Interspecies nuclear transfer embryos reconstructed from cat somatic cells and bovine ooplasm

This study was conducted to investigate the developmental capacity of domestic cat-bovine reconstructed embryos via interspecies somatic cell nuclear transfer (iSCNT) and to observe the mitochondrial DNA (mtDNA) content of the iSCNT embryos. The iSCNT embryos were generated using mixed-breed domestic cat fibroblasts as donor cells and enucleated bovine oocytes as the recipient cytoplasm. When the developmental capacities of iSCNT embryos and parthenogenic bovine embryos were compared, there was no difference (P>0.05) in the rates of cleavage and development to the 8-cell stage (86.6 vs. 84.0% and 32.2 vs. 36.2%, respectively). However, in contrast to development of parthenogenic embryos to the morula and blastocyst stages, no iSCNT embryos (0/202) developed beyond the 8-cell stage. For mtDNA analysis, iSCNT embryos at the 1-cell, 2-cell, 4-cell and 8-cell stages were randomly selected. Both cat and bovine mtDNA quantification analysis were performed using quantitative PCR. The levels of both cat and bovine mtDNA in cat-bovine iSCNT embryos varied at each stage of development. The cat mtDNA concentration in the iSCNT embryos was stable from the 1-cell to 8-cell stages. The bovine mtDNA in the iSCNT embryos at the 8-cell stage was significantly lower than that at the 4-cell stage (P<0.05). No difference in the proportions of cat mtDNA in the iSCNT embryos was found in any of the observed developmental stages (1- through 8-cell stages). In conclusion, bovine cytoplasm supports domestic cat nucleus development through the 8-cell stage. The mtDNA genotype of domestic cat-bovine iSCNT embryos illustrates persistence of heteroplasmy, and the reduction in mtDNA content might reflect a developmental block at the 8-cell stage.     

Mitochondrial and DNA damage in bovine somatic cell nuclear transfer embryos

The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H₂O₂ and ˙OH radical levels, mitochondrial morphology and membrane potential (ΔΨ), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H₂O₂ (35.6 ± 1.1 pixels/embryo) and ˙OH radical levels (44.6 ± 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 ± 1.5 and 23.8 ± 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The ΔΨ of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 ± 0.04 vs. 1.21 ± 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 ± 26.4 µm vs. 425.6 ± 25.0 µm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels.     

体细胞核移植生产转基因牛胚胎的研究

为了优化利用体细胞核移植生产转基因牛早期胚胎的体系,以携带绿色荧光蛋白-新霉抗性双标记基因的pMSCV质粒转染的胎牛耳成纤维细胞为供体,以体外成熟的牛卵母细胞为受体构建克隆胚,研究供体细胞的传代次数对转基因胚的影响和重构胚在不同电场条件下（电场强度、直流电脉冲次数）融合效率及其在不同体外培养系统中的发育效果。结果表明：传代5次的细胞做核供体较有利于转基因胚的发育;在电场条件为电场强度1.6 kV/cm、直流脉冲1次的融合率最高;负压单层细胞共培养体系中的转基因囊胚发育率较好,与未转基因体细胞核移植胚的发育相比无显著差异（P〉0.05）。     

Effect of donor cell type on nuclear remodelling in rabbit somatic cell nuclear transfer embryos

Cloned rabbits have been produced for many years by somatic cell nuclear transfer (SCNT). The efficiency of cloning by SCNT, however, has remained extremely low. Most cloned embryos degenerate in utero, and the few that develop to term show a high incidence of post-natal death and abnormalities. The cell type used for donor nuclei is an important factor in nuclear transfer (NT). As reported previously, NT embryos reconstructed with fresh cumulus cells (CC-embryos) have better developmental potential than those reconstructed with foetal fibroblasts (FF-embryos) in vivo and in vitro. The reason for this disparity in developmental capacity is still unknown. In this study, we compared active demethylation levels and morphological changes between the nuclei of CC-embryos and FF-embryos shortly after activation. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized and cloned rabbit embryos revealed that there was no detectable active demethylation in rabbit zygotes or NT-embryos derived from either fibroblasts or CC. In the process of nuclear remodelling, however, the proportion of nuclei with abnormal appearance in FF-embryos was significantly higher than that in CC-embryos during the first cell cycle. Our study demonstrates that the nuclear remodelling abnormality of cloned rabbit embryos may be one important factor for the disparity in developmental success between CC-embryos and FF-embryos.     

