Effects of fusion/activation methods on development of embryos produced by nuclear transfer of porcine fetal fibroblast

The present studies were carried out to investigate the effects of intensity of dc pulse, number of dc pulse and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. In experiment 1, different fusion/activation intensity (two dc pulses of 0.4, 0.8, 1.2, 1.6 and 2.0 kV/cm for 30 micros, respectively) was carried out to investigate development of embryos. In experiment 2, the reconstructed oocytes were fused and activated with one, two or three dc pulses of 1.2 kV/cm for 30 micros. In experiment 3, reconstructed oocytes were equilibrated in TCM-199 medium for 0-6 h, respectively, and fused/activated with one dc pulse of 1.2 kV/cm for 30 micros. The reconstructed embryos were cultured in PZM-3 medium containing 0.3% BSA. When oocytes were fused with donor cell by two dc pulses of 0.4 kV/cm for 30 micros, the rates of cleavage and blastocyst formation were significantly lower (32.9% and 2.5%) than those of fused by 0.8 kV/cm (59.0% and 17.4%) or 1.2 kV/cm (63.3% and 18.4%), respectively. One dc pulse of 1.2 kV/cm for 30 micros was enough to fuse and activate embryos to develop to blastocyst (24.8%). Equilibration for 2-3 h in TCM-199 before fusion/activation was beneficial for improving the developmental ability of embryos produced by nuclear transfer (25.6-23.3% at blastocysts).     

人-牛异种克隆胚构建及其线粒体来源

通过人-牛异种核移植技术获得异种克隆囊胚, 便于在不消耗人类卵母细胞的情况下从异种克隆胚中分离出人类干细胞。通过透明带下注射法将人胎儿成纤维细胞和牛耳成纤维细胞分别注入去核牛卵母细胞中构建异种和同种胚胎, 并比较两者之间的融合率、卵裂率、8-细胞发育率以及囊胚率。并对处于2-细胞、4-细胞、8-细胞、桑椹胚、囊胚阶段的异种克隆胚的线粒体DNA来源进行检测。结果表明, 异种克隆胚体外各个阶段的发育率均低于同种克隆胚, 尤其是8-细胞到囊胚阶段的发育率, 以及囊胚率都显著低于同种克隆胚(P<0.05)。异种克隆胚在2-细胞到桑椹胚阶段检测到人、牛线粒体DNA共存, 囊胚阶段只检测到牛线粒体DNA。结果表明: 牛卵母细胞可以重编程人胎儿成纤维细胞, 完成异种克隆胚植入前的胚胎发育, 异种克隆胚由于核质相互作用的不谐调, 影响其发育能力, 使其囊胚率显著低于同种克隆胚。牛线粒体DNA存在于植入前异种胚胎发育的各个阶段。异种克隆胚胎用于人类胚胎干细胞分离具有可行性。     

Expression of enhanced green fluorescent protein in porcine- and bovine-cloned embryos following interspecies somatic cell nuclear transfer of fibroblasts transfected by retrovirus vector

Interspecies somatic cell nuclear transfer (iSCNT) has emerged as an important tool for studying nucleo-cytoplasmic interactions and cloning of animals whose oocytes are difficult to obtain. This study was designed to explore the feasibility of employing transgenic fibroblasts as donor cells for iSCNT. The study examined the chromatin morphology, in vitro development, and expression of an enhanced green fluorescent protein (EGFP) gene in porcine- and bovine-cloned embryos produced by iSCNT of fetal fibroblast transfected with a pLNbeta-EGFP retroviral vector. Parthenogenetic and transfected or nontransfected intraspecies SCNT embryos were used as controls for comparison. Analysis of data revealed that xenogenic oocyte was able to reprogram somatic cells of different genus and supports their in vitro development to the blastocyst stage. However, the developmental rates of transgenic iSCNT embryos to the blastocyst stage were significantly lower than those of intraspecies SCNT embryos. The reduction in development rates was however, not due to integration of the transgene as the lower (P < 0.05) development rates of the intraspecies SCNT porcine or bovine embryos did not differ between transgenic and nontransgenic groups. Expression of EGFP was observed in 100% of blastocysts and mosaicism was not observed. Furthermore, after iSCNT of porcine or bovine donor nuclei into xenogenic ooplasm, patterns of nuclear remodeling in reconstructed embryos were similar. In conclusion, our data demonstrated the feasibility of producing transgenic iSCNT embryos. To our knowledge, this is the first report of transgenic cloned embryo production by iSCNT approach. In the future, this may provide a powerful research tool for studying developmental events in domestic animals and provide marked cell lines for other genetic manipulations.     

Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry 'foreign' DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6?. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8-16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8-16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine-bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.     

Development of bovine-ovine interspecies cloned embryos and mitochondria segregation in blastomeres during preimplantation

The objective of the study was to investigate interspecies somatic cell nuclear transfer (iSCNT) embryonic potential and mitochondrial DNA (mtDNA) segregation during preimplantation development. We generated bovine-ovine reconstructed embryos via iSCNT using bovine oocytes as recipient cytoplasm and ovine fetal fibroblast as donor cells. Chromosome composition, the total cell number of blastocyst and embryonic morphology were analyzed. In addition, mtDNA copy numbers both from donor cell and recipient cytoplasm were assessed by real-time PCR in individual blastocysts and blastomeres from 1- to 16-cell stage embryos. The results indicated the following: (1) cell nuclei of ovine fetal fibroblasts can dedifferentiate in enucleated bovine ooplasm, and the reconstructed embryos can develop to blastocysts. (2) 66% of iSCNT embryos had the same number of chromosome as that of donor cell, and the total cell number of iSCNT blastocysts was comparable to that of sheep parthenogenetic blastocysts. (3) RT-PCR analysis in individual blastomeres revealed that the ratio of donor cell mtDNA: recipient cytoplasm mtDNA remained constant (1%) from the one- to eight-cell stage. However, the ratio decreased from 0.6% at the 16-cell stage to 0.1% at the blastocyst stage. (4) Both donor cell- and recipient cytoplasm-derived mitochondria distributed unequally in blastomeres with progression of cell mitotic division. Considerable unequal mitochondrial segregation occurred between blastomeres from the same iSCNT embryos.     

The fate of mitochondria in Ibex-hirus reconstructed early embryos

Inter-species nuclear transfer could be used to preserve North Goat (Capra ibex), an endangered species. We established the culture conditions for ibex-hirus reconstructed embryos and optimized the method for DNA extractions of a single cell and early cloned embryo. By using mitochondria-specific probes of ibex and hirus respectively we found that mitochondria of donor cells can co-exist with recipients in 1-cell and 2-cell stages of the reconstructed embryos but not in the following developmental stages.     

Interspecies somatic cell nucleus transfer with porcine oocytes as recipients: A novel bioassay system for assessing the competence of canine somatic cells to develop into embryos

Interspecies somatic cell nucleus transfer (iSCNT) could be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos. To examine this possibility, we performed canine iSCNT using porcine oocytes, allowed to mature in vitro, as recipients. Canine fibroblasts from the tail tips and dewclaws of a female poodle (Fp) and a male poodle (Mp) were used as donors. We demonstrated that the use of porcine oocytes induced blastocyst formation in the iSCNT embryos cultured in porcine zygote medium-3. In Fp and Mp, the rate of blastocyst formation from cleaved embryos (Fp: 6.3% vs. 22.4%; and Mp: 26.1% vs. 52.4%) and the number of cells at the blastocyst stage (Fp: 30.7 vs. 60.0; and Mp: 27.2 vs. 40.1) were higher in the embryos derived from dewclaw cells than in those derived from tail-tip cells (P < 0.05). The use of donor cells of any type in later passages decreased the rate of blastocyst formation. Treatment with trichostatin-A did not improve the rate of blastocyst formation from cleaved dewclaw cell-derived embryos but did so in the embryos derived from the tail-tip cells of Fp. Only blastocysts derived from dewclaw cells of Mp developed outgrowths. However, outgrowth formation was retrieved in the embryos derived from dewclaw cells of Fp by aggregation at the 4-cell stage. We inferred that iSCNT performed using porcine oocytes as recipients could represent a novel bioassay system for evaluating the developmental competence of canine somatic cells.     

Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 μM 2',3'-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.     

Interspecies nuclear transfer reveals that demethylation of specific repetitive sequences is determined by recipient ooplasm but not by donor intrinsic property in cloned embryos

DNA methylation/demethylation of donor genomes in recipient ooplasm after nuclear transfer occurs in a species-specific way. In cloned rabbit and bovine embryos, repetitive sequences maintain the donor-type methylation status, but typical demethylation of repetitive sequences takes place in cloned porcine embryos. To clarify whether the demethylation is controlled by donor nucleus intrinsic property or by recipient ooplasm, we used interspecies somatic cell nuclear transfer (iSCNT) model to examine the methylation status of repetitive sequences in pig-to-rabbit and rabbit-to-pig interspecies embryos. We found that no demethylation of pig repetitive sequences was observed in pig-to-rabbit iSCNT embryos, while the examined rabbit repetitive sequence Rsat IIE was demethylated in rabbit-to-pig iSCNT embryos. These results indicate that demethylation of donor repetitive sequences is determined by ooplasm but not by donor intrinsic property and that ooplasm from different species have different capabilities to demethylate genes.     

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.     

The Fate of Mitochondria in Ibex-hirus Reconstructed Early Embryos

Nuclear transfer can be used to maintain limited popu-lations of highly endangered species [1–3] . Especiallywhen the oocytes of these species are difficult to obtain,inter-species nuclear transfer seems more appropriate forthis goal. The main methods are as follows: somatic cellis injected directly or fused by electroporation with re-cipient enucleated oocyte. Therefore, nDNA as well asmtDNA ought to be transferred totally or partially to theoocyte. Mitochondria provides adenosine triphos…     

Improvement of canine somatic cell nuclear transfer procedure

The purpose of the present study on canine somatic cell nuclear transfer (SCNT) was to evaluate the effects of fusion strength, type of activation, culture media and site of transfer on developmental potential of SCNT embryos. We also examined the potential of enucleated bovine oocytes to serve as cytoplast recipients of canine somatic cells. Firstly, we evaluated the morphological characteristics of in vivo-matured canine oocytes collected by retrograde flushing of the oviducts 72 h after ovulation. Secondly, the effectiveness of three electrical strengths (1.8, 2.3 and 3.3 kV/cm), used twice for 20 micros, on fusion of canine cytoplasts with somatic cells were compared. Then, we compared: (1) chemical versus electrical activation (a) after parthenogenetic activation or (b) after reconstruction of canine oocytes with somatic cells; (2) culture of resulting intergeneric (IG) embryos in either (a) mSOF or (b) TCM-199. The exposure time to 6-DMAP was standardized by using bovine oocytes reconstructed with canine somatic cells. Bovine oocytes were used for SCNT after a 22 h in vitro maturation interval. The fusion rate was significantly higher in the 3.3 kV/cm group than in the 1.8 and 2.3 kV/cm treatment groups. After parthenogenesis or SCNT with chemical activation, 3.4 and 5.8%, respectively, of the embryos developed to the morula stage, as compared to none of the embryos produced using electrical activation. Later developmental stages (8-16 cells) were transferred to the uterine horn of eight recipients, but no pregnancy was detected. However, IG cloned embryos (bovine cytoplast/canine somatic cell) were capable of in vitro blastocyst development. In vitro developmental competence of IG cloned embryos was improved after exposure to 6-DMAP for 4 h as compared to 0, 2 or 6h exposure, although the increase was not significantly different among culture media. In summary, for production of canine SCNT embryos, we recommend fusion at 3.3 kV/cm, chemical activation, culture in mSOF medium and transfer of presumptive zygotes to the oviduct of recipient animals. The feasibility of IG production of cloned canine embryos using bovine cytoplasts as recipient of canine somatic cells was demonstrated.     

