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范晶晶
, 郑秋闿
FAN Jing-jing, ZHENG Qiu-kai
文献标识码: 1671-9964(2016)01-0011-05
文章编号: 1671-9964(2016)01-0011-05
收稿日期: 2015-02-1
网络出版日期: 2016-01-20
版权声明: 2016 上海交通大学期刊中心 版权所有
基金资助:
作者简介:
作者简介: 范晶晶(1981-), 女, 博士, 讲师, 研究方向:动物生殖与发育研究, E-mail:fanjingjing2008@163.com
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摘要
本研究主要探讨了牛磺酸对昆明(KM)小鼠体外胚胎发育及对胚胎着床的作用。在mKSOM(modified potassium simplex optimized medium)胚胎体外培养液中分别添加不同浓度牛磺酸, 培养24、72 h后分别统计4-细胞率、囊胚率、囊胚总细胞数; 采用子宫角注射的方法, 研究牛磺酸转运抑制剂β-丙氨酸对孕D9胚胎着床数的影响。结果表明, 添加10 mmol/L牛磺酸培养液组的胚胎4-细胞率高于对照组(P<0.05), 囊胚率与囊胚总细胞数明显高于对照组(P<0.01)。子宫角注射β-丙氨酸(20 μg/mL)显著降低D9着床的胚胎数。本研究初步明确了牛磺酸对小鼠体外胚胎发育及对胚胎着床的影响, 为进一步研究胚胎发育和着床提供参考。
关键词：
Abstract
The effects of taurine on development and implantation of Kunming mouse early embryos were investigated.The embryos were cultivated in modified potassium simplex optimized medium(mKSOM)with different concentration of taurine.The percentage of 4-cell, blastocysts and average total cell number of blastocysts were calculated after 24 h and 72 h.Cornua uteri injection technique was used to investigate the effects of β-alaline(β-ala, taurine transport inhibitor)on embryo implantation.The results showed that there were significant differences between 10 mmol/L taurine treatment and control in the percentage of 4-cell(P<0.05), blastocysts and total cell number of blastocysts(P<0.01).Meanwhile, injection of β-ala(20 μg/mL)into uterine horn significantly reduced the number of implanted embryos on D9.This study explicated the effects of taurine on the development and implantation of early Kunming mouse embryos, providing a useful reference for further uncovering of mechanism of embryo development and implantation.
Keywords：
牛磺酸是雌性动物子宫液、输卵管液中含量最丰富的氨基酸成分之一[1, 2], 大鼠每克输卵管含有高达10 μmol/L的牛磺酸[3]。研究发现在人和大鼠妊娠的子宫内牛磺酸含量也呈逐渐升高趋势[4]。如果怀孕和哺乳过程中缺乏牛磺酸会出现胎儿生长发育停滞、畸形发育、视网膜退化、心肌损害和中枢神经系统的机能紊乱[5]。随着动物胚胎工程如胚胎移植、体外受精、克隆、转基因等技术的发展与应用, 胚胎体外培养的质量成为提高效率的关键, 它不仅影响胚胎移植后在母体子宫的着床率及妊娠率, 而且能影响胎儿将来的生长和发育, 因此选择或建立适合的胚胎体外培养体系获得高质量体外培养胚胎显得尤为重要。由于动物种类、品系和选用的胚胎培养液的类型不同, 牛磺酸的体外最佳添加浓度也有所不同。本实验筛选出了在昆明(KM)小鼠胚胎体外培养液mKSOM(modified potassium simplex optimized medium)中牛磺酸的最佳添加浓度, 观察比较了不同浓度牛磺酸对胚胎体外发育的影响。另外进行体内着床抑制实验, 采用子宫角注射牛磺酸的选择性转运抑制剂β-丙氨酸(β-ala)来研究牛磺酸对胚胎着床的作用。