猪体细胞核移植胚体外发育初步研究

采用胞质内注射法进行猪体细胞核移植，对去核、激活和培养等关键技术过程进行研究。结果表明：（1）点压法、挤压法对卵母细胞的去核率明显高于盲吸法（三者分别为62．5％、64．6％、50．7％，P〈0．05）。对早成熟的卵母细胞（36-44h）进行去核可明显提高去核效率（P〈0．05），在36-38h、39-41h、42-44h去核率分别为60．9％、67．8％、64．3％，而45-48h为48．4％。（2）体细胞预激活有助于提高核移胚卵裂率（28．0％、20．1％，P〈0．05）。钙离子载体A23187单独或与6-二甲基氨基嘌呤（6-DMAP）联合作用能使猪体细胞核移胚激活继续发育。（3）核移胚以胚胎培养液NCSU23及卵丘单层共培养体系进行分别培养，核移胚卵裂率无明显差异（30．06％、31．5％，P〉0．05）。但NCSU23培养4细胞后发育能力更高（13．5％、3．9％，P〈0.05）。     

Microtubule distribution in somatic cell nuclear transfer bovine embryos following control of nuclear remodeling type

This study was conducted to evaluate the microtubule distribution following control of nuclear remodeling by treatment of bovine somatic cell nuclear transfer (SCNT) embryos with caffeine or roscovitine. Bovine somatic cells were fused to enucleated oocytes treated with either 5 mM caffeine or 150 µM roscovitine to control the type of nuclear remodeling. The proportion of embryos that underwent premature chromosome condensation (PCC) was increased by caffeine treatment but was reduced by roscovitine treatment (p < 0.05). The microtubule organization was examined by immunostaining β- and γ-tubulins at 15 min, 3 h, and 20 h of fusion using laser scanning confocal microscopy. The γ-tubulin foci inherited from the donor centrosome were observed in most of the SCNT embryos at 15 min of fusion (91.3%) and most of them did not disappear until 3 h after fusion, regardless of treatment (82.9-87.2%). A significantly high proportion of embryos showing an abnormal chromosome or microtubule distribution was observed in the roscovitine-treated group (40.0%, p < 0.05) compared to the caffeine-treated group (22.1%). In conclusion, PCC is a favorable condition for the normal organization of microtubules, and inhibition of PCC can cause abnormal mitotic division of bovine SCNT embryos by causing microtubule dysfunction.     

In vitro development of canine somatic cell nuclear transfer embryos in different culture media

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.     

Development potential of transgenic somatic cell nuclear transfer embryos according to various factors of donor cell

The present study was conducted to establish an efficient production system for bovine transgenic somatic cell nuclear transfer (SCNT) embryos, the effect of various conditions of donor cells including cell type, size, and passage number on the developmental competence of transgenic SCNT embryos were examined with their expression rates of a marker gene. An expression plasmid for human prourokinase was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human prourokinase target gene into a pcDNA3 plasmid. Three types of bovine somatic cells including two adult cells (cumulus cells and ear fibroblasts) and fetal fibroblasts were prepared and transfected with the expression plasmid using a liposomal transfection reagent, Fugene6, as a carrier. In Experiment 1, three types of bovine cells were transfected at passages 2 to 4, and then trypsinized and GFP-expressing cells were randomly selected and used for SCNT. Developmental competence and rates of GFP expression in bovine transgenic SCNT embryos reconstructed with cumulus cells were significantly higher than those from fetal and ear fibroblasts. In all cell types used, GFP expression rates of SCNT embryos gradually decreased with the progression of embryo development. In Experiment 2, the effect of passage number of cumulus cells in early (2 to 4) and late (8 to 12) passages was investigated. No significant differences in the development of transgenic SCNT embryos were observed, but significantly higher GFP expression was shown in blastocysts reconstructed with cumulus cells at early passage. In Experiment 3, different sizes of GFP-expressing transfected cumulus cells [large (>30 microm) or small cell (<30 microm)] at passages 2 to 4 were used for SCNT. A significant improvement in embryo development and GFP expression was observed when small cumulus cells were used for SCNT. Taken together, these results demonstrate that (1) adult somatic cells as well as fetal cells could serve as donor cells in transgenic SCNT embryo production and cumulus cells with small size at early passage were the optimal cell type, and (2) transgenic SCNT embryos derived from adult somatic cells have embryonic development potential.     

