应用FRAP技术分析小鼠嵌合体和正常胚胎发育中细胞间隙连接介导通讯

应用激光扫描共聚焦显微镜的光漂白恢复(fluorescence redistribution after photobleaching,FRAP)技术分析小鼠嵌合体胚胎和正常胚胎的卵裂球之间细胞间隙连接介导通讯(gap junctional inter-cellular communica-tion,GJIC),结果发现:8-细胞期嵌合体胚胎的光漂白恢复率(24.3%)明显低于正常胚胎(64.2%),提示GJIC的降低可能是影响嵌合体胚胎发育率降低的因素之一;囊胚的光漂白恢复率也较低(22.7%),提示随着细胞分化,GJIC的水平有所降低。     

小鼠2-细胞胚胎单个卵裂球体外培养及其发育形成囊胚的玻璃化冷冻保存技术的研究

单个卵裂球分离和培养是生产同卵双胎或一卵多胎动物的一种有效方法。在羊和牛已经获得了来源于2-细胞和4-细胞期胚胎的单个卵裂球的活体后代;在兔也获得来源于4-细胞胚胎的单个卵裂球后代;在猪已获得8-细胞单个卵裂球的后代。但多数的研究者发现,小鼠单个卵裂球培养后的体外发育率、移植妊娠率和产仔率都很低。上述均为新鲜胚移植结果,至于分离后卵裂球发育成的囊胚再进行玻璃化冷冻保存尚未见报道。本实     

慢病毒载体介导的绿色荧光蛋白转基因标记法研究猪嵌合体制作

目的:通过建立慢病毒载体感染猪胚胎体系实现胚胎标记,进而研究不同发育阶段猪孤雌胚胎之间的嵌合能力,为进一步研究猪早期胚胎发育以及细胞分化奠定基础.方法:首先,通过显微注射的方法把2×109I.U./ml、2×108I.U./ml和2×107I.U./ml三个梯度的表达绿色荧光的慢病毒载体分别注射到猪1-细胞胚胎和2-细胞胚胎的透明带下,进行胚胎的GFP转基因标记,在荧光显微镜下观察比较卵裂率、阳性胚胎率、囊胚率、阳性囊胚率和囊胚细胞数.然后,采用凹窝聚合法对同步发育胚胎在不同阶段(2-细胞,4-细胞,8-细胞)进行嵌合,2-细胞胚胎与不同发育阶段(2-细胞、4-细胞、8-细胞)胚胎进行嵌合以及2-细胞胚胎卵裂球互换制作嵌合体胚胎,发育到囊胚时在荧光显微镜下检测胚胎的嵌合状态.结果:2×109I.U./ml的慢病毒感染猪2-细胞胚胎组中,体外受精和孤雌胚胎感染阳性率( 80.00％、76.36％)和阳性囊胚率(90.74％、89.56％)都显著高于其它滴度组(P＜0.05),另外,慢病毒感染的两种胚胎与对照组对卵裂率、囊胚率和囊胚细胞数三个指标没有显著影响(P＞0.05).2-细胞胚胎之间嵌合囊胚率和2-细胞卵裂球互换嵌合囊胚率( 53.85％、62.50％)显著高于2-细胞胚胎与4-细胞胚胎的嵌合率(18.60％,P＜0.05),在同步发育胚胎中8-细胞胚胎之间的嵌合率(75.00％)高于4-细胞胚胎之间和2-细胞胚胎之间的嵌合率( 65.00％、53.80％).结论:2×109I.U./ml的慢病毒感染2-细胞期胚胎效率最高,另外,慢病毒感染对猪胚胎发育没有明显影响.8-细胞间的嵌合率比较高;发育同步胚胎间的嵌合率高于发育非同步胚胎间的嵌合率.     

使用激光微束研究哺乳动物早期胚胎分化

近年来，一些研究者用显微操作方法证实， 哺乳动物早期胚胎至少直至8一细胞期，每个卵 裂球仍然是全能的，即还没有开始分化。1965 年Daniel用激光微束照射破坏2一细胞期兔胚中 一个卵裂球后，另一卵裂球在培养条件下发育 至桑堪胚期。此外，还有Tarkowski和Wroblewska (1967）的卵裂球分离实验，Mintz (1965) 卵裂球重聚合实验以及最近Willadsen (1979) 羊胚卵裂球分离实验等。     