实验采用清洁级8周龄KM小鼠, 体重20~25 g, 购自青岛市实验动物和动物实验中心, 生产许可证号:SCXK(鲁)20130010。饲养条件为人工控温22~26 ℃, 14L∶10D光照, 自由饮水采食。
丙酮酸钠、60%乳酸钠、葡萄糖、4-羟乙基哌嗪乙磺酸(HEPES)、乙二胺四乙酸二钠(EDTA·Na2)、牛磺酸(Taurine, Tau)、牛血清白蛋白(BSA)、石蜡油、Hochest33258等均为美国Sigma公司产品; 孕马血清促性腺激素(PMSG)和人绒毛膜促性腺激素(hCG)购自宁波市激素制品有限公司。
CO2培养箱(Thermo公司)、Mili-Q超纯水仪、体视显微镜(Olympus公司)、倒置显微镜(Leica公司)、生物安全超净工作台(济南空气净化设备厂)。
小鼠超数排卵参考强苏静报道的方法和剂量[6], 雌性KM小鼠于15:00腹腔注射6 U PMSG, 48 h后腹腔注射6 U hCG, 然后与性成熟公鼠合笼(雌∶雄=2∶1), 第2天早上检查阴栓, 见栓当天记为第1天(D1)。
对有阴栓的小鼠于D2上午10:00颈椎脱臼处死, 无菌条件下取输卵管置于HFM(HEPES缓冲的KSOM培养液)工作液中。在实体显微镜下用眼科镊撕开输卵管膨大部, 释放出2-细胞胚胎, 在mKSOM培养液中洗涤数次。
将发育良好的2-细胞胚胎移入过夜平衡的在含不同浓度Tau(0、5、10 mmol/L), 10 mmol/L Tau+10 mmol/L β-丙氨酸(β-ala, 为牛磺酸的选择性转运抑制剂)、10 mmol/L β-细胞的mKSOM培养液中培养, 每滴培养液放10个2-细胞胚胎, 置于37 ℃, 5% CO2饱和湿度的培养箱中培养至囊胚期, 培养24、72 h后分别统计4-细胞率和囊胚率。
将囊胚放在1%多聚甲醛中固定3 min, PBS冲洗后用Hochest33342(5 μg/mL)染色2 min, 50%甘油封片, 荧光显微镜下观察。呈现蓝色荧光的为囊胚细胞核, 统计囊胚细胞总数来评价不同浓度牛磺酸对小鼠胚胎体外发育的影响。
为了研究牛磺酸在着床过程中的作用, 怀孕雌鼠于D4上午通过子宫角注射牛磺酸的选择性转运抑制剂β-ala来进行体内着床抑制实验。采用外科手术, 小鼠麻醉后, 暴露右侧子宫角, 用微量注射器缓缓注入3 μL的浓度分别为20、200 μg/mL的β-ala, 每组5只作为实验组。每只小鼠的左侧子宫角作为内对照, 注射同样体积的0.9%生理盐水。另取5只小鼠, 两侧子宫角都注射同样剂量的生理盐水作为正常对照。实验重复3次。受试动物于孕D9剖检, 采集子宫, 统计每侧子宫角着床的胚胎数。
所有数据用Mean±SE表示, 用Student’s t-test 检验各组之间的差异, P<0.05为差异显著。
在mKSOM培养液中分别添加5、10 mmol/L Tau、10 mmol/L Tau+10 mmol/L β-ala、10 mmol/L β-ala, 胚胎发育到囊胚的状态见图1。用Hochest33342对囊胚染色, 荧光显微镜下囊胚细胞核呈现蓝色荧光见图2。各组从2-细胞胚胎发育到4-细胞和囊胚的百分率及囊胚总细胞数见表1、图3, 结果显示小鼠胚胎在mKSOM培养液中未出现2-细胞发育阻滞现象, 对照组有77%的胚胎发育到4-细胞, 在mKSOM培养液中添加10 mmol/L Tau组的4-细胞率(87%)与对照组(77%)相比差异显著(P<0.05), 囊胚率(80%)、囊胚总细胞数(50±1.84)与对照组(62%, 39±2.18)相比都差异极显著(P<0.01)。10 mmol/L牛磺酸转运抑制剂β-ala和相同浓度牛磺酸共同添加时, 2-细胞胚胎发育到4-细胞和囊胚的百分率及囊胚总细胞数与对照组相比差异不显著。
图1 牛磺酸对小鼠胚胎体外培养囊胚率的影响
Fig.1 Effect of taurine on mouse embryo blastocyst ratio in vitro
表1 牛磺酸对昆明小鼠胚胎在mKSOM培养液中体外发育的影响
Tab.1 Effect of taurine on the development of KM mouse embryos in mKSOM
