/L）和放线菌酮（cycloheximide,CHX：10 mg/L）对猪卵母细胞激活发育的效果。结果表明：（1）9%EH激活处理10 min效果好于15 min;（2）使用9%EH激活处理10 min,再结合CHX、6-DMAP、Sr^2＋、CHX＋Sr^2＋、Sr^2＋＋6-DMAP、CHX＋6-DMAP或CHX＋6-DMAP＋Sr^2＋组处理3～4 h,以EH＋6-DMAP组效果最好,分裂率及囊胚率分别达到82.86%和22.86%;（3）在使用化学激活（Ion＋6-DMAP组和9%乙醇10 min＋6-DMAP组）和电激活（50 V/mm,50μs,2t）的方法中,Ion法激活猪卵母细胞效果较好,囊胚率达到37.50%;（4）卵母细胞包被的卵丘细胞层数不同对卵母细胞成熟激活有显著的影响,卵丘细胞层数4～6层和多于6层的卵丘-卵母细胞复合体孤雌激活的分裂率和囊胚率分别为（68.99%,32.56%）和（75.36%,37.68%）,2组之间差异不显著（P〉0.05）;但其显著高于其他组（P〈0.05）,这2组细胞在猪孤雌激活发育研究中是最佳的实验研究材料。     

水牛卵母细胞孤雌激活及孤雌胚与体外受精胚发育比较

对MII期水牛卵母细胞进行人工诱导激活可帮助人们间接判断体外成熟卵母细胞质量的优劣,且卵母细胞的充分激活也是提高核移植效率的关键因素之一。本试验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27h或30h的囊胚发育率（19.0%or 17.7%）明显高于体外成熟21h或24h的囊胚发育率（12.3%or 13.8%）;Ion联合6-DMAP激活水牛卵母细胞的效果优于其他激活方法;在相同条件下,孤雌激活胚的囊胚发育率显著高于体外受精胚（13.0%vs.21.7%）。     

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.     

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。     

单宁酸对猪卵母细胞体外成熟及胚胎发育能力的影响

研究旨在探讨单宁酸对猪卵母细胞体外成熟质量及其胚胎发育能力的影响。在猪卵丘卵母细胞复合体（COCs）体外成熟培养液中添加不同浓度（0、1、10、100 μg/mL）单宁酸培养42 h后,检测COCs的扩散程度和卵丘细胞扩散指数,统计COCs的体外成熟率,检测成熟卵母细胞内谷胱甘肽（glutathione,GSH）、活性氧（reactive oxygen species,ROS）和生长分化因子9（growth differentiation factor 9,GDF9）的水平,并统计孤雌激活及体外受精胚胎48和168 h的卵裂率、囊胚率及囊胚总细胞数。结果显示,与对照组相比,10 μg/mL单宁酸组卵丘细胞扩散指数显著提高（P<0.05）,100 μg/mL单宁酸组显著降低（P<0.05）;1和10 μg/mL单宁酸组卵母细胞成熟率差异不显著（P>0.05）,100 μg/mL单宁酸组卵母细胞成熟率显著降低（P<0.05）;1和10 μg/mL单宁酸组GSH和GDF9水平显著提高（P<0.05）,ROS水平显著降低（P<0.05）。孤雌胚胎和体外受精胚胎发育能力结果显示,与对照组相比,各单宁酸组卵裂率差异不显著（P>0.05）,10 μg/mL单宁酸组孤雌胚胎囊胚率及体外受精胚胎囊胚率显著提高（P<0.05）,100 μg/mL单宁酸组孤雌胚胎囊胚细胞数及体外受精胚胎囊胚细胞数均显著低于其他各组（P<0.05）。以上结果表明,10 μg/mL单宁酸可通过提高卵丘细胞扩散能力及GSH和GDF9水平、降低卵母细胞内ROS水平,改善猪卵母细胞成熟质量,提高孤雌胚胎及体外受精胚胎的发育能力。     

哺乳动物卵母细胞孤雌激活的研究

研究哺乳动物卵母细胞的激活及孤雌胚发育,对了解哺乳动物早期胚胎发育、深入细致地研究核移植技术和动物克隆技术、建立生产孤雌哺乳动物品系及加速家畜育种进程等均具有重要意义。本文主要从卵母细胞孤雌激活的机制及影响因素、不同动物卵母细胞的激活、提高卵母细胞孤雌激活效果的技术途径以及对孤雌激活的研究意义和发展前景等方面作了详尽论述,为同类研究提供参考。     

