IVF-20培养液用于大鼠体外受精的实验研究

目的探讨人用体外受精液用于实验大鼠体外受精的可行性,为实验大鼠的胚胎保种提供参考。方法选用人体外受精IVF-20培养液作为大鼠精子获能和受精培养液,对SD、Wistar、GK、F344等四种不同品系的实验大鼠进行体外受精;用mR1ECM培养液对体外受精所得胚胎进行体外培养实验。同时,将所得2-细胞胚胎移植给经假孕处理的受体鼠。结果四个品系大鼠体外受精后的卵裂率分别可达83.2%、72.6%、87.8%和71.6%。体外受精卵裂胚能够在体外进一步发育,SD大鼠囊胚发育率为16.7%,与体内受精胚胎在体外培养的囊胚发育率(24.5%)差异不显著。80枚2-细胞胚胎移植给2只受体鼠后均妊娠成功,产下正常仔鼠10只,产仔率12.5%。结论用于人体外受精的IVF-20培养液同样适合于实验大鼠精子的体外获能和受精,可以在多种品系获得较高的卵裂率,所得卵裂胚能够在体外发育到囊胚,并且所得卵裂胚在移植给受体鼠后能够产下正常仔鼠。     

小鼠母源因子对早期胚胎发育的影响

在脊椎动物中发育过程中,卵母细胞要经历MII期停滞、受精、早期胚胎发育的启动、胚胎基因组的转录激活、并指导完成个体的发育过程。同时,核移植过程中,分化的细胞核在去核的卵母细胞中能够重编程到胚胎早期的状态并能完成个体的发育过程。在这些发育过程中母源因子都发挥了极其的重要作用。在小鼠胚胎发育研究中发现,小鼠的基因组激活发生在2细胞期,这一时期标志着合子的发育由卵母细胞控制向胚胎控制的过渡,期间发生一系列复杂的生化过程。体外培养的小鼠的胚胎的发育阻断也易发生的2细胞时期。因此对卵母细胞及早期胚胎母源因子的研究,将有利于了解早期体外培养胚胎和克隆胚胎发育失败的原因,为提高体外培养和克隆胚胎发育的成功率提供理论的基础。     

外源中心体在哺乳动物胚胎发育中的遗传机制

中心体是一个非膜包被的半保留细胞器,由一对相互垂直的圆柱形中心粒及其周围大量的高电子密度的蛋白质-中心体基质(pericentriolar material,PCM)组成.在所有哺乳动物细胞中,中心体(centrosome)作为主要的微管组织中心(microtubule organizing centers,MTOCs),起到组装和稳定微管的关键功能.在大多数哺乳动物精子形成过程中,精子保留了近端中心粒,失去了大部分的中心体旁蛋白和远端中心粒,而在卵母细胞形成过程中两个中心粒被逐渐降解,主要的中心体旁蛋白被保留了下来,弥散于卵胞质中.受精后,在卵母细胞中精子中心粒被进一步降解,来源于卵母细胞和精子的中心体旁蛋白形成受精卵的MTOCs在胚胎分裂过程中行使功能.但在小鼠等啮齿类动物精子形成过程中,两个中心粒全部被降解,因此受精卵中的MTOCs主要由来源于卵母细胞中心体旁蛋白组成.在大多数哺乳动物核移植胚胎中.外源中心粒在胚胎1-细胞期即被降解,而是来源于供体细胞和受体卵母细胞的中心体旁蛋白形成重构胚的MTOCs指导纺锤体形成,中心粒是在囊胚期才从头合成的.在灵长类中,来源于精子的中心粒能与PCM一起组成典型的中心体在胚胎分裂过程中行使功能,但在其核移植胚胎中,体细胞中心体和去核卵母细胞中剩余的中心体旁蛋白不能有效的组装形成功能性中心体,这可能是灵长类哺乳动物体细胞克隆失败的一个关键原因. 成过程中,两个中心粒全部被降解,因此受精卵中的MTOCs主要由来源于卵母细胞中心体旁蛋白组成.在大多数哺乳动物核移植胚胎中.外源中心粒在胚胎1-细胞期即被降解,而是来源于供体细胞和受体卵母细胞的中心体旁蛋白形成重构胚的MTOCs指导纺锤体形成,中心粒是在囊胚期才从头合成的.在灵长类中,来源于精子的中心粒能与PCM一起组成典型的中心 在胚胎分裂过程中行使功能,但在其核移植胚胎中,体细胞中心体和去核卵母细胞中剩余的中心体旁蛋白不能有效的组装形成功能性中心体,这可能是灵长类哺乳动物体细胞克隆失败的一个关键原因. 成过程中,两个中心粒全部被降解,因此受精卵中的MTOCs主要由来源于卵母细胞中心体旁蛋白组成.在大多数哺乳动物核移植胚胎中.外源中心粒在胚胎1-细胞期即被降解,而是来源于供体细胞和受体卵母细胞的中心体旁蛋白形成重构胚的MTOCs指导纺锤体形成,中心粒是在囊胚期才从头合成的.在灵长类中,来源于精子的中心粒能与PCM一起组成典型的中心 在胚胎分裂过程中行使功能,但在其核移植胚胎中,体细胞中心体和去核卵母细胞中剩余的中心体旁蛋白不能有效的组     