| 牛磺酸/ (mmol·L-1) Tau | β-丙氨酸/ (mmol·L-1) β-ala | 2-细胞胚胎数 No. of 2-细胞 | 胚胎数 No.of embryos | 囊胚细胞数 No. of cell (Mean±SE) | 囊胚细胞数 范围 Range | |||
|---|---|---|---|---|---|---|---|---|
| 发育至4细胞个数(比例) No. of 4-cell(ratio) | 发育至囊胚个数 (比例)No. of blastocysts(ratio) | |||||||
| - | - | 435 | 338(77%) | 270(62%) | 39±2.18 | 27-66 | ||
| 5 | - | 293 | 219(75%) | 187(64%) | 40±2.13 | 23-68 | ||
| 10 | - | 376 | 327(87%)* | 300(80%)** | 50±1.84** | 25-83 | ||
| - | 10 | 408 | 261(64%) | 204(58%) | 35±2.55 | 23-52 | ||
| 10 | 10 | 312 | 221(71%) | 184(59%) | 41±3.36 | 29-67 | ||
图2 不同浓度牛磺酸mKSOM培养液中的囊胚总细胞的荧光染色
Fig.2 Fluoresence staining of mouse blastocysts in mKSOM mediums containing different concentration of taurine
为了研究牛磺酸在小鼠胚胎着床过程的作用, 进行了体内抑制功能的研究。由表2可见, 孕D4子宫角注射牛磺酸的选择性转运抑制剂β-ala呈剂量依赖性的显著降低D9着床的胚胎数(图4)。当β-丙氨酸在20 μg/mL时, 右侧子宫角胚胎着床数(4.67±0.55)与对照组(10.67±0.43)相比具有显著性差异(P<0.01), 当β-丙氨酸在200 μg/mL时, 右侧子宫角胚胎着床数(1.67±0.63)与对照组相比差异极显著(P<0.01), 而注射生理盐水的对照组与正常组的胚胎着床数无显著差异。
人们为了提高体外培养时胚胎的质量, 往往是模拟胚胎体内发育的环境, 最终建立最佳的培养体系。研究显示, 牛磺酸在小鼠输卵管液中的含量为233~401 μmol/L, 达总游离氨基酸含量的59%[7]。
图3 牛磺酸对小鼠体外培养胚胎发育的影响
Fig.3 Effect of taurine added to mKSOM on mouse embryo development in vitro
图4 孕D4子宫角注射不同浓度牛磺酸转运抑制剂β-丙氨酸对着床的影响
Fig.4 Effect of intrauterine injection of β-alanine at different concentrations in uterus on D4 of pregnancy
表2 宫角注射β-丙氨酸对孕D9小鼠胚胎着床数的影响
Tab.2 The number of embryos after intrauterine injection of β-alanine on D9 of pregnancy
| 孕D9胚胎数No. of embryos on D9(Mean±SE) | |||
|---|---|---|---|
| 生理盐水 Normal saline | β-丙氨酸(20 μg·mL-1) β-ala | β-丙氨酸(200 μg·mL-1) β-ala | |
| 11.67±0.67 | 左侧Left | 12.66±0.89 | 11.00±0.58 |
| 1.67±0.63** | 右侧Right | 10.67±0.43 | 4.67±0.55** |
用5 U PMSG处理C57/BL小鼠48 h后体内输卵管液中牛磺酸浓度能达到6.64 mmol/L[2]。由于动物种类、品系和选用的体外胚胎培养液的类型不同, 体外牛磺酸最佳添加浓度也有所不同。Spindle报道, 对于CD-1小鼠在胚胎体外无BSA的TE培养液中牛磺酸的最适添加浓度为24 mmol/L[8]。Damoulin报道, 在B6D2小鼠胚胎T6体外培养液中添加5或10 mmol/L牛磺酸时培养效果最佳[9]。Li等人发现在兔胚胎体外培养时的牛磺酸最佳浓度为2.5 mmol/L[10]。Manjunatha等人研究结果显示在基础培养液(TCM-199 + 10% SS)中添加1 mmol/L牛磺酸能显著提高水牛胚胎体外发育能力[1]。袁水桥等人在体外受精牛2-细胞胚胎基础培养液(TCM-199 + 10% FBS)中分别添加7 mmol/L牛磺酸可显著提高桑椹胚率和囊胚率[12]。