葡萄糖对小鼠胚胎体外发育的影响

通过在CZB培养液中添加不同浓度葡萄糖及在胚胎发育的不同阶段加入葡萄糖,对小鼠胚胎进行体外培养,以探讨葡萄糖在小鼠早期胚胎体外发育中的作用。其结果表明,小鼠胚胎在含糖CZB与在无糖CZB中培养比较,4-细胞发育率无差异;各浓度葡萄糖组囊胚率显著高于无糖组,其中3.0mmol/L浓度组囊胚细胞数显著高于其余组;实验二：2-细胞至4-细胞、4-细胞至桑椹胚前添加葡萄糖囊胚率显著提高。上述结果证明,在小鼠胚胎体外培养中加入葡萄糖不会导致2-细胞阻滞;葡萄糖浓度增加至10mmol/L对小鼠胚胎无毒性作用,其最适浓度为3.0mmol/L;2-细胞至4-细胞、4-细胞至桑椹胚前添加葡萄糖是必要的。关键词 葡萄糖;小鼠;2-细胞阻滞;胚胎;体外发育     

昆明小鼠原核胚在不同培养液中的体外发育

目的优化昆明小鼠原核胚胎体外培养系统,提高胚胎发育率.方法小鼠经超排获得原核期胚胎,制备小鼠输卵管上皮共培养系统,使用M16、CZB和KSOM培养液进行体外培养,并对体内和体外发育的囊胚细胞计数.结果在KSOM和CZB中添加胎牛血清能显著提高胚胎囊胚发育率(14.71%对85.71%;6.45%对10.81%);输卵管上皮共培养可以提高胚胎的卵裂率和囊胚发育率,同时提高胚胎质量和同步发育,小鼠胚胎在KSOMFBS中囊胚发育率达85.19%,显著高于CZB和M16.结论在小鼠输卵管上皮共培养条件下,KSOMFBS能够很好支持昆明小鼠原核期胚胎体外发育.     

葡萄糖对ICR小鼠胚胎体外发育的影响

研究葡萄糖在小鼠早期胚胎体外发育中的作用。实验1将6—8周龄的ICR雌鼠超数排卵后与公鼠交配,收集1-细胞放入含0(对照组)、0.5、1、3、5、10mmol/L葡萄糖的CZB中培养;实验2将从超排的ICR雌鼠输卵管内收集的1-细胞放入无糖CZB中培养,分别于1细胞、2细胞、4细胞、桑椹胚阶段移入含3.0mmol/L葡萄糖(最适浓度)的CZB中,培养24h后又移回到无糖CZB中(桑椹胚阶段除外)继续培养以及整个胚胎培养过程均在含糖CZB中,对照组胚胎培养全程均在无糖CZB中。每组胚胎于37℃、5%CO2培养箱中培养120h,每24h在倒置显微镜下观察胚胎发育情况,分别计算2-细胞率、4-细胞率、桑椹胚率、囊胚率和孵化率,并进行囊胚细胞计数。结果显示,小鼠胚胎在含糖CZB中与在无糖CZB中4-细胞发育率无差异;含糖CZB中囊胚率显著高于对照组;3.0mmol/L浓度组囊胚细胞数显著高于其余组;2-细胞至4-细胞、4-细胞至桑椹胚前添加葡萄糖囊胚率显著高于对照组,1-细胞至2-细胞、桑椹胚及其以后阶段添加葡萄糖囊胚率与对照组无差异。实验证实,在ICR小鼠胚胎体外培养中加入葡萄糖不会导致2-细胞阻滞;葡萄糖浓度增至10mmol/L对ICR小鼠胚胎无毒性作用;ICR小鼠胚胎体外培养的最适葡萄糖浓度为3.0mmol/L;2-细胞至4-细胞、4-细胞至桑椹胚前添加葡萄糖是必要的。     

三种抗氧化剂对小鼠体外受精胚胎发育的影响

本实验以昆明小鼠为实验对象,CZB为基础培养液,在其中分别添加不同浓度的三种抗氧化剂-槲皮素（quercetin）、维生素C和维生素E,比较它们对小鼠体内受精1-细胞胚胎体外发育的影响,测定其克服小鼠胚胎体外发育阻滞、促进胚胎发育的最佳浓度。然后将三种抗氧化剂分别加入体外受精胚胎培养液中,观察三种抗氧化剂对小鼠体外受精胚胎发育的作用,     