Recent progress in bovine somatic cell nuclear transfer

Bovine somatic cell nuclear transfer (SCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization. However, the full‐term developmental rate of SCNT embryos is very low, owing to the high embryonic and fetal losses after embryo transfer. In addition, increased birth weight and postnatal mortality are observed at high rates in cloned calves. The low efficiency of SCNT is probably attributed to incomplete reprogramming of the donor nucleus and most of the developmental problems of clones are thought to be caused by epigenetic defects. Applications of SCNT will depend on improvement in the efficiency of production of healthy cloned calves. In this review, we discuss problems and recent progress in bovine SCNT.     

Effects of cycloheximide treatment on in-vitro development of porcine parthenotes and somatic cell nuclear transfer embryos

This study aimed to verify the beneficial effect of cycloheximide (CHX) treatment on the development of porcine somatic cell nuclear transfer (NT) embryos, which were constructed with enucleated oocytes and cumulus cells by using a single direct current (DC) pulse. In the first experiment, a single DC pulse applied to the induction of fusion and activation of NT embryos gave a high fusion rate. However, cleavage and subsequent development of fused couplets (NT embryos) to the blastocyst stage were poor. Experiment 2 was conducted to determine whether CHX treatment could enhance metaphase II (M II) oocyte activation and improve the subsequent parthenogenetic development. After giving the DC pulse and incubation with or without CHX, M II oocytes incubated with CHX showed higher cleavage and development to blastocysts compared with those incubated without CHX (P < 0. 05). Experiment 3 was carried out to verify the beneficial effect of CHX on the development of NT embryos. The NT embryos treated with the DC pulse and CHX treatment showed higher cleavage and subsequent development compared with those treated with the DC pulse alone (P < 0.05) . The present study demonstrates that CHX treatment enhances the electrical stimulus-induced activation of oocytes and NT embryos, and improves the subsequent development of parthenotes and NT embryos. The results indicate that protein synthesis inhibition treatment required for the induction of oocyte activation promotes the development of NT embryos.     

细胞松弛素B对水牛体细胞核移植效果的影响

以水牛耳皮成纤维细胞为供体细胞,采用电融合方法,探讨细胞松弛素B(CB)对水牛体细胞核移植效果的影响.体外成熟培养22～24 h的水牛卵母细胞去核后.将经0.1 mg/L Aphidicolin(APD)+0.5%FBS培养2～9 d的水牛耳皮成纤维细胞注射到卵周隙中再经电融合(100 V/mm,15μs,电脉冲3次)构建核移植重构胚.重构胚经化学激活后(5 μmol/L)离子霉素5 min,2 mmol/L 6-DMAP 3 h)培养,7～9 d评定其胚胎发育能力.结果显示,在含CB(3 mg/L)的融合液中进行电融合后,核移植的融合率、重组胚的存活率、卵裂率和囊胚率与对照组(不含CB)相比均无显著差异(P>0.05);核移植重组胚激活前用含CB(6 mg/L)的培养液培养1 h,其激活后的存活率(97.52%)和体外囊胚发育率(22.09%)均显著地高于未经CB处理的重组胚的存活率(93.87%)和囊胚率(13.25%,P<0.05);重组胚经离子霉素激活5 min后,在6-DMAP+CB中培养3 h的分裂率明显低于放在6-DMAP中培养3 h的分裂率(65.37% vs 78.92%,P<0.05),但囊胚发育率无显著差异(11.19% vs 10.96%,P>0.05).这表明水牛体细胞核移植电融合时,融合液中不添加CB,而核移植重组胚激活前经CB培养处理后,有利于胚胎的进一步发育,但激活后用CB培养处理会降低胚胎的发育率.     