兔核移植胚胎起始发育的超微结构研究

对兔核移植胚胎起始发育的超微结构变化进行电镜观察,并与供体桑椹胚细胞,受体卵母细胞及同期正常受精胚胎的超微结构进行比较,“原核”期兔核移植胚胎的超微结构明显不同于供体桑椹胚细胞及受体卵母细胞的超微结构,而与同期正常受精胚胎相似,但有些核移植胚胎中皮质反应,及核仁和线粒体中出电子致密的网眼结构,与正常受精卵存在差别,分裂至２－细胞期时,与正常２－细胞胚超微结构更相似,结果提示,兔胚胎细胞核移植后,供     

整合人lactoferrin基因的山羊体细胞支持核移植克隆胚的体外发育

克隆了人lactoferrin基因和山羊[[beta]]-casein基因5′端调控区, 构建了人lactoferrin的乳腺表达载体, 并将该载体利用脂质体介导转染了奶山羊胎儿成纤维细胞, 获得了稳定整合人lactoferrin基因的转基因体细胞克隆17个, 其中PCR和Southern Blot检测阳性的细胞克隆14个, 阳性率82.4%。以转基因体细胞为供体细胞进行了核移植, 获得了能够体外发育的山羊转基因克隆胚胎, 体内成熟卵母细胞来源的核移植囊胚率为64.8%, 体外成熟卵母细胞来源的核移植囊胚率为51.7%, 证明了山羊转基因体细胞能够支持克隆胚的进一步发育。     

Molecular Cell | 首次揭示SCNT胚胎早期发育过程中Cohesin对初级合子基因组激活相关基因表达的阻碍

体细胞核移植(somatic cell nuclear transfer，SCNT)技术可将体细胞重编程为全能细胞，但研究人员对这一过程中染色体3D构象的重塑却知之甚少。清华大学生命科学学院颉伟研究组与华中农业大学动科动医学院苗义良研究组通过少量细胞全基因组染色质构象捕获技术(sisHi-C)检测了小鼠SCNT胚胎和体外受精(in vitro fertilization, IVF)胚胎卵裂期染色体构象变化(2020年6月23日在线发表，doi:10.1016/j.molcel.2020.06.001)。研究发现胚胎在卵裂期发育过程中拓扑相关结构域(topologically associating domain, TADs)相较其供体细胞或MII卵显著减弱，但SCNT胚胎在1-细胞期TADs明显强于相应时期的体外受精胚胎，2-细胞期之后两种胚胎TADs的变化趋同，2-细胞期之后TADs减弱，至8-细胞期TADs重新增强。研究人员进一步在1-细胞期敲低了TADs关键蛋白Cohesin (黏连蛋白)，发现Cohesin敲低的SCNT胚胎1-细胞期TADs和对照组相比明显减弱，胚胎发育至囊胚的比例显著增加。转录组测序发现Cohesin敲低后的染色体构象变化促进了初级合子基因组激活相关基因的表达，从而促进了SCNT胚胎的体外发育。该研究结果表明染色体构象的异常可能是SCNT胚胎发育能力较IVF胚胎低的重要原因，为提高SCNT胚胎发育能力提供了新思路。     