郑云胜等人在KSOM培养液中添加7 mmol/L牛磺酸可显著提高体外受精绵羊胚胎桑椹胚和囊胚的发育比例[13]。Devreker等人证明在含0.5%人血清的EBSS培养液中添加5 mmol/L牛磺酸能促进2~4-细胞胚胎发育成囊胚[14]。本实验选用的是KM小鼠和mKSOM体外培养液, 结果显示当培养液中的牛磺酸添加浓度为10 mmol/L时, 第5天的囊胚率(80%)与囊胚总细胞数(50±1.84)与对照组(62%, 39±2.18)差异极显著(P<0.01)。这也与以前所报道的牛磺酸能促进多种动物早期胚胎的发育相一致。多种研究结果显示, 牛磺酸对体外胚胎发育的促进作用可能通过以下几种方式实现:一是通过渗透压调节保护细胞膜[10]; 二是抗氧化作用, 它可以抑制高氧条件下产生的氧化性的毒素物质, 从而对胚胎起到保护作用[15-16]; 三是可以通过促进细胞增殖来实现胰岛素样作用[17]; 四是抵抗K+浓度过高引起的细胞损伤[9]。
本研究采用子宫角注射方法[18], 通过给着床前(D4)子宫腔注射牛磺酸选择性转运抑制剂β-ala, 检测了牛磺酸对着床的作用。子宫角注射法比传统所使用腹腔注射的药物剂量少, 并且更加直接的反应药物对胚胎植入的影响[19], 这种方法已用来阻断多种因子的功能作用, 如白介素-1, 白血病抑制因子, 表皮生长因子等。实验结果显示, 当D4子宫角注射3 μL β-ala(20 μg/mL)时, D9时子宫角胚胎着床数(4.67±0.55)与对照组(10.67±0.43)相比具有显著性差异(P<0.01), 表明牛磺酸可以影响胚胎着床。胚胎着床是一个涉及到子宫与胚胎间紧密对话的复杂发育过程。胚胎、母体、胚胎和母体之间的对话中任一环节出现异常都会导致着床失败。实验中宫内注射牛磺酸转运抑制剂β-ala所产生的抑制着床效应可能是妨碍了滋养层细胞的侵入和子宫的容受性, 具体机制还有待于进一步研究。
The authors have declared that no competing interests exist.
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Temporal effects of taurine on mouse preimplantation development in vitro [J].DOI:10.1111/j.1365-2559.1992.tb00983.x URL PMID: 1587949 [本文引用: 1] 摘要
Previously it has been shown that significantly more 2-cell mouse embryos reach the blastocyst stage when cultured in medium supplemented with taurine. In this study, in-vitro fertilized zygotes from a hybrid mouse strain were used to examine the temporal effects of 10 mM taurine on embryonic development in vitro during the preimplantation period. Taurine exerted its beneficial effect exclusively during the first 2 days post-insemination. The effect of taurine on blastocyst formation appeared to be restricted mostly to the period 20-48 h after fertilization, during which time mouse embryos are at the two-cell stage. Although more blastocysts were found when embryos were cultured in taurine-containing medium from 5 to 20 h post-insemination, this difference was not significant compared to the number of blastocysts when embryos were cultured without taurine. Taurine did not appear to affect the two-cell block of mouse embryos from random-bred strains.