Effects of repetitive ionomycin treatment on in vitro development of bovine somatic cell nuclear transfer embryos

To artificially activate embryos in somatic cell nuclear transfer (SCNT), chemical treatment with ionomycin has been used to induce transient levels of Ca(2+) and initiate reprogramming of embryos. Ca(2+) oscillation occurs naturally several times after fertilization (several times with 15- to 30-min intervals). This indicates how essential additional Ca(2+) influx is for successful reprogramming of embryos. Hence, in this report, the experimental design was aimed at improving the developmental efficiency of cloned embryos by repetitive Ca(2+) transients rather than the commonly used ionomycin treatment (4 min). To determine optimal Ca(2+) inflow conditions, we performed three different repetitive ionomycin (10 μM) treatments in reconstructed embryos: Group 1 (4-min ionomycin treatment, once), Group 2 (30-sec treatment, 4 times, 15-min intervals) and Group 3 (1-min treatment, 4 times, 15-min intervals). Pronuclear formation rates were checked to assess the effects of repetitive ionomycin treatment on reprogramming of cloned embryos. Cleavage rates were investigated on day 2, and the formation rates of blastocysts (BLs) were examined on day 7 to demonstrate the positive effect of repeated ionomycin treatment. In Group 3, a significant increase in BL formation was observed [47/200 (23.50%), 44/197 (22.33%) and 69/195 (35.38%) in Groups 1, 2 and 3, respectively]. Culturing embryos with different ionomycin treatments caused no significant difference among the groups in terms of the total cell number of BLs (164.3, 158.5 and 145.1, respectively). Additionally, expression of the anti-apoptotic Bcl-2 gene and MnSOD increased significantly in Group 3, whereas the expression of the pro-apoptotic Bax decreased statistically. In conclusion, the present study demonstrated that repeated ionomycin treatment is an improved activation method that can increase the developmental competence of SCNT embryos by decreasing the incidence of apoptosis.     

Propagation of elite rescue dogs by somatic cell nuclear transfer

The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6‐dimethylaminopyridine (DMAP) (SOF‐DMAP) or porcine zygote medium (PZM‐5) with 1.9 mmol/L DMAP (PZM‐DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM‐DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF‐DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6‐DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies.     

Epigenetic modulation on cat‐cow interspecies somatic cell nuclear transfer embryos by treatment with trichostatin A

This study aimed to determine the acetylation at lysine 9/18/23 of histone H3 (H3K9ac/H3K18ac/H3K23ac; H3K9/18/23 ac) and the di‐methylation at lysine 9 of histone H3 (H3K9me2) during early embryogenesis among trichostatin A (TSA)‐treated interspecies somatic cell nuclear transfer (iSCNT) cat‐cow (TSA‐iSCNT) embryos, TSA‐untreated iSCNT cat‐cow control (control) embryos and bovine in vitro fertilization (IVF) embryos, because TSA‐iSCNT embryos can develop to blastocysts. Compared to the control embryos, higher expressions of H3K9/18/23 ac were observed in TSA‐iSCNT embryos and IVF embryos at most following stages (2 h post‐fusion / post‐insemination (PF/PI) to eight‐cell stage). At 6 h PF/PI the expression of H3K9/23 ac in TSA‐iSCNT embryos and IVF embryos were lower than those in control embryos, and the expression of H3K18ac was no difference among the three groups. The expression of H3K9/23 ac increased in TSA‐iSCNT embryos and IVF embryos at pronuclear (PN) stages. The expression of H3K9me2 in TSA‐iSCNT embryos resembled that of IVF embryos at 2 h PF/PI to PN stages, and these expression levels were greater than those of control embryos. These results suggest that treatment of iSCNT embryos with TSA modifies the patterns of histone modification at certain lysine residues in a manner that is comparable with that seen in IVF during early embryogenesis.     