几种克服昆明小鼠2一细胞胚胎发育阻滞的培养液研究

比较了几种培养液对克服昆明小鼠胚胎２－细胞发育阻滞的效果。实验１的结果表明，在Ｍ１６及ＣＺＢ培养液基础上，加减几种成分得到的改进Ｍ１６培养液（用ｍＭ１６表示）和改进的ＣＺＢ培养液（用ｍＣＺＢ表示）均能有效克服２－细胞阻滞现象。除去Ｍ１６和ＣＺＢ培养液中的葡萄糖和磷酸盐后添加５．５５ｍｍｏｌ／Ｌ牛磺酸、１００或１１０μｍｏｌ／Ｌ ＥＤＴＡ、２ｍｍｏｌ／Ｌ谷氨酰胺和２％必需氨基酸（ＥＡＡ）及１％非必需     

牛体外受精卵在两种共同培养系统中发育的研究

为了使牛体外受精卵能通过体外早期发育阻滞期,我们建立了卵丘细胞单层(A)和输卵管上皮单层(B)两种共同培养系统。A1共同培养实验是在本实验室进行的,A2和B共同培养实验均在日本岗山大学农学部动物繁殖学研究室进行,牛输卵管组织的分离使用0.76％EDTA—PBS溶液,共同培养系统均使用含10％小牛血清的TCM—199(Earle's salts)作为培养液。培养的卵泡卵母细胞体外成熟率为100％,体外受精率为99—100％。在A1共同培养实验中,越过阻滞期发育到16—细胞以上的胚胎占卵裂胚的35.7％,与A2共同培养实验中越过阻滞期的发育率(40.1％)无显著差异(P<0.05)。A1和A2共同培养实验,在卵裂基础上得到的桑椹胚和囊胚发育率分别为23.7％和27.9％。每百枚培养的卵母细胞,在A1共同培养实验中可获得桑椹胚和囊胚15.1枚,在A2共同培养实验中可获桑椹胚和囊胚的20.5枚。B共同培养实验中桑椹胚和囊胚发育率为54.1％,显著高于A1或A2共同培养实验的相应发育率(P<0.001),使用B共同培养系统每百枚培养的卵母细胞可以获得37枚桑椹胚和囊胚。     

小鼠体外受精、胚胎培养及胚胎快速冷冻的研究

目的　为扩大胚胎来源并获取特定胚龄胚胎 ,建立小鼠冷冻胚胎库。方法　运用超数排卵、体外受精与胚胎培养及胚胎冷冻技术系统研究了小鼠受精卵的体内发育与运行规律。卵母细胞的体外成熟与受精、单细胞胚胎培养及胚胎快速冷冻。结果　 (1)注射hCG后 12～ 2 0h受精卵发育至原核期 ,4 2～ 4 8h为 2 细胞期 ,4 8～ 6 0h为 4 细胞期 ,6 0～ 6 8h为 8 细胞期 ,以上各期受精卵均处于输卵管中 ;75～ 78h为桑椹胚 ,78～ 80h为致密桑椹胚 ,90～ 92h为早期囊胚 ,92～ 96h为囊胚 ,以上各期均处于子宫角中。 (2 )培养液中添加促性腺激素 (FSH与hCG) ,能显著提高卵母细胞的体外受精率 ,添加FCS和激素组的体外受精率又显著高于单独添加激素组 ,FCS还能显著提高胚胎发育。 (3)在培养液中添加EDTA ,能有效克服小鼠胚胎的 2 细胞阻断 ,其 2 细胞胚的发育率达 10 0 % ,8 细胞胚发育率达 5 5 %以上 ;牛、羊上皮细胞培养液上清也能有效克服 2 细胞阻断。添加乳酸钠和丙酮酸钠可使 2细胞与 8细胞期胚的发育率显著提高。 (4)以D PBS +甘油 +蔗糖为冷冻液 ,以D PBS +蔗糖为稀释液 ,对小鼠胚胎进行快速冷冻 ,桑椹胚的存活率为 6 9 3% ,早期囊胚的存活率为 6 0 4 %。结论　研究为将生物技术应用于小鼠 ,扩大卵子和胚胎来源     