转人t-PA指形区缺失基因的山羊体细胞核移植及其继代核移植

为了探索转基因体细胞核经连续核移植后的发育潜力,以转人组织型纤溶酶原激活剂(t-PA)指形区缺失基因的山羊胎儿成纤维细胞为核供体,MII期的卵母细胞质为核受体,利用胞质内注射法构建原代核移胚胎(G0),并进行了原代核移植胚胎的继代核移植研究。比较原代和继代核移植胚胎在体外发育能力上的差异;在G1、G2代核移植试验过程中,比较了供体胚胎细胞的发育阶段对核移植胚胎体外发育的影响。结果表明,原代核移植胚胎的卵裂率(76.45%±1.17%)与继代核移植胚胎的卵裂率(72.18%±1.97%,76.05%±2.38%,75.99%±2.84%)无显著性差异(P>0.05)。但原代核移植胚胎的桑葚胚率(47.20%±2.93%)、囊胚率(11.00%±1.42%)显著高于G1、G2、G3代核核移植胚胎的桑葚胚率(34.99%±2.66%,28.23%±2.00%,23.34%±1.99%)、囊胚率(3.87%±0.67%,2.08%±1.66%,0);在G1、G2中,当用16-细胞期核移植胚胎作为核供体时的桑葚胚率(29.57%±1.53%,24.43%±1.87%)、囊胚率(1.96%±1.31%,2.01%±1.34%)低于用32~64-细胞时期的核移植胚胎的桑葚胚率(34.32%±1.31%,29.76%±1.66%)、囊胚率(3.86%±1.03%,3.48%±0.34%),但无显著性差异(P>0.05)。由此得出结论:转基因体细胞核移植胚胎不宜进行多代克隆;胞质内注射法构建核移植胚胎,用32~64-细胞期的胚胎作为核供体构建的核移植胚胎的体外发育率高于用16-细胞期的胚胎作为核供体构建的核移植胚胎的体外发育率。     

牛体细胞克隆胚胎类ES细胞集落的筛选及其核移植

对第7d的牛体细胞克隆囊胚进行体外增殖培养,分离筛选类ES细胞,并对其进行了传代培养,接种在饲养层上的体细胞克隆囊胚细胞,在传代的24h内增殖形成小集落,2～3d有雀巢状的集落出现,筛选形态相同的细胞集落进行传代培养,4～5代后,皿底出现多个大小不等的多细胞单层集落,将传4～5代的细胞集落接种到无饲养层的4孔培养皿中培养,24h出现多细胞单层集落,4～7d长满皿底,并形成上皮样细胞,呈网状,将其作为核供体细胞进行核移植实验。结果有80%（40/50）核-质融合的移核重构胚发生卵裂,5%（2/40）发育至桑椹胚期,2.5%（1/40）发育至囊胚期,92.5%（37/40）停止在2～4细胞期,结果表明：采用牛体细胞克隆胚胎的类ES细胞进行核移植,具发育形成早期胚胎的潜能。     

