乙醇和液体石蜡对早期小鼠胚胎体外发育的影响

哺乳动物早期胚胎体外培养技术是研究早期胚胎发育和胚胎工程的基本手段。目前最常用的方法是采用液体石蜡覆盖的液滴培养法。该方法中所用液体石蜡和CO2质量的好坏对体外培养胚胎的发育有严重影响。本文就此对几种中国产液体石蜡和含气体乙醇的CO2作为胚胎体外培养条件对胚胎发育的影响进行了研究。取成熟小鼠受精后的2细胞和8细胞胚胎分别用于两组实验。一、不同品牌液体石蜡对胚胎发育的影响。体外培养采用液体石蜡覆盖液滴培养法。所用液体石蜡均经过水洗。CO2为经过一次水滤的工厂粗制品。其乙醇含量经气相色谱仪测定约为0．18％。二、不同浓度乙醇对胚胎发育的影响。采用试管培养法。采用乙醇浓度不同的培养液。表1为五个不同厂家的液体石蜡对早期胚胎体外培养的影响。其中：I、II（上海、北京）号两种产品符合胚胎体外培养的要求,可使2细胞后期胚胎发育到囊胚的比率达到92％以上,对细胞没有毒害作用（图1A和图2A、B）。其余,则不符合要求,对细胞有毒害作用（图1B）,甚至用无水乙醇和水先后各洗涤3至4遍,亦无改善。由于影响液体石蜡质量的硝基萘可溶于乙醇而被清除,说明这些不合格的液体石蜡中可能还含有其它对胚胎发育有毒害作用的未知因素,有待进一步查明。商品液体石蜡经水洗能够提高其胚胎培养效率。即使是上海产品（I号）,如果不经水洗,2细胞胚胎也只能发育到桑椹阶段。但是,水洗后的液体石蜡需静置至油水彻底分离,才能正常使用。另外,这5种液体石蜡的比重各不相同。比重相关最大的I号和II号产品均符合培养要求,说明液体石蜡的比重对胚胎的正常发育没有影响。表2为各种乙醇浓度对8细胞胚胎发育的影响。当乙醇浓度达到0．1％时,即可使8细胞胚胎发育为囊胚的比率下降为73．9％;达到0．8％以上时,胚胎甚至不能发育。可见,培养液中若含有乙醇将会严重影响胚胎的体外培养。这除了由于水滤降低了CO2中气体乙醇的含量外,还可能是由于液体石蜡覆盖培养液滴后,对气体乙醇的渗入有一定阻作用所致。因此,在胚胎体外培养中不必非使用价格昂贵的纯CO2不可。     

小鼠2—细胞期经电融合后的早期胚胎发育

小鼠２－细胞期经电融合后的早期胚胎发育小鼠,电融合,细胞数,核型,四倍体胚胎小鼠２细胞期经电融合后的早期胚胎发育ＥＡＲＬＹＤＥＶＥＬＯＰＭＥＮＴＯＦＭＯＵＳＥＥＭＢＲＹＯＳＰＲＯＤＵＣＥＤＢＹＥＬＥＣＴＲＯＦＵＳＩＯＮＡＴ２ＣＥＬＬＳＴＡＧＥ关键...     

一种早期胚胎体外培养新方法的建立

建立一种不依赖于液体石蜡的早期胚胎体外培养方法——套皿法,并比较了4种不同处理方法体外培养胚胎的效果。结果显示,采用套皿法进行胚胎培养,盖液体石蜡和不盖液体石蜡皿中的胚胎在各阶段的发育率差异不显著。与套皿法相比,用单皿微滴覆盖法(Brinster法)培养的胚胎在各阶段的发育率显著降低。不覆盖液体石蜡单皿中的胚胎则阻断于二细胞阶段。实验设计的套皿法是一种有效的早期胚胎体外培养方法,为早期胚胎体外培养提供了一种新方法。     