早期转基因兔胚的培养及外源基因滞留的研究

用显微注射法导入外源Smtpgh基因的早期免胚进行体外培养,并用PCR技术对单个胚中的外源基因进行了检测,以探讨SMTPGH基因在早期免胚中的滞留情况。研究结果表明早期转基因免胚在Tcl99+10％Fcs培养液中有75％发育到囊胚期;其外源基因在8细胞期前没有丢失,随后有逐渐丢失的现象。到胚胎的发育后期,其外源基因的滞留率接近于整合率。     

兔输卵管上皮细胞解除大鼠早期胚胎发育阻滞的研究

本研究利用兔输卵管上皮细胞(ROEc), 与异种动物大鼠的受精卵共培养,结果大量出现突破2细胞发育阻滞的现象。体外受精卵和体内受精卵的2细胞发育阻滞突破率分别为62%和73%;利用ROEC 条件培养液培养大鼠的体外受精卵,2细胞发育阻滞的突破率达68%,且能顺利发育至桑椹胚和囊胚。将ROEC直接培养在含~(35)S-甲硫氨酸的CZB 培养液中,经SDS-聚丙烯酰胺凝胶电泳及放射自显影,发现在该条件培液中出现了分子量分别为135Kd,68Kd,55Kd,51Kd 和44Kd 由ROEC 分泌的多肽,其中68 Kd 区带最显著,而135 Kd区带非常弱。用(125)~I-酰化剂标记示踪发现,与ROEC 共培养24 h 的大鼠胚胎透明带上含有68Kd 蛋白,并发现55 Kd 蛋白与透明带结合的痕迹。这些蛋白很可能就是胚胎发育调控由母型向合子型过渡并突破早期发育阻滞的关键因子,在功能上似无种属特异性。     

小鼠原核期受精卵体外培养系统的筛选

为了筛选出最佳的小鼠原核期受精卵的体外发育培养系统,分别进行了四个试验。试验I:在体外分别用自配的M16、mM16、KSOM、mKSOM、CZB进行体外发育培养,进而筛选出一种最佳的体外培养系统;试验Ⅱ、Ⅲ、Ⅳ分别:探讨了血清、PVA、rhLIF对小鼠胚胎发育的影响。结果,试验I中胚胎发育到2-细胞的比率差异不明显,但是在mM16和mKSOM中,发育到4-细胞的比率94.7%,90.7%(91/96;78/86)和发育到桑椹胚/囊胚的比率分别为78.1%,67.4%(75/96;58/86)均明显高于其他三种培养液;试验II用10?S代替M16中BSA时,胚胎的发育率均下降,即使在mM16中桑椹胚/囊胚率仅为4.8%(12/35)与对照组M16(40.5%)差异显著(p<0.05);试验Ⅲ用PVA取代mM16和mKSOM中的BSA其体外发育率显著下降,胚胎均无一例发育到桑椹胚/囊胚;试验Ⅳ:rhLIF能提高胚胎在体外的发育率可使mM16培养的胚胎囊胚率、囊胚脱出率分别达到84%(47/56)、39.2%(22/56)。结论:在不添减其他成分前提下,只在M16中添加0.1mMolEDTA、0.5mMol牛磺酸、1000IU/mlrhLIF便可获得84%的囊胚率,同时证明在M16或mM16添加血清都会降低其体外发育率;PVA还不能有效的取代mM16、mKSOM中的血清。     

An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro

One-cell CF-1 x B6SJLF1/J embryos, which usually exhibit a 2-cell block to development in vitro, have been cultured to the blastocyst stage using CZB medium and a glucose washing procedure. CZB medium is a further modification of modified BMOC-2 containing an increased lactate/pyruvate ratio of 116, 1 mM-glutamine and 0.1 mM-EDTA but lacking glucose. Continuous culture of one-cell embryos in CZB medium allowed 83% of embryos to develop beyond the 2-cell stage of which 63% were morulae at 72 h of culture, but blastocysts did not develop. However, washing embryos into CZB medium containing glucose after 48 h of culture (3-4-cell stage) was sufficient to allow development to proceed, with 48% of embryos reaching the blastocyst stage by 96 h of culture. Exposure of embryos to glucose was only necessary from the 3-4-cell stage through the early morula stage since washing back into medium CZB without glucose at 72 h of culture still promoted the development of 50% of embryos to the blastocyst stage. The presence of glucose in this medium for the first 48 h of culture (1-cell to 4-cell stage) was detrimental to embryo development. Glutamine, however, exerted a beneficial effect on embryo development from the 1-cell to the 4-cell stage although its presence was not required for development to proceed during the final 48 h of culture. Blastocysts which developed under optimum conditions contained an average of 33.7 total cells. The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.     