兔体细胞核移植的初步研究

实验以兔胎儿成纤维细胞为核供体,对兔体细胞核移植技术的融合,激活和发育等环节进行了初步研究。实验通过比较不同电场强度对兔2细胞胚胎卵裂球融合以及卵母细胞激活的影响,证实200和260V/mm的电场强度可有效地诱导2细胞胚胎的融合和兔卵母细胞的孤雌激活。然后将200和260V／mm电场强度用于体细胞核移植,融合率分别为44．4％和48．4％,卵裂率分别为58．8％和53．8％,桑椹胚／囊胚发育率分别为5．9％和5．5%。但112枚核移植胚胎移植到5只受体后没有幼子出生。结果表明,实验中所建立的程序至少可以支持兔体细胞克隆胚胎的早期发育。     

不同冷冻保护剂对早期卵裂期小鼠胚胎冷冻后成活率和发育的影响

为了评价利用不同冷冻保护剂冷冻早期卵裂期胚胎的效果,用小鼠为实验动物,采用慢速冷冻、快速融解的冷冻技术,比较丙二醇、二甲基亚砜和甘油作冷冻保护剂对小鼠2-细胞、4-细胞、8-细胞胚胎冷冻后胚胎存活率和囊胚形成率的影响。发现以丙二醇和蔗糖为冷冻保护剂冷冻4-细胞、8-细胞胚胎,解冻后胚胎成活率和囊胚形成率显著高于以二甲基亚砜或甘油为冷冻保护剂。结果表明,丙二醇是一种冷冻早期卵裂期小鼠胚胎有效的冷冻保护剂。     

体细胞来源及培养代数对核移植重构胚发育的影响

为探讨体细胞来源及培养代数对核移植重构胚发育的影响,实验采用电融合法将小鼠2—细胞胚胎卵裂球、胚胎干细胞(ES)、胎儿成纤维细胞、耳成纤维细胞、尾尖成纤维细胞、睾丸支持细胞和精原细胞以及不同培养代次的胎儿成纤维细胞进行了核移植。结果显示：2—细胞胚胎卵裂球供核重构胚发育最好,囊胚率为7．4％;ES细胞重构胚虽然发育率低,但仍有囊胚出现,比例为0．7％;胎儿成纤维细胞重构胚最高发育阶段为桑椹胚,比例为0．2％;精原细胞重构胚只能发育到8-细胞阶段,比例为0．3％;其他几类细胞重构胚则仅能发育至4-细胞阶段。不同培养代数的胎儿成纤维细胞重构胚除第3代外都可发育到8-细胞阶段,且发育率差异不显著,但第一代细胞重构胚2-细胞发育率(40．7％)显著低于2、3和4代细胞重构胚。结果表明：不同分化程度的细胞核移植后,重新编程的难易程度是不一样的,分化程度越高则重新编程越难;未调整细胞周期的ES细胞由于多数处于S期,所以重构胚发育率很低;体外培养传代有利于体细胞核移植后重新编程。     

激光显微照射金鱼受精卵对其胚胎发育的影响

2-细胞期胚胎经激光显微照射(功率为90毫瓦)其中一个卵裂球时能产生明显的损伤光斑,受照射的卵裂球立即停止发育。另一未受照射的卵裂球仍能正常卵裂直至孵化幼鱼,所获得的幼鱼在形态上与正常幼鱼相同。8-细胞或囊胚期胚胎经激光显微照射(功率为372毫瓦)时,则可在受照射的卵裂球上产生明显伤斑,并在照射部位溢出部分细胞内含物,绝大部分胚胎发育成不正常的胚体和各种畸形幼鱼。     

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。     

一种用FRAP测定细胞间隙连接介导通讯的方法

本文探讨了利用荧光漂白恢复技术(FRAP)测定体外培养小鼠大脑皮层神经胶质细胞间隙连接通讯的方法。经细胞培养、荧光染色、荧光激发、荧光漂白、激光扫描及计算 机分析等处理,结果各所选神经胶质细胞内荧光均得到不同程度的恢复,即经扫描15.3分钟后,1—5号细胞内荧光相对强度分别上升了14％、28％、43％、17.5%、12.5％,三次平均上升了18.57±10.06％,荧光恢复速率平均为1.223±0.785％/min(n=14)。表明该方法可用来测定细胞间隙连接通讯。     

Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment

The development of nuclear-transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight- and 16-cell stages (oocytes and zygotes) or with pronuclei-containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight-cell stages were also transferred into pseudopregnant does. Development of nuclear-transfer oocytes was distinctly better than that of nuclear-transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight- and 16-cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four-cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four-cell embryos developed from enucleated zygotes receiving eight-cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16-cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte- and zygote-derived nuclear-transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16-cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight-cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight-cell nucleus in the development of reconstituted zygotes.     