Developmental competence of bovine embryos reconstructed by the transfer of somatic cells derived from frozen tissues.

The ability of frozen-thawed fetal skin was examined to generate viable cell lines for nuclear transfer. Fetal skin frozen at -35 degrees C or -80 degrees C in the presence of 5% DMSO were used as tissue explants to generate somatic cells. The resultant confluent cells were then used as donors for nuclear transfer (NT). Of the bovine NT embryos reconstructed from the somatic cells, 78% to 81% showed cleavage, 43% to 48% reached the stage of morula formation and 34% to 35% reached blastocyst formation. There were no significant differences in development (P>0.05) when the NT embryos were compared with those reconstructed from fresh somatic-cell-derived skin tissues (75%, 45%, and 38%, for cleavage, and development to morula and blastocyst stages, respectively). The results indicated that cell lines derived from bovine fetal skin cryopreserved by a simple method could be used as donors in nuclear transfer. The resulting embryos showed similar development in vitro to those reconstructed from unfrozen fetal-skin-derived somatic cells.     

Modification of maturation condition improves oocyte maturation and in vitro development of somatic cell nuclear transfer pig embryos

This study examined effects on the developmental competence of pig oocytes after somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) of : 1) co-culturing of oocytes with follicular shell pieces (FSP) during in vitro maturation (IVM); 2) different durations of maturation; and 3) defined maturation medium supplemented with polyvinyl alcohol (PVA; control), pig follicular fluid (pFF), cysteamine (CYS), or β-mercaptoethanol (β-ME). The proportion of metaphase II oocytes was increased (p < 0.05) by co-culturing with FSP compared to control oocytes (98% vs. 94%). However, blastocyst formation after SCNT was not improved by FSP coculture (9% vs. 12%). Nuclear maturation of oocytes matured for 39 or 42 h was higher (p < 0.05) than that of oocytes matured for 36 h (95-96% vs. 79%). Cleavage (83%) and blastocyst formation (26%) were significantly higher (p < 0.05) in oocytes matured for 42 h than in other groups. Supplementation of a defined maturation medium with 100 µM CYS or 100 µM β-ME showed no stimulatory effect on oocyte maturation, embryo cleavage, or blastocyst formation after PA. β-ME treatment during IVM decreased embryo cleavage after SCNT compared to pFF or PVA treatments, but no significant difference was found in blastocyst formation (7-16%) among the four treatment groups. The results indicated that maturation of oocytes for 42 h was beneficial for the development of SCNT embryos. Furthermore, the defined maturation system used in this study could support in vitro development of PA or SCNT embryos.     

Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development

Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm‐transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro‐fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.     

Abnormal distribution of chromosomes in the first division of nuclear transferred mouse embryos

The majority of somatic cell nuclear transferred (SCNT) embryos die before or after implantation. Many studies have focused on morphological remodeling of the donor nucleus and its associated cytoskeletal structures in the early events of nuclear transfer. However, little is known about the 2-cell stage of SCNT embryos after the first division. In this study, we compared the morphological status of chromosomal division during the 1-cell stage to the 2-cell stage in SCNT embryos with that in intracytoplasmic sperm injection (ICSI) embryos. The microtubules and cytoplasmic asters, which are related to chromatin segregation, disappeared at the pronuclear stage, although formation of the first mitotic spindle was normal in both the SCNT and ICSI embryos. However, nuclear fragmentation was observed in 30% of the 2-cell SCNT embryos and 12% of the 2-cell ICSI embryos. Nuclear fragmentation was present in both blastomeres of these embryos. No apoptotic DNA fragmentation was observed in TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assays for either the SCNT or ICSI embryos. In both the SCNT and ICSI embryos, the distribution of chromosomes in the first mitotic spindle was disturbed during the process of division from the 1-cell stage to the 2-cell stage. These results suggest that loss of SCNT embryos just before or after implantation may be due to an abnormal chromosome distribution at the 2-cell stage.