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| [8] |
Beneficialeffects of taurine on mouse zygotes developing in protein-free culture medium [J].DOI:10.1016/0093-691X(95)00275-D URL PMID: 16727773 [本文引用: 1] 摘要
ABSTRACT The objectives of this study were to determine if mouse zygotes from outbred mice can develop in simple culture medium in the absence of bovine serum albumin (BSA), and if taurine can be used as a medium supplement to improve development. Zygotes from 2 stocks of outbred mice (CD-1 and CF-1) were cultured in simple embryo culture medium (TE medium) lacking BSA and with or without taurine (24 mM), or in regular TE medium with BSA. The presence of BSA had little or no effect on development, but development to post-blastocyst endpoints was enhanced when CD-1 zygotes were cultured in medium containing taurine. In addition, when CD-1 blastocysts were transferred to pseudopregnant animals, embryos cultured in the presence of taurine developed into fetuses more often than those cultured in medium without taurine, and their weights were higher than those of embryos cultured in regular TE medium with BSA. These beneficial effects of taurine do not appear to be the nonspecific effects of a fixed nitrogen source, because the addition of glycine to BSA-free TE medium did not have similar beneficial effects. It was concluded that mouse zygotes from outbred mice do not require BSA for their preimplantation development in culture and that the presence of taurine in preimplantation culture medium is beneficial not only for preimplantation development of the zygotes, but also for their post-blastocyst development.
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| [9] |
Positive effect of taurine on preimplantation development of mouse embryos in vitro [J].DOI:10.1530/jrf.0.0940373 URL PMID: 1593540 [本文引用: 2] 摘要
The effect of various taurine concentrations in modified Tyrode's medium on in vitro fertilization of mouse oocytes was examined. No significant difference in fertilization rate was found at concentrations of 0, 0.1, 1, 5, 10 and 20 mM taurine. In a second series of experiments, the effect of taurine on preimplantation embryonic development after fertilization in vitro was studied. At concentrations of 1, 5, 10 and 20 mM taurine, significantly more two-cell embryos reached the blastocyst stage compared with medium without taurine. Culture in the presence of 5 mM or 10 mM taurine resulted in blastocysts with the highest mean number of cells. The positive effect of taurine on embryonic development was found to be more pronounced both in a second medium (human tubal fluid medium) which has a higher potassium concentration than Tyrode's medium, and in a modified Tyrode's medium with an increased potassium concentration. In addition to these in vitro studies, it is reported that taurine comprised about 59% of the total free amino acid content in mouse oviduct flushings, compared with 17% in mouse serum.
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| [10] |
Concentration and composition of free amino acids and osmolalities of porcine oviductal and uterine fluid and their effects on development of porcine IVF embryos [J].DOI:10.1002/mrd.20682 URL PMID: 17342727 [本文引用: 2] 摘要
Abstract The concentration of free amino acids and the osmolalities in porcine oviductal (OF) and uterine fluids (UFs) on day 3 (D3) and day 5 (D5) were measured by HPLC and Vapor Pressure Osmometer, respectively. Based on these measurements we designed new media based on PZM3 by modifying the amino acid composition and osmolality. The effectiveness of the modified PZM3 on the development of porcine IVF embryos was then investigated. A total of 24 free amino acids were measured, including 20 protein and 4 nonprotein amino acids (β-alanine, taurine, ornithine, and citrulline). There was no significant difference in the total concentration of amino acids among D3OF (13.0665± 3.63 mmol/L), D3UF (10.5465±655.16 mmol/L), or D5UF (10.2365±656.69 mmol/L). But the total concentration of amino acids in D5OF (5.8965±651.47 mmol/L) was significantly lower than the three fluids above. Some individual amino acids varied significantly depending on where they were collected and from which day. The blastocyst rates of porcine IVF embryos were not improved when embryos were cultured in PZM3 with amino acids at D3OF (PZM3-D3OF, 20.365±657.9%) or D5UF (PZM3-D5UF, 14.365±6510.7%) concentrations or in PZM3-D3OF for the first 48 (20.565±6515.1), 72 (25.665±6510.4), and 96 (18.765± 10.0) hr and then transferred into PZM3-D5UF compared with PZM3 with Sigma amino acid solution (PZM3-SAA) (30.865±659.1%). However, when IVF embryos were cultured in PZM3-D5UF, the average nuclear number per blastocyst (57.665±658.3) was increased compared to PZM3-SAA (40.565±653.5). The osmolalities in D3OF, D3UF, D5OF, and D5UF were 31865±658, 32065±6532, 321, and 29365±658 mOsM, respectively. When the IVF embryos were cultured in PZM3-SAA and PZM3-D3OF at a variety of osmolalities (150–360 mOsM), higher blastocyst rates were obtained at 270–300 mOsM in the PZM3-SAA group (24.6–33.9%) and 270–290 mOsM in PZM3-D3OF group (22.4–24.2%). The blastocyst rate gradually decreased when the osmolality was increased or decreased in both groups. When the embryos were cultured in PZM3-SAA at 330 mOsM for the first 72 hr and then transferred to 250 mOsM (33.365±653.4%), the blastocyst rate was higher than original PZM3 (21.265±652.2%) (288 mOsM). Mol. Reprod. Dev. 74: 1228–1235, 2007. 08 2007 Wiley-Liss, Inc.