Oct4对鼠-猪异种核移植胚胎早期发育的影响

目的探讨Oct4转录因子能否促进鼠-猪异种核移植胚胎的早期发育。方法RT-PCR获得小鼠Oct4基因,构建pEGFP-N1-Oct4-EGFP融合质粒及pEGFP-N1-Oct4-EGFP终止质粒,pEGFP-N1质粒为阴性对照,脂质体法转染小鼠NIH3T3细胞,阳性克隆经RT-PCR,荧光显微镜验证正确后,移入去核的猪卵母细胞,观察并记录发育率。结果未转染的NIH3T3阴性对照组、转染Oct4-EGFP的小鼠NIH3T3细胞和转染pEGFP-N1组均能够支持猪异种核移植胚胎的早期发育,但转染pEGFP-N1-Oct4-EGFP实验组重构胚的卵裂率和8细胞发育率与转染pEGFP-N1组和未转染的NIH3T3组的重构胚发育率差异不显著。结论NIH3T3细胞能够支持鼠-猪异种核移植胚胎早期发育,Oct4对鼠猪异种核移植胚胎的发育并没有表现出促进作用,可能也受到NIH3T3来源的异种核移植胚胎本身发育率低以及本实验室核移植显微操作水平的限制,具体的机制尚需进一步探讨。     

转人t-PA指形区缺失基因的山羊体细胞核移植及其继代核移植

为了探索转基因体细胞核经连续核移植后的发育潜力,以转人组织型纤溶酶原激活剂（t-PA）指形区缺失基因的山羊胎儿成纤维细胞为核供体, MII期的卵母细胞质为核受体,利用胞质内注射法构建原代核移胚胎(G0),并进行了原代核移植胚胎的继代核移植研究。比较原代和继代核移植胚胎在体外发育能力上的差异;在G1 、G2代核移植试验过程中,比较了供体胚胎细胞的发育阶段对核移植胚胎体外发育的影响。结果表明,原代核移植胚胎的卵裂率(76.45%±1.17%)与继代核移植胚胎的卵裂率(72.18%±1.97%,76.05%±2.38%,75.99%±2.84%)无显著性差异(P>0.05)。但原代核移植胚胎的桑葚胚率(47.20%±2.93%)、囊胚率(11.00%±1.42%)显著高于G₁、G₂、G₃代核核移植胚胎的桑葚胚率(34.99%±2.66%,28.23%±2.00%,23.34%±1.99%)、囊胚率(3.87%±0.67%,2.08%±1.66%,0);在G₁、G₂中,当用16-细胞期核移植胚胎作为核供体时的桑葚胚率(29.57%±1.53%, 24.43%±1.87%)、囊胚率(1.96%±1.31%, 2.01%±1.34%)低于用32～64-细胞时期的核移植胚胎的桑葚胚率(34.32%±1.31%, 29.76%±1.66%)、囊胚率(3.86%±1.03%, 3.48%±0.34%),但无显著性差异 (P>0.05)。由此得出结论：转基因体细胞核移植胚胎不宜进行多代克隆;胞质内注射法构建核移植胚胎,用32～64-细胞期的胚胎作为核供体构建的核移植胚胎的体外发育率高于用16-细胞期的胚胎作为核供体构建的核移植胚胎的体外发育率。     

Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.     

Nuclear lamin antigen and messenger RNA expression in bovine in vitro produced and nuclear transfer embryos

The nuclear lamina is a complex meshwork of nuclear lamin filaments that lies on the interface of the nuclear envelope and chromatin and is important for cell maintenance, nucleoskeleton support, chromatin remodeling, and protein recruitment to the inner nucleolus. Protein and mRNA patterns for the major nuclear lamins were investigated in bovine in vitro fertilized (IVF) and nuclear transfer embryos. Expression of lamins A/C and B were examined in IVF bovine germinal vesicle (GV) oocytes, metaphase II oocytes, zygotes, 2-cell, 8-cell, 16-32-cell embryos, morulae, and blastocysts (n = 10). Lamin A/C was detected in 9/10 immature oocytes, 10/10 zygotes, 8/10 2-cell embryos, 4/10 morulae, 10/10 blastocysts but absent during the maternal embryonic transition. Lamin B was ubiquitously expressed during IVF preimplantation development but was only detected in 4/10 GV oocytes. Messenger RNA expression confirms that the major lamins, A/C and B1 are expressed throughout preimplantation development and transcribed by the embryo proper. Lamin A/C and B expression were observed (15 min, 30 min, 60 min, 120 min) following somatic cell nuclear transfer using adult fibroblasts and at the 2-cell, 8-cell, 16-32-cell, morula and blastocyst stage (n = 5). Altered expression levels and localization of nuclear lamins A/C and B was determined in nuclear transfer embryos during the first 2 hr post fusion, coincidental with only partial nuclear envelope breakdown as well as during the initial cleavage divisions, but was restored by the morula stage. This mechanical and molecular disruption of the nuclear lamina provides key evidence for incomplete nuclear remodeling and reprogramming following somatic cell nuclear transfer.     