Nuclear-cytoplasmic "tug of war" during cloning: effects of somatic cell nuclei on culture medium preferences of preimplantation cloned mouse embryos

Cloning by somatic cell nuclear transfer is critically dependent upon early events that occur immediately after nuclear transfer, and possibly additional events that occur in the cleaving embryo. Embryo culture conditions have not been optimized for cloned embryos, and the effects of culture conditions on these early events and the successful initiation of clonal development have not been examined. To evaluate the possible effect of culture conditions on early cloned embryo development, we have compared a number of different culture media, either singly or in sequential combinations, for their ability to support preimplantation development of clones produced using cumulus cell nuclei. We find that glucose is beneficial during the 1-cell stage when CZB medium is employed. We also find that potassium simplex optimized medium (KSOM), which is optimized to support efficient early cleavage divisions in mouse embryos, does not support development during the 1-cell or 2-cell stages in the cloned embryos as well as other media. Glucose-supplemented CZB medium (CZB-G) supports initial development to the 2-cell stage very well, but does not support later cleavage stages as well as Whittten medium or KSOM. Culturing cloned embryos either entirely in Whitten medium or initially in Whittens medium and then changing to KSOM at the late 4-cell/early 8-cell stage produces consistent production of blastocysts at a greater frequency than using CZB-G medium alone. The combination of Whitten medium followed by KSOM resulted in an increased number of cells per blastocyst. Because normal embryos do not require glucose during the early cleavage stages and develop efficiently in all of the media employed, these results reveal unusual culture medium requirements that are indicative of altered physiology and metabolism in the cloned embryos. The relevance of this to understanding the kinetics and mechanisms of nuclear reprogramming and to the eventual improvement of the overall success in cloning is discussed.     

Development of 1-cell embryos from different strains of mice in CZB medium

One-cell embryos from several different strains of mice have been cultured to the blastocyst stage in CZB medium. CZB medium can be used to culture CF1 x B6SJLF1/J 1-cell embryos to the blastocyst stage provided glucose is introduced into the medium on Day 3 of culture. The amount of glucose required for embryo development was titrated using a concentration range of 5.5 to 49.5 mM. With the exception of the highest concentration, all glucose levels tested supported 65-85% development to the morula and blastocyst stages. Variations of CZB medium were tested for their ability to support the development of 1-cell embryos from 4 strains of mice. For embryos from CF1 and DBA/2J (both x B6SJLF1/J) mice, which exhibit a "2-cell block" to development in vitro, CZB medium containing glutamine with the addition of glucose on Day 3 supported optimum development from the 1-cell stage to morula and blastocysts (79% and 87%). For embryos from B6D2F1/J and CD1 female mice (both x B6SJLF1/J males), which do not exhibit a "2-cell block" to in vitro development, optimum development to morula and blastocyst stages (95% and 50%) was in CZB medium containing both glutamine and glucose from the start of culture.     

模拟微重力环境对昆明小鼠早期胚胎体外发育的影响

90年代初,美国航空航天局(NASA)设计研制出一种转壁式生物反应器(Rotating Wall Vessel Bioreactor,RWVB)。采用RWVB进行基地试验时,意外地发现离体细胞在RWVB中呈现高密度聚集,并形成较大的组织样结构。RWVB的核心结构是由两个同心圆柱体构成的旋转培养装置。将细胞与培养液置入内、外圆柱体之间,整个装置绕水平纵轴旋转,使培养物长时间保持悬浮状态。由于在旋转过程中     

M16添加牛磺酸和EDTA支持昆明白小鼠体外受精并发育至囊胚

在以往研究工作的基础上证明了通过添加2.5mmol/L的牛磺酸和0.1mmol/L的EDTA至M16培养液中,可支持昆明白小鼠的体外受精（IVF）,并支持受精卵突破2－细胞阻滞发育至囊胚期。本研究进一步证明了牛磺酸和EDTA在昆明小鼠早期胚胎发育和克服2－细胞阻滞中起关键作用。 Abstract:The results of our early experiments show that the 2-cell block can be overcome by culturing zygote in modified M16 ,modified CZB and TE medium.Our research shows that the taurine and EDTA play key role in overcoming 2-cell block in Kunming mouse.The results show that the addition of taurine and EDTA to M16 medium can support the IVF and develoment to blastocyst in vitro in Kunming strain mice.This is the first report that M16 medium added with taurine and EDTA can be used in both IVF and culture medium to overcome the 2-cell block of embryo development in Kunming strain mouse.     