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| [11] |
Effect of taurine and melatonin in the culture medium on buffalo in vitro embryo development [J].DOI:10.1111/j.1439-0531.2007.00982.x URL PMID: 18507802 摘要
Contents This study was carried out to investigate the effect of supplementing culture medium with different concentrations of taurine and melatonin, on buffalo oocyte in vitro meiotic maturation and embryo development. In experiment 1, oocytes were matured in vitro and the cleaved embryos were cultured in the same following seven culture medium; (i) control (TCM 199+10% SS); (ii) control+0.5m m taurine; (iii) control+1m m taurine; (iv) control+3m m taurine; (v) control+5μ m melatonin; (vi) control+10μ m melatonin and (vii) control+50μ m melatonin. In experiment 2, based on the results of experiment 1, to examine the synergistic effect of antioxidants, the oocytes were matured in culture medium (TCM199+10% SS), supplemented with both taurine at 1m m and melatonin at 10μ m concentration and the cleaved embryos were cultured in the same medium. Supplementation of taurine at 1m m concentration in the culture medium resulted in a higher (p<0.05) transferable embryo (TE) yield when compared with control (20.6% vs 14.1%). Supplementation of melatonin at 10 and 50μ m concentration in the culture medium resulted in a higher (p<0.05) meiotic maturation rate (90.3% and 88.8% respectively) and TE yield (28.4% and 27.2% respectively), than the other treatments. In experiment 2, the TE yield did not improve by supplementing the culture medium with both taurine and melatonin, when compared with melatonin alone. In conclusion, the results of this study demonstrated that, enriching the culture medium with taurine and melatonin, improves in vitro embryo production efficiency in buffaloes. In particular, a high TE yield was obtained by enriching the culture medium with 10μ m melatonin.
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| [12] |
牛磺酸在牛胚胎体外发育中的作用研究 [J].DOI:10.3969/j.issn.1000-1700.2007.04.023 URL [本文引用: 1] 摘要
研究了牛体外受精后早期胚胎体外发育时,向其基础培养液中加入牛磺酸对胚胎桑椹胚率、囊胚率 和孵化囊胚率的影响,以探寻牛磺酸克服牛胚胎“体外发育阻滞”现象的作用。试验结果表明：以TCM-199＋10%FBS为基础培养液（对照组）,再加入 7,14mM的牛磺酸（试验组）,试验组与对照组的桑椹胚率分别为48.1%、47.4%和43.2%;囊胚率分别为26.4%、22.3%和 21.0%;孵化囊胚率分别为21.8%、18.7%和0%。IVF后2细胞、4～8细胞及8～16细胞期,在基础培养液中分别添加7mM的牛磺酸时,桑 椹胚率分别为48.7%、57.1%和52.0%,囊胚率分别为25.1%、30.7%和27.9%,孵化囊胚率分别为25.9%、28.6%和 25.0%;而添加14mM牛磺酸组时,桑椹胚率分别为50.4%、56.4%和55.4%,囊胚率分别为26.5%、31.3%和27.7%,孵化囊胚 率分别为27.5%、31.1%和29.9%。体外发育培养液中添加7mM牛磺酸可显著提高桑椹胚率和囊胚率（p〈0.05）,在4～8细胞期添加 14mM牛磺酸最为合适。
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| [13] |
氨基酸和牛磺酸对绵羊体外受精胚胎体外培养的影响 [J].DOI:10.3969/j.issn.0258-7033.2004.03.007 URL [本文引用: 1] 摘要
本文在KSOM培养液中添加牛磺酸和NEAA、EAA,研究氨基酸对绵羊胚胎体外培养的影响.研究表明:①牛磺酸、NEAA可以显著提高胚胎的桑椹胚和囊胚发育率;②EAA能促进胚胎由囊胚向孵化囊胚发育;③NEAA和EAA可促进胚胎的囊胚发育和孵化囊胚的发育.