Mouse cloning and somatic cell reprogramming using electrofused blastomeres

Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.     

Differential effect of recipient cytoplasm for microtubule organization and preimplantation development in rat reconstituted embryos with two-cell embryonic cell nuclear transfer

In the present study, we examined the developmental ability of enucleated zygotes, MII oocytes, and parthenogenetically activated oocytes at pronuclear stages (parthenogenetic PNs) as recipient cytoplasm for rat embryonic cell nuclear transfer. Enucleated zygotes as recipient cytoplasm receiving two-cell nuclei allowed development to blastocysts, whereas the development of embryos reconstituted with MII oocytes and parthenogenetic PNs was arrested at the two-cell stage. Previous observations in rat two-cell embryos suggested that the distribution of microtubules is involved in two-cell arrest. Therefore, we also examined the distribution of microtubules using immunofluorescence. At the two-cell stage after nuclear transfer into enucleated zygotes, microtubules were distributed homogeneously in the cytoplasm during interphase, and normal mitotic spindles were observed in cleaving embryos from the two- to four-cell stage. In contrast, embryos reconstituted with MII oocytes and parthenogenetic PNs showed aberrant microtubule organization. In enucleated zygotes, fibrous microtubules were distributed homogeneously in the cytoplasm. In contrast, dense microtubules were localized at the subcortical area in the cytoplasm and strong immunofluorescence intensity was observed at the plasma membrane, while very weak intensity was detected in the central part of enucleated MII oocytes. In enucleated parthenogenetic PNs, high-density and fibrous microtubules were distributed in the subcortical and central areas, respectively. Pre-enucleated parthenogenetic PNs also showed lower intensity of microtubule immunofluorescence in the central cytoplasm than zygotes. In conclusion, the results of the present study showed that zygote cytoplasm is better as recipient than MII oocyte and parthenogenetic PNs for rat two-cell embryonic cell nuclear transfer to develop beyond four-cell stage. Furthermore, microtubule organization is involved in the development of reconstituted embryos to overcome the two-cell arrest.     

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.     

Effects of donor oocytes and culture conditions on development of cloned mice embryos

Mice have been successfully cloned from somatic and embryonic stem (ES) cells using the "Honolulu method." In the present study, different donor oocytes and different culture conditions were compared to evaluate the developmental potential of nuclear transfer embryos reconstructed with an inbred ES cell line HM-1. Oocytes were recovered from two different F1 donors B6D2F1 (C57BL/6 x DBA/2) and B6CBAF1 (C57BL/6 x CBA). There was no effect of oocyte origin on development of cloned embryos to the morulae/blastocyst stage (B6D2F1 44.1% vs. B6CBAF1 45.0%), and the transferred embryos could develop to term. Two culture conditions were compared to show their ability to support development to the morulae/blastocyst stage of reconstructed embryos with B6D2F1 oocytes. The total cell number in the cloned blastocysts cultured in M16 with 20% oxygen was much higher than that observed in CZB with 20% oxygen. Low oxygen concentration during culture of nuclear transfer embryos in CZB medium showed no beneficial effect on pre-implantation development, no embryos developed to term after transfer to surrogate mothers. Our results demonstrated that not only B6D2F1, but B6CBAF1 oocytes, can be used for nuclear transfer. M16 medium is superior for culture of nuclear transfer embryos and low oxygen concentration with CZB medium during culture shows no benefit on development of cloned embryos.     