模拟微重力环境对昆明小鼠早期胚胎体外发育的影响（简报）

In order to investigate the development of Kunming mouse preimplantation embryos cultured in vitro under simulated microgravtiy, one-cell and 4-cell embryos of Kunming mouse (Fertilization In Situ) were cultured in CZB or KSOM media under simulated microgravity or normal gravity environment respectively. The results showed that under normal gravity, the percentage of passing 2-cell development block embryos was not different in CZB with in KSOM, but the percentage of blastocysts was lower in CZB than that in KSOM significantly. The percentages of passing 2-cell development block embryos and blastocysts in CZB or KSOM were lower under simulated microgravity than those under normal gravity. It is suggested that the frequency of early embryonic lethality is possibly increased by simulated microgravity.     

One-minute exposure of 4-cell mouse embryos to glucose overcomes morula block in CZB medium

One-cell mouse embryos that block at the 2-cell stage can progress to the morula stage in CZB medium, but fail to cavitate and then swell and lyse. A 1-min exposure to 27 mM glucose at the 4-cell stage (～42 hr) will support a high frequency of development to the blastocyst stage (75%) in the same medium. A glucose exposure is beneficial anytime between 30 and 54 hr of culture (67–73% blastocysts). Of a group of additional sugars and glucose analogues tested for their ability to replace glucose, only galactose was equivalent in promoting embryo development to the blastocyst stage (64% blastocysts). © 1994 Wiley-Liss, Inc.     

昆明白小鼠1细胞胚胎体外培养系统的研究

研究发现在有或者没有磷酸盐的条件下,葡萄糖均抑制昆明白小鼠ｌ－４细胞期胚胎的体外发 育。在不含葡萄糖和磷酸盐的ＨＥＣＭ－ｌ中,桑椹率为４０．０５％（７４／１６８）,而对照Ｇ－ＨＥＣＭ－１仅为 ８．１４％（７／８６）;不含葡萄糖含有磷酸盐的ＣＺＢ中,桑椹率为６７．１１％（９３／１５２）,而对照ＴＡＬＰ仅 ６· ６７％（６／９０）。用不含葡萄糖而含有１． ０ｍｍｏ１／Ｌ谷氨酸肢和０． １１ｍｍｏｌ／Ｌ ＥＤＴＡ的ＣＺＢ液,与兔输 卵管上皮单层培养细胞（ＲＯＥＣ）协同培养小鼠１细胞胚,７３．３３％（１１０／１５０）胚胎发育至桑椹胚, 但没有观察到囊胚形成、用上述ＣＺＨＲＯＥＣ系统培养小鼠１细胞胚４８小时（３－４细胞）,再移入 ＴＣＭ１９９＋１０％ＦＣＳ＋ＲＯＥＣ系统,有７６．７４％（６７／８６）胚胎发育至桑椹胚,９６／小时后,４０．７０％ （３５／８６）发育至囊胚。     

Effects of the culture density of mouse zygotes on the development in vitro and in vivo

Different numbers of CD-1 mouse zygotes(1, 5, 10, 20, 40 and 60) were cultured in 10 mul M16 medium, in M16 medium+EDTA, in M16 dedium+SOD+thioredoxin, and in CZB medium, respectively. When the zygotes, regardless of the number, were cultured with M16, no blastocysts could be obtained. The suitable ratio of embryos to 1 mul of M16 medium+EDTA or M16 medium+SOD+thioredoxin was 1:1 or 2:1. Medium volume from 1 to 10 mul did not affect blastocyst development when the embryo density was 1:1. However, blastocysts obtained from zygotes cultured singly had fewer cell numbers and showed inferior development to live fetuses after transfer to recipients. When CZB medium was used, suitable embryo density was not clear. The ratio of embryos to volume of culture medium was shown to be an important factor for in vitro culture of mouse zygotes.