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| [14] |
Van den Bergh M, Biramane J, et al.Effects of taurine on human embryo development in vitro [J].DOI:10.1093/humrep/14.9.2350 URL PMID: 10469709 [本文引用: 1] 摘要
Glutamine and taurine are reported to be beneficial for mouse embryo development in vitro, and we have recently shown that glutamine improves human blastocyst formation in vitro. This randomized study compared the development of supernumerary human embryos in the presence of 1 mmol/l glutamine and/or 5 mmol/l taurine from the 2-4-cell stage to the blastocyst stage. Blastocyst development and cell numbers were similar in the presence of glutamine or taurine: 52.6% and 58.3% of the embryos reached the blastocyst stage, respectively. Pyruvate uptake was similar in the presence of glutamine or taurine throughout development, as was lactate production after the 8-cell stage. Before this stage, lactate production was 4-fold higher in the presence of taurine (P < 0.001). The proportion of embryos reaching the blastocyst stage was similar with glutamine alone or with glutamine and taurine (62.5% and 47.2% respectively), as were the blastocyst cell numbers (63.0 +/- 4.6 and 61.0 +/- 5.1 respectively). In conclusion, taurine supports development of 2-4-cell human embryos to the blastocyst stage, although it does not further augment the beneficial effects of glutamine.
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| [15] |
Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings [J].URL PMID: 3281591952098529535152922232222112846618 [本文引用: 1] |
| [16] |
Glutamine and hypotaurine improves intracellular oxidative status and in vitro development of porcine preimplantation embryos [J].DOI:10.1017/S0967199407004273 URL PMID: 17967211 [本文引用: 1] 摘要
We previously developed an in vitro-production system for porcine embryos and reported that the addition of glutamine (Gln) and (HT) during in vitro culture improved embryo . This study examined the effects of Gln and HT on in vitro , oxidative status and DNA damage of porcine preimplantation embryos. Porcine zygotes produced by in vitro maturation (IVM) and in vitro (IVF) were cultured until day 2 (day 0 = day of IVF) in porcine zygote medium (PZM) including 2 mM Gln and 5 mM HT, namely PZM-5. On day 2, the cleaved embryos were selected and cultured for 24 h in PZM-5 to which one of the following substances was added: (1) none (control); (2) Gln; (3) HT; or (4) Gln + HT. After 24 h of culture in each medium, the embryos were then returned to PZM-5 and cultured until day 5. Day-5 blastocyst yield was significantly higher in the Gln and Gln + HT groups (p < 0.05) than in the control and HT groups. In addition, Gln + HT significantly increased the total number of cells in blastocysts (p < 0.05) compared with the control. Although the number of cells and the GSH levels in day-3 cleaved embryos did not differ among treatments, addition of Gln, HT or Gln + HT significantly (p < 0.05) reduced the content and the extent of DNA damage compared with the control. These results indicate that the presence of Gln and HT in PZM-5 from day 2 to day 3 promotes the of porcine embryos by improvement of oxidative status.