Preimplantation and postimplantation development of rat embryos cloned with cumulus cells and fibroblasts

In this report we demonstrate the successful in vitro culture of fertilised embryos from 1-cell to blastocyst stage, albeit in a strain-dependent fashion. We report procedures for the enucleation of rat oocytes; nuclear transfer by injection of nuclei (NT) from adult rat cumulus cells, rat primary embryonic fibroblasts and genetically modified rat fibroblasts; and activation resulting in advanced preimplantation development. Blastocyst stage rat embryos were obtained after in vitro culture of nuclear transfer zygotes at similar frequencies with each of these nuclear donor cell types. Transfer of NT embryos to surrogate mothers leads to implantation of 24% of the zygotes. These results suggest that the nuclei of cultured rat cells, even following genetic modification, can be reprogrammed to support early embryonic development, which is a prerequisite to cloning the rat.     

Influence of cell cycle stage at nuclear transplantation on the development in vitro of mouse embryos

Nuclei were transplanted from embryos of mice at different stages of the 1st and 2nd cell cycle to oocytes enucleated at various times after fertilization. After transfer of pronuclei, a greater proportion of embryos developed to blastocysts if donor and recipient embryos were at the same stage of the cell cycle (synchronous transfer = 94%, asynchronous transfer = 76%). By contrast, when 2-cell blastomere nuclei were fused to the cytoplasm of enucleated zygotes, there was a significant effect of both cytoplast and karyoplast cell cycle stage on the development of the reconstituted embryos. Karyoplasts and cytoplasts derived from embryos at later stages of the cell cycle had greater potential to support development to blastocysts in vitro. It is suggested that the secretion of stage-specific messengers and the timing of nuclear membrane breakdown are the main factors causing the karyoplast and cytoplast effects, respectively.     

Heterogeneous nuclear-transferred-embryos reconstructed with camel (Camelus bactrianus) skin fibroblasts and enucleated ovine oocytes and their development H-M

This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and ovine oocytes as recipient cytoplasts for investigating the developmental potential of the reconstructed embryos. Serum-starved adult camel skin fibroblast cells were used as donor somatic cells. Ovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electrofusion. The fused oocytes were activated by 5mM/ml inomycin with 2mM/ml 6-dimethylaminopurine (6-DMAP). The activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum (FCS) for 168h. A total of 300 enucleated ovine oocytes were available for xenonuclear embryo reconstruction. The results showed that 71% of the nuclear transfer couplets were successfully fused, 55% of the fused oocytes cleaved within 48h after activation, 82% of the cleaved oocytes developed to 2-16-cell embryo stages and 18% of the cleaved nuclear transfer zygotes developed to the morula stage. This study demonstrated that the xenonuclear transfer camel embryos can undergo the first embryonic division and subsequent development to morula stage in vitro.     

Factors influencing chromosome condensation and development of cloned rat embryos

Factors influencing premature chromosome condensation (PCC) in transferred rat nuclei have been examined. Chromosome condensation of rat cumulus cell nuclei did not occur when the cell nuclei were injected into enucleated rat oocytes. By contrast, chromosome condensation did occur after transfer to enucleated mouse oocytes or intact rat oocytes. In the first serial NT experiment, rat somatic cell nuclei were injected into enucleated mouse oocytes, and the reconstructed oocytes were activated by strontium chloride. From these reconstructed embryos, karyoplasts containing pronucleus-like vesicles were transferred into pronuclear zygote-derived cytoplasts by a DC pulse. Transfer of a total of 340 serial NT zygotes into recipient females, including 206 two-cell embryos, resulted in only seven implantation sites. In the second serial NT experiment, rat somatic cell nuclei were injected into intact rat oocytes; the recipient metaphase-plate was then aspirated under UV light from the NT oocytes in which PCC of injected nuclei was observed. After activation of the NT oocytes, karyoplasts were introduced into zygote-derived cytoplasts. Transfer of a total of 115 serial NT zygotes, including 37 two-cell embryos, resulted in four implantation sites but no live offspring. These results establish a mean of inducing chromosome condensation in rat oocytes and demonstrate that reconstructed rat zygotes can be prepared by serial NT procedures. Developmental competence of these embryos remains to be clarified.