小鼠胚胎体外培养条件的研究

为了探讨小鼠胚胎在体外发育过程中的最佳方案,我们将从超排昆明小鼠取出的５４３枚受精卵经过不同的培养液（Ｍ１６、Ｍ１６＋ＯＥＣ、Ｇｌｕ－ＦｒｅｅＭ１６和Ｇｌｕ－ＦｒｅｅＭ１６＋ＯＥＣ）及不同的培养微环境处理后,观察胚胎体外发育的过程。结果表明,葡萄糖在胚胎发育早期（２细胞期）有比较明显的发育阻断作用,同种输卵管上皮（ＯＥＣ）共同培养能够有效地抑制这种阻断作用;葡萄糖对胚胎８－１６细胞期及以后阶段的发育具有十分重要的作用,而此时ＯＥＣ的作用则不明显;除了葡萄糖外,在早期可能还有其它一些象磷酸盐、重金属离子的物质也能起胚胎发育阻断作用。同时还发现培养微环境的稳定也是胚胎发育的重要保证条件,石蜡油的封盖能够保持某种特定微环境而支持胚胎的体外发育。     

γ—氨基丁酸抑制缺氧所致神经元钙超载

本文以体外分散培养的新生大鼠海马ＣＡ１区神经细胞为标本,分别采用激光扫描共聚集显微镜动态监测单个细胞［Ｃａ＾２＋］ｉ和膜片箝全细胞记录的电生理技术检测细胞的ＮＭＤＡ电流和电压依赖性Ｃａ＾２＋电流等方法,较为深入地研究了抑制性神经递质γ－氨基丁酸（ＧＡＢＡ）及ＧＡＢＡ－Ａ受体激动剂蝇蕈醇对急性缺氧时海马ＣＡ１神经元［Ｃａ＾２＋］ｉ升高过程的影响方式及其作用机制。结果表明：对照组细胞缺氧后比缺氧前［Ｃ     

ICAM在着床期小鼠PBLC及EC/DC中表达的研究(英文)

细胞粘附分子（ＣＡＭ）可介导细胞间及细胞与间质之间的相互作用并传导信息,参与机体胚胎发育、免疫调节、炎症反应、组织修复及肿瘤转移等生理和病理过程。细胞间粘附分子１（ＩＣＡＭ１）是主要的ＣＡＭ分子之一,可表达于活化的细胞,内皮细胞等。人膜是母体与胚胎滋养层直接接触的特殊组织、已发现蜕膜细胞在着床过程中参与了局部免疫耐受的形成,但对着床期ＩＣＡＭ１在蜕膜细胞表达的动态研究鲜见报道。本研究采用免疫荧光、多参数流式细胞术,分别从整体和局部角度、着床过程中外周血淋巴细胞（ＰＢＬＣ）及子宫内膜／蜕膜（ＥＣ／ＤＣ）细胞ＩＣＡＭ１的不同表达特点进行了动态观察和对比性分析。结果发现,ＩＣＡＭ１在着床期ＰＢＬＣ及ＥＣ／ＤＣ中的表达均存在明显的动态变化（Ｔａｂ．１;Ｆｉｇｓ．１＆２）。ＩＣＡＭ１在ＰＢＬＣ中的表达于妊娠第一天（Ｄ１）即开始降低,Ｄ２降至最低;与此不同,ＩＣＡＭ１在ＥＣ／ＤＣ中的表达于Ｄ２开始降低,Ｄ４降至最低,Ｄ５开始恢复,但尚未恢复到对照水平。结果表明,ＩＣＡＭ１在蜕膜局部的表达调节方式不同于外周血;ＩＣＡＭ１表达阳性的外周血,淋巴细胞和蜕膜细胞均代表着活化的功能性细胞,这些细胞表面ＩＣＡＭ     