不同哺乳动物卵母细胞孤雌激活的研究进展

卵母细胞孤雌激活是核移植的关键技术之一，卵母细胞激活率的高低直接影响核移植的效率。作者主要对卵母细胞孤雌激活的机制、激活途径及不同哺乳动物卵母细胞孤雌激活进行概括性的分析。     

Efficient Transgene Expression in IVF and Parthenogenetic Bovine Embryos by Intracytoplasmic Injection of DNA–Liposome Complexes

Transgenic animals constitute an important tool with many biotechnological applications. Although there have been advances in this field, we propose a novel method that may greatly increase the efficiency of transgenic animal production and thereby its application. This new technique consists of intracytoplasmic injection of liposomes, in bovine oocytes and zygotes, to introduce exogenous DNA. In the first experiment, we evaluated embryo development and EGFP expression in In Vitro Fertilization (IVF) embryos injected with different concentrations of exogenous DNA–liposome complexes (0.5, 5, 50, 500 ng pCX‐EGFP/μl). The highest EGFP‐embryos rates were obtained using 500 ng pCX‐EGFP/μl. In the second experiment, we evaluated embryo development and EGFP expression following the injection of DNA–liposome complexes into pre‐fertilized oocytes and presumptive zygotes, 16 and 24 h post‐fertilization. Approximately 70% of the cleaved embryos and 50% of the blastocysts expressed EGFP, when egfp–liposome was injected 16 h post‐fertilization. The percentages of positive embryos for the 24‐h post‐fertilization and pre‐fertilization groups were 30.1 and 6.3, respectively. Blastocysts that developed from injected zygotes were analysed by PCR, confirming the presence of transgene in all embryos. Finally, we examined the embryo development and EGFP expression of parthenogenetic embryos that resulted from the injection of egfp–liposome complexes into pre‐activated oocytes, and 3 and 11 h post‐activated oocytes. The group with the highest expression rate (48.4%) was the one injected 3 h post‐activation. In summary, this study reports the efficient, reproducible and fast production of IVF and parthenogenetic embryos expressing EGFP, by the intracytoplasmic injection of liposomes to introduce the foreign DNA.     

Parthenogenetic Activation of Pig Oocytes Using Pulsatile Treatment with a Nitric Oxide Donor

The nitric oxide donor (+)–S‐nitroso‐N‐acetylpenicillamine (SNAP) is capable of inducing parthenogenetic activation in pig oocytes matured in vitro. However, quite a long exposure to the nitric oxide donor, exceeding 10 h, is necessary for successful oocyte activation. Repeated short‐term treatment with 2 mm SNAP significantly increased the activation rates despite the fact that the overall exposure time to the nitric oxide donor did not exceed 4 h. With regard to the activation rate, 12 repeated treatments lasting 10 min each were found to be the most efficient regimen (63.3%). The continuous exposure to the nitric oxide donor for the same overall time induced parthenogenetic activation in 12.5% oocytes (2‐h continuous treatment with 2 mm SNAP). The development of parthenogenetic embryos increased after repeated short‐term treatment with SNAP. After continuous treatment with 2 mm SNAP for 10 h, only 6.7% of the oocytes cleaved, and none developed beyond the 4‐cell stage. Thirty‐minute treatment repeated four times with 2 mm SNAP induced cleavage in 37.5% of the oocytes, 18.3% developed to the morula stage, and 6.7% reached the blastocyst stage. Based on the results, it is concluded that pulsatile treatment can significantly improve parthenogenetic activation rate when compared with the continuous treatment using nitric oxide donors.     

小鼠卵母细胞孤雌激活试验

应用两种激活方法(乙醇和离子霉素 6-DMAP)激活和三种培养液(CZB，KSOM，M199)对90只小鼠卵母细胞进行了孤雌激活研究。结果显示，离子霉素结合6-DMAP的激活效果高于乙醇；三种培养液中，以CZB激活效果较为理想。在随后的卵母细胞发育率方面，CZB、KSOM和M199三者之间的差异极为明显，以CZB最高，KSOM次之，M199的发育率最低。     

Effect of exposure duration of ovaries and oocytes at ambient temperature on parthenogenetic development of porcine follicular oocytes