Polarization of blastomeres in the cleaving rabbit embryo

Cellular polarization is believed to be a crucial event in the differentiative divergence of the two cell lineages leading to the blastocyst in rodent embryos. This study was undertaken to determine if rabbit embryos exhibited cellular polarization prior to blastocyst formation and to define the embryonic stage at which polarization was first apparent. Polarity was assayed by observation of the pattern of binding of FITC-Con A to dissociated blastomeres from three stages of rabbit embryos. Scanning electron microscopy on the dissociated cells confirmed the fluorescence results. Fifty-one percent of blastomeres in 38- to 66-cell rabbit embryos exhibited an intense pole of FITC-Con A binding and a single pole of microvilli. Only 2% of blastomeres at the 17- to 34-cell stage were similarly polarized and none were polarized at the 8- to 16-cell stage. In addition, during attempts to remove the mucin coat and zona pellucida from the rabbit embryos prior to their dissociation, it was found that the protease sensitivity of these coats also changed at the 38- to 66-cell stage. Prior to this time, although the mucin coat disappeared after 30 min in 0.5% pronase, the zona required approximately 1.5-2.5 hr in pronase for even partial removal. At the 38- to 66-cell stage, pronase dissolved the mucin coat within 10 min and the zona pellucida within 20 min. The zona was resistant to 0.1% proteinase K at all stages examined.     

Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo

Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived embryos (相似文献   

Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.     

Archenteron-Forming Capacity in Blastomeres Isolated from Eight-Cell Stage Embryos of the Starfish, Asterina pectinifera

Starfish blastomeres are reported to be totipotent up to the 8-cell stage. We reinvestigated the development of blastomeres of 8-cell stage embryos with a regular cubic shape consisting of two tiers of 4 blastomeres. On dissociation of the embryo by disrupting the fertilization membrane at the 8-cell stage, each of the 4 blastomeres of the vegetal hemisphere gave rise to an embryo that gastrulated, whereas blastomeres from the animal hemisphere did not. By injection of a cell lineage tracer into blastomeres of 8-cell stage embryos, we found that only those of the vegetal hemisphere formed cells constituting the archenteron. Next, we compressed 4-cell stage embryos along the animal-vegetal axis so that all the blastomeres in the 8-cell stage were in a single layer. When these 8 blastomeres were then dissociated, an average of 7 of them developed into gastrulae. By cell lineage analysis, all the blastomeres in single-layered embryos at the 8-cell stage were shown to have the capacity to form cells constituting an archenteron. Taken together, these findings indicate that the fate to form the archenteron is specified by a cytoplasmic factor(s) localized at the vegetal hemisphere, and that isolated blastomeres that have inherited this factor develop into gastrulae.     

Effect of blastomere sex and fluorescent labelling on the development of bovine chimeric embryos reconstituted at the four-cell stage

The development rate of bovine chimeric embryos reconstituted at the 4-cell stage is relatively low. If chimerism is to be used as an approach in producing transgenic livestock, it is important to investigate whether this rate is affected by the sex of the blastomeres being combined and if all blastomeres survive equally well. In Experiment 1, blastomeres from 4-cell stage embryos were inserted into surrogate zonae pellucidae either in pairs to reconstitute 4-cell chimeras, or as the original sets of four to make handled controls. The development of chimeras with one pair of blastomeres labelled with PKH26-GL was also investigated. The rate of development into blastocysts was similar in chimeras with unlabelled blastomeres (23%) and in those in which one pair of blastomeres was labelled (26%) and was lower (P < 0.001) than in the handled and IVF control groups (43 and 58%, respectively). Labelled cells were distributed approximately evenly between ICM and trophoblast. In Experiment 2, the effect of sex differences between pairs of blastomeres in chimeras was investigated; chimeras were reconstituted from pairs of blastomeres taken from 4-cell embryos in which the remaining pair was sexed by PCR. No significant differences according to the sex of constituent blastomeres were detectable (mixed sex, 27%; males, 24%; females, 21%; P > 0.05). These results suggest that, in addition to the negative effects of micromanipulation, factors other than the sex of the blastomeres are involved in the reduced rate of development of chimeric bovine embryos. They also confirm the usefulness of PKH26-GL labelling for tracking the progeny of cleaving bovine blastomeres at least to the blastocyst stage.     

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.