小鼠2,4,8—细胞胚胎卵裂球体外培养的研究

以ＣＺＢ为基础培养液,培养小鼠２、４、８－细胞胚胎的卵裂球,研究葡萄糖、牛磺酸和胰岛素对１／２卵裂球体外发育的影响及１／４、１／８和２／８卵裂球的体外发育规律。２－细胞胚胎卵裂球在ＣＺＢ中和在添加牛磺酸的ＣＺＢ中培养,其囊胚发育率（分别为９２％、８９％）无显差异（Ｐ＞０．０５）。胰岛素在少量葡萄糖存在的情况下,不影响卵裂球的囊胚发育率;在无葡萄糖时,卵裂球的囊胚发育率（３８％）显降低。牛磺酸在     

草鱼早期胚胎胚盘细胞表面微区元素的研究

本文应用扫描电镜Ｘ射线能量色散分析技术,对草鱼早期胚胎胚盘区细胞的表面元素进行了分析,结果表明：（１）草鱼胚胎细胞表面含有Ｎａ,Ｍｇ,Ａｌ,Ｓ,Ｃｌ,Ｋ和Ｃａ（２）七种元素在胚胎各期的相对含量是不同的,并且各种元素在胚胎发育中的变化曲线也是不同的。     

乙醇对着床前小鼠胚胎体外发育的影响

用含不同浓度乙醇的Whitten氏培养液对小鼠2细胞、4细胞、8细胞和桑椹期胚胎分别进行体外培养,研究了乙醇对小鼠不同发育时期胚胎体外发育的影响。首先利用含0、0．1％、0．5％、1．0％、1．5％、2．0％、3．0％、5．0％和10．0％乙醇的Whitten氏培养液对2细胞胚胎进行培养,发现小鼠2细胞胚胎对培养液中乙醇浓度的耐受极限在1．5％左右。然后又用含1％和3％乙醇的Whitten氏培养液分别对小鼠2细胞、4细胞、8细胞和桑椹期胚胎进行培养。结果发现：含1％乙醇的培养液对于8细胞胚胎和桑椹胚的囊胚形成有促进作用,而在2细胞和4细胞胚胎中则影响不明显。3％乙醇则对各期胚胎均有不同程度的抑制作用,但随着胚胎发育其对乙醇的耐受力逐渐增强。     

山羊体外受精胚胎序贯培养的研究

目的：根据早期胚胎不同发育阶段的营养需求设计体外培养体系,以建立适于山羊早期胚胎体外发育的序贯培养方法。方法：对屠宰场来源山羊卵巢卵母细胞进行体外成熟和体外受精后移入添加不同胚胎发育影响因子的培养体系中进行体外培养,显微镜观察、统计各阶段胚胎发育情况,并对培养的胚胎进行移植。结果：BSA和EGF对山羊体外受精卵具有明显促卵裂作用,培养系统中添加EGS、EGF、HTAU或p—Me可有效支持8-细胞期山羊胚胎克服发育阻滞而提高桑葚胚发育率;在不同胚胎期添加各发育影响因子进行序贯培养的卵裂率、≥8-细胞率和桑葚胚发育率分别为45．2％、60．6％和23．4％,序贯培养的胚胎移植后产羔率为11．1％。结论：宜根据早期胚胎各时期代谢特点和营养需求对山羊胚胎进行序贯培养。     

The influence of insulin on the in vitro development of mouse and bovine embryos

To further investigate the role of insulin during preimplantation embryo development, we compared the effects of insulin on the development of mouse and bovine preimplantation embryos and on cell proliferation during culture in vitro in simplex media. The influence of insulin on the development of mouse zygotes was determined during cultivation in mSOF medium, alone or supplemented with glucose. Similarly, the effects of insulin on the bovine preimplantation embryo development were studied in mSOF medium. The addition of insulin into mSOF medium enhanced significantly the number of cells per mouse blastocyst. Moreover, when mSOF medium was supplemented with insulin and 0.2 mmol x l(-1) glucose, the percentage of hatched blastocysts and the mean cell number of mouse blastocysts were significantly higher. Insulin had no significant effect on the development of bovine embryos, produced by in vitro fertilization of in vitro matured oocytes. Neither the rates of developing embryos nor the mean number of cells in blastocysts were different in comparison with control embryos. Our results suggest that the in vitro development of mouse embryos could be enhanced by the addition of insulin to the culture medium and is further improved by the addition of glucose. In contrast to this our results indicate that insulin has no detectable beneficial effect on the preimplantation development of bovine embryos in mSOF medium.     