This study was conducted to evaluate the effects of exposing porcine ovaries to 30-33 C during transportation for 4 h and subsequently room temperature (25 C) for 6 h of storage on in vitro maturation (IVM) and subsequent parthenogenetic development of oocytes collected from the ovaries. After IVM, oocytes having a tight oopalsm membrane and no signs of degeneration were exposed to Dulbecco's phosphate-buffered saline (DPBS) with 7% ethanol (v/v) for 7 min to induce parthenogenetic activation. Moreover, we also determined whether exposure of the collected oocytes to room temperature for 1, 2 and 4 h in DPBS or porcine follicular fluid (pFF) affected parthenogenetic development. When porcine ovaries were stored after transportation, oocytes collected from the stored ovaries showed a significantly higher rate of degeneration after 65 h of IVM (58.4%) and a significantly lower rate of cleavage after parthenogenetic activation (40.1%) than oocytes collected from ovaries immediately after transportation (38.9% and 47.4%, respectively). However, there was no significant difference in developmental rates to the morula and blastocyst stages between these two groups (14.4% and 14.3%, respectively). The duration of preservation, 1, 2, and 4 h, of oocytes in DPBS did not affect parthenogenetic development. In contrast, when preserved for 4 h in pFF, the developmental rates of the oocytes were significantly decreased. This suggested that some factor(s) in follicular fluid affects the developmental rate of oocytes with the passage of time in ambient conditions. These results suggest that even after 6 h storage of ovaries, oocytes having normal morphology after IVM have the same rate of parthenogenetic development as oocytes collected from ovaries just after 4 h of transportation, except for a lower cleavage rate, and that exposure of oocytes to room temperature for 4 h in DPBS does not affect their parthenogenetic developmental competence.     

Activation of Zona‐Free Buffalo (Bubalus bubalis) Oocytes by Chemical or Electrical stimulation,and Subsequent Parthenogenetic Embryo Development

Parthenogenetic activation using zona‐free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. The aim of this study was to compare the efficacy of different methods for the activation of zona‐free buffalo oocytes in terms of their effects on the developmental competence of parthenogenetic embryos. The effects of zona removal on parthenogenetic activation and in vitro developmental competence of metaphase II oocytes were also examined. All activation methods were followed by incubation of 2 mm 6‐dimethylaminopurine (6‐DMAP) for 4 h. Out of three different pulse strengths (1.2, 2.1 or 3.3 kV/cm) used, 2.1 kV/cm resulted in the highest blastocyst rate (25.3%). On comparing different chemical agents and electric pulse, highest blastocyst rate was observed for calcium ionophore (CaI) (28.6%) followed by ethanol (25.0%), electric pulse (22.5%) and combined CaI and ethanol treatment (16.7%) although differences among them were not significant. Furthermore, a significantly reduced developmental potential was observed in zona‐free oocytes when compared to zona‐intact ones up to the blastocyst stage (44.3% vs 27.1%). In conclusion, zona‐free buffalo oocytes can be successfully activated for parthenogenetic development using chemical or electrical stimulation. Out of different agents examined, CaI followed by 6‐DMAP resulted in the highest blastocyst rate.     

Effects of ghrelin on in vitro development of porcine in vitro fertilized and parthenogenetic embryos

The objective of the present study was to investigate the effect of addition of ghrelin to in vitro culture medium on preimplantation development of porcine in vitro fertilized and parthenogenetic embryos. In Experiment 1, we sought to compare the in vitro developmental competence of IVF and parthenogenetic embryos. No significant (P<0.05) differences were detected for cleavage rate or blastocyst rate between the in vitro fertilization (IVF)- and parthenogenetic activation-derived embryos. In Experiment 2, parthenogenetic embryos were cultured in Porcine Zygote Medium-3 containing various concentrations of ghrelin. The blastocyst rate was remarkably (P<0.05) increased when 5 ng/ml (PA-5) and 500 ng/ml (PA-500) of ghrelin was added to in vitro culture medium compared with the other groups. Total cell number per blastocyst was slightly promoted in the ghrelin treatment groups compared with the controls. However, the ratio of inner cell mass (ICM) cell number/total cell number was significantly reduced in the PA-50 group compared with the controls (P<0.05). In Experiment 3, we cultured in vitro fertilized embryos in Porcine Zygote Medium-3 supplemented with ghrelin at different dosages. The rate of blastocyst formation was markedly (P<0.05) elevated when 500 ng/ml ghrelin was added to culture medium (IVF-500) compared with the controls. Increased total cell numbers (P<0.05) were observed when in vitro fertilized embryos were cultured in IVF-50 and IVF-500 compared with the controls. However, the ratio of ICM cell number/total cell number was decreased in the ghrelin treatment groups compared with the controls (P<0.05). Taken together, the results suggest that ghrelin can enhance blastocyst formation of porcine in vitro fertilized and parthenogenetic embryos while exerting a negative effect on the structural integrity of the blastocysts.