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| [17] |
In vitro development of equine nuclear transfer embryos:effects of oocyte maturation media and amino acid composition during embryo culture [J].DOI:10.1017/S0967199403001102 URL PMID: 12625532 [本文引用: 1] 摘要
This study was conducted to evaluate the effects of insulin-like growth factor I (IGF-I) and other media factors during oocyte maturation, and the presence of different compositions of amino acids in embryo culture medium, on the development of equine embryos. Oocytes recovered from slaughterhouse-derived ovaries were matured in vitro for 24 h and those with a polar body were subjected to intracytoplasmic sperm injection (ICSI) or nuclear transfer with adult fibroblasts (NT). For ICSI embryos, there were no significant differences in rates of morphological cleavage, cleavage with normal nuclei or average nucleus number at 96 h post-ICSI between the absence and presence of IGF-I in maturation medium, or between embryos cultured in G1.2 or a modified CZB medium (CZB-C). Embryos produced by interspecies NT (equine donor cells into bovine cytoplasts) also showed no difference in cleavage rate or average nucleus number whether cultured in G1.2 or in CZB-C. The rates of cleavage, cleavage with normal nuclei and average nucleus number of equine NT embryos were not significantly different among oocytes matured in M199 with FSH in the presence or absence of IGF-I, or in EMMI medium, which contains IGF-I, epidermal growth factor, steroid hormones, FSH and LH. There were no differences in development of equine NT embryos cultured in any of three amino acid treatments (with or without non-essential amino acids, or containing taurine, hypotaurine and cysteine only). The cleavage rate and average nucleus number of parthenogenetically activated oocytes (treated similarly to NT oocytes but not enucleated or subjected to donor cell injection) were significantly (p < 0.05) higher than those for NT embryos. These results indicate that the presence of IGF-I or of EMMI medium during in vitro maturation of equine oocytes does not have a beneficial effect on their developmental competence as assessed at 96 h. Presence or absence of non-essential amino acids in embryo culture medium does not affect development of NT embryos within the first 96 h of culture. Factors associated with enucleation or nuclear transfer decrease the developmental competence of equine NT embryos. CZB-C medium may be used for culture of equine embryos with results similar to those obtained with G1.2 medium, thus providing a base medium that may be modified for further study of culture requirements of equine embryos.
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| [18] |
DOI:10.3969/j.issn.1006-060X.2013.09.002 URL [本文引用: 1] 摘要
以小白鼠为试验动物,随机分为 未妊组(不作处理的未妊小鼠)、妊娠抑制组(注射L-NAME)和妊娠去抑制组(注射L-NAME+SNP),采用子宫角注射法,通过分析胚胎附植不同时 期金属蛋白酶-9(MMP-9)基因mRNA的表达水平的变化,研究一氧化氮影响胚胎的附植机理。结果表明:MMP-9在性成熟未妊小鼠及正常妊娠小鼠的 胚胎附植前期,附植期和附植后期均有不同程度的表达。注射NOS抑制剂后,子宫内膜MMP-9 mRNA水平在附植前期(检测不到表达)和附植期(0.47±0.15)均受到明显抑制,极显著低于妊娠对照则同期附植过程的MMP-9 mRNA表达水平(P0.01);而抑制组附植后期的子宫内膜MMP-9 mRNA水平(0.55±0.10)则与未妊组附植后期的水平相类似(P0.05)。L-NAME和SNP合并注射时MMP-9的表达水平正常,说明NO 可以通过局部调节MMP-9的表达而参与胚胎植入过程的调节。
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| [19] |
整合素αVβ3在小鼠胚胎植入中的作用 [J].
<p>整合素是一类由α , β 亚基构成的异二聚体黏附分子, 能够调节细胞间的黏附和细胞通讯. 近期研究表明, 整合素α Vβ 3在胚胎“植入窗口”期能够参与母胎相互作用, 是子宫内膜接受性的标记分子, 同时决定胚胎侵入性. 就此作用机理作了进一步研究. 间接免疫荧光和激光共聚焦扫描 (confocal) 结果显示, 整合素α Vβ 3在小鼠胚泡中明显表达. 妊娠第3天小鼠子宫角注射α Vβ 3抗血清显著降低妊娠第8天子宫角的着床胚胎数 (<em>P</em> < 0.001). 在共培养体系中, 1∶100 和1∶200稀释度的整合素α Vβ 3抗血清对小鼠胚泡在子宫上皮细胞单层上的黏附和扩展有显著抑制作用. 相关分析显示, 整合素α Vβ 3抗血清的这种抑制作用具浓度/稀释度依赖性. 由此可见, 整合素α Vβ 3是小鼠胚胎植入子宫内膜中的必需因子, 它在“植入窗口”期的小鼠胚泡中明显表达, 能够通过作用于胚泡滋养层在子宫上皮细胞的黏附和扩展途径来调节胚胎植入过程.</p>
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