Effects of oxygen toxicity on early development of mouse embryos.

To examine the effects of oxygen toxicity on embryonic development, mouse pronuclear embryos were cultured under low oxygen conditions with or without superoxide dismutase (SOD), and the blastulation rate was compared with that of embryos cultured under standard conditions. The blastulation rate of mouse pronuclear embryos cultured under standard conditions was only 1.5% (2/131). This rate was increased significantly, to 28.5% (43/151), when the embryos were cultured under low oxygen conditions; and to 31.0% (35/113) when SOD (500 micrograms/ml) was added to the medium under standard conditions; the rate was increased to 75.2% (115/153) when the embryos were cultured under low oxygen conditions in the presence of SOD. The minimum effective concentration of SOD in the culture medium was 50 micrograms/ml under conditions of 5% O2. The blastulation rate was significantly decreased after 1-hr exposure of pronuclear embryos to room atmospheric oxygen concentration (20% O2), and subsequent culture under 5% O2 with SOD did not result in an improved blastulation rate. Culture with SOD under 5% O2 promoted the development of two-cell stage embryos to the blastocyst stage. When two-cell stage embryos were collected 48 hr after hCG and cultured for 66 hr, their blastulation rate was similar to that of embryos collected from mice 114 hr after hCG. These results suggested that embryonic development in vitro is greatly affected by atmospheric oxygen throughout the early embryonic stages and that this harmful effect can be prevented by culturing embryos under low oxygen conditions and in the presence of SOD.     

Buffalo rat liver cells produce factors that support preimplantation development of mouse embryos cultured in vitro

To examine the effects of buffalo rat liver (BRL) cells on the preimplantation development of mouse embryos in vitro, we first cultured two-cell mouse embryos alone in serum-free Dulbecco modified Eagle medium. As expected, the embryos did not develop to subsequent stages. However, when cocultured with BRL cells, the embryos developed to the blastocyst stage efficiently. Direct contact of embryos with BRL cells was not necessary for development: the medium conditioned by BRL cells contained soluble factors that supported the preimplantation development of mouse embryos. Embryos cultured with BRL-conditioned medium that was replaced at various intervals had a further increased rate of development to the blastocyst stage. This finding indicated that the activities of the factors were maintained only briefly. Seven proteins between 35 and 44 kDa that were detected in the medium were highly beneficial to the development of the embryos. Follistatin-related protein and pigment epithelium-derived factor are believed to be the factors supporting embryo development. The other five proteins also may improve the environment for the development of mouse embryos cultured in vitro.     

Quick freezing of one-cell mouse embryos using ethylene glycol with sucrose

One-cell mouse embryos were frozen by direct plunging into liquid nitrogen (LN(2)) vapor after equilibration in 3 M ethylene glycol with 0.25 M sucrose (freezing medium) for 5 to 40 minutes. After thawing, the embryos were cultured in vitro and the effects of the equilibration period and dilution method were examined. No significant difference was observed in the in vitro survival of embryos when 0.5 or 1.0 M sucrose was used for the dilution of the cryoprotectant for each equilibration period. The highest survival rate (67.2%) was obtained when the embryos were equilibrated for 10 minutes, and the cryoprotectant diluted with either 0.5 or 1.0 M sucrose after thawing. Shorter (5 minutes) or prolonged (40 minutes) equilibration of embryos in the freezing medium yielded significantly lower survival rates. Dilution by direct transfer of the frozen-thawed embryos into PB1 resulted in lower survival rates than when 0.5 or 1.0 M sucrose was used. The in vitro development to the blastocyst stage of one-cell mouse embryos frozen after 10 minutes equilibration in the freezing medium and diluted after thawing in 0.5 M sucrose was significantly lower than the control (68.0 vs 92.7%). However, transfer of the blastocysts developing from frozen-thawed one-cell mouse embryos into the uterine horns of the recipients resulted in fetal development and implantation rates similar to the control.     