Segregation of donor cell mitochondrial DNA in gaur-bovine interspecies somatic cell nuclear transfer embryos, fetuses and an offspring

The fate of foreign mitochondrial DNA (mtDNA) following somatic cell nuclear transfer (SCNT) is still controversial. In this study, we examined the transmission of the heteroplasmic mtDNA of gaur donor cells and recipient bovine oocytes to an offspring and aborted and mummified fetuses at various levels during the development of gaur-bovine interspecies SCNT (iSCNT) embryos. High levels of the donor cell mtDNA were found in various tissue samples but they did not have any beneficial effect to the survival of iSCNT offspring. However, the factors on mtDNA inheritance are unique for each iSCNT experiment and depend on the recipient oocyte and donor cell used, which might play an important role in the efficiency of iSCNT.     

Blastocysts derived from adult fibroblasts of a rhesus monkey ( Macaca mulatta) using interspecies somatic cell nuclear transfer

In non-human primates, it is difficult to collect sufficient numbers of oocytes for producing identical embryos by somatic cell nuclear transfer (SCNT). Because of this factor, inter-species SCNT (iSCNT) using heterospecific oocytes is an attractive alternative approach. The objective of this study was to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissue from a 6-year-old male rhesus monkey was collected by biopsy and fibroblasts were isolated. Immature cumulus-oocyte complexes from cow ovaries were collected and matured in vitro in Medium 199. The enucleated oocytes were reconstructed with rhesus monkey fibroblasts and iSCNT embryos were cultured in modified synthetic oviduct fluid in an atmosphere of 5-5.5% CO2 under various conditions (37-39 °C and 5-20% O2) to examine the effects of in vitro culture conditions. Most embryos were arrested at the 8- or 16-cell stage and only three blastocysts were derived in this way using iSCNT from a total of 1153 cultured activated embryos (0.26% production rate). Two of the three blastocysts were used for counting nuclear numbers using bisbenzimide staining, which were 51 and 24. The other iSCNT-derived blastocyst was used to analyse mitochondrial DNA (mtDNA) by PCR, and both rhesus monkey and cow mtDNA were detected. Although the development rate was extremely low, this study established that iSCNT using two phylogenetically distant species, including a primate, could produce blastocysts. With improvements in the development rate, it may be possible to produce rhesus monkey iSCNT-derived embryonic stem cell lines for studies on primate nucleus and cow mitochondria interaction mechanisms.     

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。     

TSA和VPA处理对食蟹猴-猪异种体细胞核移植胚胎早期发育的影响

食蟹猴-猪异种体细胞核移植(Interspecies somatic cell nuclear transfer,iSCNT)研究旨在由iSCNT胚胎建立具有与人类相似遗传背景的胚胎干细胞(ESCs),作为医学和基础科学研究的实验材料。文章探讨了两种组蛋白脱乙酰化酶抑制剂(HDACi)Trichostatin A(TSA)和Valproic acid(VPA)处理浓度、时间与培养液(PZM-3和HECM-10)组合对食蟹猴-猪iSCNT胚胎早期发育的影响。结果表明,在PZM-3中添加10 nmol/L TSA处理48 h组的囊胚率显著高于对照组(22.78%vs 9.86%,P<0.05)。但是,不管在PZM-3或是HECM-10中,添加2～10mmol/L VPA处理均不能提高iSCNT胚胎早期发育能力。文章证明了TSA处理可以提高食蟹猴-猪iSCNT胚胎早期发育能力。     