Sucrose dilution of glycerol from mouse embryos frozen rapidly in liquid nitrogen vapour

The toxic effects of sucrose and the conditions of in-straw glycerol removal after freezing and thawing were studied using Day-3 mouse embryos. At 20 degrees C, exposure to less than or equal to 1.0 M-sucrose for periods up to 30 min had no adverse effects on freshly collected embryos. At 25 and 36 degrees C, however, greater than or equal to 1.0 M-sucrose significantly reduced the developmental potential (P less than 0.001). In the freezing experiments the embryos were placed in 0.5 ml straws containing 40 microliters freezing medium separated by an air bubble from 440 microliters sucrose solution. The straws were frozen rapidly in the vapour about 1 cm above the surface of liquid nitrogen. The post-thaw viability was substantially better after sucrose dilution at 20 degrees C than at 36 degrees C. Mixing the freezing medium with the sucrose diluent immediately after thawing further improved the rate of survival relative to mixing just before freezing (P less than 0.001). The best survival was obtained when the freezing medium contained 3.0 M-glycerol + 0.25 M-sucrose; it was mixed with the diluent after thawing and the glycerol was removed at 20 degrees C. Under such conditions the sucrose concentration in the diluent had no significant effect on the rate of development (0.5 M, 69%; 1.0 M, 73%; 1.5 M, 64%). The results show that during sucrose dilution the temperature should be strictly controlled and suggest that intracellular and extracellular concentrations of glycerol are important in the cryoprotection of embryos.     

Development of preimplantation embryos of the golden hamster in a defined culture medium

Eight-cell embryos were recovered from mated golden hamsters that had been superovulated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Embryos were cultured for 24 or 32 h in a defined medium (modified Tyrode's solution) designed for fertilization of hamster oocytes in vitro. This medium was supplemented in some experiments with amino acids (glutamine, phenylalanine, methionine and isoleucine) and with vitamins (Eagle's Minimum Essential Medium vitamin supplement). At the end of the culture period, the numbers of embryos developing to the blastocyst stage were recorded. In other experiments, the effects of varying the osmotic pressure (225, 250, 275 and 300 m0smol/kg) and the pH (6.8 and 7.4) of the culture medium on blastocyst formation were examined. A difference was found between the ability of early 8-cell embryos (approx. 54 h post-egg activation) and late 8-cell embryos (approx. 62 h post-egg activation) to develop in culture. In the unsupplemented culture medium, only 2% of early 8-cell embryos developed to the blastocyst stage compared with 22% of late 8-cell embryos. A marked effect of the four amino acids on development was found. In the presence of amino acids 36% of early 8-cell embryos developed into blastocysts (18-fold increase). The amino acids also increased the percentage of late 8-cell embryos that developed into blastocysts from 22% to 66%. These data suggest that an important metabolic change may occur in hamster embryos during a critical period at the 8-cell stage of development. No additional effect on development was observed when vitamins were included in the culture medium. No significant effect of either osmotic pressure of pH of the culture medium on development was found. When blastocysts formed from cultured 8-cell embryos were transferred surgically to pseudopregnant hamsters, about 25% developed into normal-looking fetuses and 5 normal-looking young were born, 4 of which have survived. These results represent an approach towards achieving complete preimplantation development of hamster embryos in vitro.     

Optimisation of in vitro culture conditions in B6CBF1 mouse embryos

This study was undertaken to investigate the effects of three media, volume and type of oil and frequency of observation on the in vitro development of mouse zygotes. B6CBF1 female mice (4 to 6 wk old) were superovulated using PMSG/hCG and mated with a proven fertile male of the same strain. Putative zygotes with polar bodies were collected from the oviducts of mated mice, 25-28 h after hCG injection, and were cultured in vitro. Embryo development was evaluated at either 96 h and 120 h or every 24 h for 120 h. The results obtained showed that the CZB medium was better than the KSOM and HCO3HTF media, and the use of 1 mL of paraffin oil was better than the use of 0.5 mL of paraffin oil. The effect of paraffin oil and mineral oil on embryo development was examined and the results indicated that the use of paraffin oil was better than the use of mineral oil. Repeated observations did not influence the proportion of embryos developing to blastocysts.     