Epigenetic reprogramming of Yak iSCNT embryos after donor cell pre-treatment with oocyte extracts

The treatment of donor cells with oocyte extracts before inter-species somatic cell nuclear transfer (iSCNT) is a novel method for cellular reprogramming. This study aims to evaluate the effect of pre-treatment donor cell with oocyte extracts on the early developmental competence of yak iSCNT embryos. Yak fibroblasts were reversibly permeabilized with streptolysin O, and then treated with yak oocyte extracts (YOE) or bovine oocyte extracts (BOE) prior to iSCNT. The 8-cell and blastocyst formation increased significantly compared with the control group (P<0.05) when donor cells pre-treated with YOE or BOE. The relative expression level of embryo-specific genes TBP1 and Mash2 were also up-regulated both in the blastocysts of the YOE and BOE groups. In addition, the methylation level of pluripotency-specific genes (Oct4 and Nanog) in the blastocysts of the YOE and BOE groups were similar to that of its IVF counterpart (53.1%, 48.8% vs. 40.1%; 24.8%, 26.5% vs. 35.9%). Our results suggested that pre-treatment of donor cells with oocyte extracts can improve nuclear-cytoplasmic reprogramming; thus representing a novel way to improve the efficiency of yak iSCNT.     

Nuclear totipotency of cultured rabbit morulae to support full-term development following nuclear transfer.

The rabbit was used as a model for nuclear transfer. A critical step in nuclear transfer is oocyte activation, which was evaluated in this research. Optimal field strength of an electric stimulus for activation was examined. A significantly higher activation rate in all criteria tested was achieved when oocytes were activated electrically with a field strength of 2.4 kV/cm versus 1.2 or 1.8 kV/cm. Also, electrical stimulation with combined alternating current (AC) and direct current (DC) was superior to DC stimulation alone for activation. In another study involving 586 oocytes, exposure of oocytes to cytochalasin B for 1 h followed by activation with electrical stimulation significantly improved development of the oocytes to blastocyst stage compared to oocytes without cytochalasin B pre-exposure (38% vs 26%, p less than 0.05). Cytochalasin B exposure alone (control), however, had no effect on activation. Exposing oocytes to activation medium without electrical stimulation also activated some oocytes. In the nuclear transfer experiment, blastomeres from 8-cell embryos cultured for 20-24 h to the 32-64-cell stage were used as nuclear donor cells. Of 491 oocytes used, 459 (93%) survived the enucleation and fusion procedure, 370 (81%) fused, and 284 (77%) developed into 2-4-cell embryos. A total of 243 of these 2-4-cell embryos were transferred to 15 pseudopregnant recipients and produced 8 young (3%). Although the efficiency is low, this study demonstrated that rabbit morulae cultured for 20-24 h to the 32-64-cell stage as nuclear donors for transfer remain totipotent.     

Proliferation of donor mitochondrial DNA in nuclear transfer calves (Bos taurus) derived from cumulus cells

In embryos derived by nuclear-transfer (NT), fusion of donor cell and recipient oocyte caused mitochondrial heteroplasmy. Previous studies from other laboratories have reported either elimination or maintenance of donor-derived mitochondrial DNA (mtDNA) from somatic cells in cloned animals. Here we examined the distribution of donor mtDNA in NT embryos and calves derived from somatic cells. Donor mitochondria were clearly observed by fluorescence labeling in the cytoplasm of NT embryos immediately after fusion; however, fluorescence diminished to undetectable levels at 24 hr after nuclear transfer. By PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, donor mtDNAs were not detected in the NT embryos immediately after fusion (less than 3-4%). In contrast, three of nine NT calves exhibited heteroplasmy with donor cell mtDNA populations ranging from 6 to 40%. These results provide the first evidence of a significant replicative advantage of donor mtDNAs to recipient mtDNAs during the course of embryogenesis in NT calves from somatic cells.