Effects of co-culture, medium components and gas phase on in vitro culture of in vitro matured and in vitro fertilized bovine embryos

To develop an in vitro culture system for bovine oocytes and early embryos, we examined the effects of co-culture of in vitro matured and in vitro fertilized embryos with trophoblastic vesicles and cumulus cells. We also studied the effects of culture medium components and oxygen gas pressure by modifying TCM-199 medium and using a gas-tight chamber. We found that co-culture with trophoblastic vesicles or cumulus cells promoted early embryos to develop beyond the eight-cell block; 17 to 19% of the initial oocytes developed to the morula stage. The effects of removing glucose and other energy sources from the medium, adding EDTA to the medium, reducing the concentration of serum, and reducing the oxygen gas pressure on the development of embryos were also examined. These modifications during the initial phase of co-culture greatly increased the rate of embryo development to the morula (36 to 38% of oocytes developed to morulae) and blastocyst stages.     

Optimization of mouse embryo culture media using simplex methods

Culture media were developed for pronuclear-stage mouse embryos using simplex optimization, which has the benefit of being able to optimize several components simultaneously. Initially, several different media were generated. All media contained the same components, yet each medium was characterized by having a different component at a high concentration. The simplex procedure identified 4 components (NaCl, pyruvate, KH2PO4 and glucose) which at high concentrations were detrimental to embryo development, compared to the other components tested. For example, all embryos cultured in a medium with high NaCl blocked at the 2-cell stage. The optimization method then adjusted each medium by lowering the concentration of the component or removing it entirely, which resulted in a significant increase in development. In an experiment comparing 8 media generated from the simplex optimization, along with 7 other media, removal of KH2PO4 resulted in the largest increase in development; 88% of embryos were greater than or equal to 4 cells on Day 3 after hCG, and 53% developed into blastocysts by Day 5. Another experiment compared 4 of the best media generated from the simplex optimization. In 3 out of the 4 media, 90% or more of the embryos were greater than or equal to 4 cells on Day 3. In 3 of the media, approximately 60% or more of the embryos developed into blastocysts. The simplex optimization procedure is an efficient method for developing culture media and determining requirements for development in vitro.     

Effects of interferon and encephalomyocarditis virus on in vitro development of preimplantation mouse embryos with and without the zona pellucida

Development of preimplantation mouse embryos, with or without the zona pellucida, in the presence of interferon (IFN) and mouse encephalomyocarditis (EMC) virus was studied using the in vitro culture method. The embryos (2- to 8-cell stages) were obtained from superovulated mice and cultured in modified Witten's medium under paraffin oil in 5% CO2 in air at 37 degrees C. Removal of the zona pellucida does not affect the subsequent development of the embryos: 90% of embryos with and 87% of embryos without the zona pellucida reached the morula-early blastocyst stages. Mouse IFN (10(4) units/ml) had no inhibitory effect on the developmental ability of the preimplantation embryos with or without the zona pellucida: 88 and 89% of the embryos in each group, respectively, reached the morula-early blastocyst stages. The preimplantation mouse embryos were sensitive to the embryotoxic effect of EMC virus: at a multiplicity of 20 infection particles per embryo the development of 43% of embryos was inhibited. The zona pellucida had no significant protective effect: Its removal changed only slightly the susceptibility of the preimplantation embryos to this virus. Pretreatment of embryos with IFN did not protect them from the embryotoxic effect of EMC virus. This work indicates that preimplantation mouse embryos appear to be resistant for both the antiviral and antiproliferative activities of IFN.