克隆种公猪生长状况和繁殖性能评价

体细胞核移植技术可以成功用于克隆性能优良的种用家畜,理论上克隆个体与供体在遗传上是完全相同的。为验证该理论,本试验检测了克隆种公猪自身在出生后的生长发育指标以及克隆种公猪与供核种公猪精液品质各项指标、情期受孕率、窝产仔数、产活仔数、断奶活仔数、仔猪初生重、仔猪断奶重和平均日增重等指标,探讨了克隆种公猪与供核种公猪在繁殖性能以及遗传性能之间的关系。结果显示,克隆种公猪与供核种公猪在各检测指标之间均不存在显著差异(P>0.05),提示克隆种公猪与供核种公猪在繁殖性能和遗传性能上无显著差异,可以作为种公猪用于扩繁。     

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.     

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.     

Effects of trichostatin A on histone acetylation and methylation characteristics in early porcine embryos after somatic cell nuclear transfer

Until now, the efficiency of animal cloning by somatic cell nuclear transfer (SCNT) has remained low. Efforts to improve cloning efficiency have demonstrated a positive role of trichostatin A (TSA), an inhibitor of deacetylases, on the development of nuclear transfer (NT) embryos in many species. Here, we report the effects of TSA on pre‐implantation development of porcine NT embryos. Our results showed that treatment of reconstructed porcine embryos with 50 nmol/L TSA for 24 h after activation significantly improved the production of blastocysts (P < 0.05), while treating donor cells with the same solution resulted in increases in cleavage rates and blastomere numbers (P < 0.05). However, TSA treatment of both donor cells and SCNT embryos did not improve blastocyst production, nor did it increase blastomere numbers. Using indirect immunofluorescence, we found that TSA treatment of NT embryos could improve the reprogramming of histone acetylation at lysine 9 of histone 3 (H3K9) and affect nuclear swelling of transferred nuclei. However, no apparent effect of TSA treatment on H3K9 dimethylation (H3K9me2) was observed. These findings suggest a positive effect of TSA treatment (either treating NT embryos or donor cells) on the development of porcine NT embryos, which is achieved by improving epigenetic reprogramming.     

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）。     

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.     

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.     

Evaluation of porcine urine-derived cells as nuclei donor for somatic cell nuclear transfer

BackgroundSomatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency.ObjectivesThis study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs.MethodsThe viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer.ResultsMost sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption.ConclusionsSow urine-derived cells could be used to produce SCNT embryos.     

影响猪体细胞核移植效果的因素

克隆技术的研究已经取得了丰硕的成果,但猪体细胞核移植还受到众多因素的制约,克隆效率仍然较低,文章针对猪体细胞核移植的各项技术环节及影响因素进行探讨与分析,以期加快猪体细胞核移植技术的研究,提高猪体细胞核移植效率,进一步促进猪体细胞核移植技术的发展和运用。     

食蟹猴-猪异种体细胞核移植的初步研究

采用食蟹猴耳部成纤维细胞作为核供体利用电融合法构建猴-猪异种核移植胚胎,研究其核重编机制并寻找适宜的培养基,初步建立食蟹猴-猪异种核移植体系。结果显示:(1)重构胚胎激活后3 h,供体核的大小基本不发生变化,但在激活后6 h大部分形成膨大的类原核。(2)食蟹猴-猪异种核移植胚胎融合率为74.2%,有70.5%的重构胚胎发生卵裂,29%的卵裂胚胎发育到桑椹胚阶段。(3)在NCSU-23+10%FCS和TCM199+10%FCS培养基中,重构胚突破4-细胞阻滞发育到8-细胞以上的比例分别为23.5%和17.0%,在NCSU-23培养基中的为19.9%,但是只有在NCSU23+10%FCS和TCM199+10%FCS培养基中重构胚胎发育到囊胚阶段(1.4%vs1.1%),尽管差异不显著。本研究首次建立了食蟹猴-猪异种体细胞克隆体系。     

单胚胎RT-qPCR对猪体细胞核移植囊胚全能性的分析

为了检测猪体细胞核移植囊胚的质量和全能性基因表达量,采用体细胞核移植技术制备克隆胚胎并于体外培养5 d后获得囊胚,对照组为从人工授精5 d后的长白母猪体内获取的体内囊胚。在高倍镜下检测两组囊胚的形态,用Hoechest 33342染色细胞核DNA,记录囊胚细胞总数;建立单胚胎cDNA的制备方法,并用qPCR检测囊胚中全能性基因(Oct4、Nanog、Sox2)的表达量。结果表明,与体内囊胚相比,猪体细胞核移植囊胚质量较差,细胞数目显著降低(分别为110±10.3和54±12.6);并且全能性基因表达量显著下降(P<0.05)。由此可见,全能性基因表达量偏低是影响猪体细胞核移植囊胚发育能力的因素之一。     

五指山猪背最长肌和半腱肌DNA甲基化检测分析

DNA甲基化作为真核生物基因组重要的表观遗传学修饰,对生物体基因的表达有重要的调控作用。为了研究五指山猪肌肉组织基因组DNA的甲基化程度,试验利用MSAP技术,使用筛选的10对引物扩增,检测五指山猪背最长肌和半腱肌的甲基化程度。结果显示:1月龄、2月龄、4月龄五指山猪背最长肌的平均甲基化率分别是44.78%、41.58%、38.92%;1月龄、2月龄、4月龄五指山猪半腱肌的平均甲基化率分别是41.70%、39.39%、38.81%。研究结果为五指山猪生长发育规律、系统选育及矮小机制等方面的研究提供表观遗传学依据。     

H19和IGF2R基因在体细胞克隆猪各组织中的甲基化状态

为了探求新生克隆猪可能的死亡原因以及是否存在不完全的DNA甲基化重编程,本试验运用亚硫酸氢盐测序法分别检测了H19基因和IGF2R基因差异甲基化区（DMR）在新生死亡克隆猪和同期正常猪心脏、肝脏、脾脏、肺脏和肾脏中的甲基化状态。结果发现,H19基因DMR在克隆猪肺脏中表现为超甲基化,极显著高于正常猪（95．20％VS46．80％P〈0．01）,且10个测序克隆中存在2处连续的全甲基化CpG位点（4-9位、12-S17位）,而在其他组织中甲基化差异不显著（P〉0．05）;IGF2R基因DMR在肝脏中处于超甲基化状态,显著高于正常猪（80．00％V839．41％P〈0．05）,而在肺脏中为去甲基化状态,板显著低于正常猪（14．71％VS66．47％P〈0．01）,在其他组织差异不显著（P〉0．05）。结果说明,在死亡克隆猪中,H19基因DMR在肺脏和IGF2R基因在肝脏与肺脏中存在不完全的DNA甲基化重编程,这可能是导致克隆动物死亡的因素之一。     

Inhibition of Suv39h1/2 expression improves the early development of Debao porcine somatic cell nuclear transfer embryos

Suppressor of variegation 3–9 homolog (Suv39h)1 and 2, Histone H3 lysine 9 trimethylation (H3K9me3)-specific methyltransferases, are mainly involved in regulating the dynamic changes of H3K9me3. Regulating Suv39h expression influences the early development of mice somatic cell nuclear transfer (SCNT) embryos, there are few reports concerning their features in domestic animals. The aim of the present study was to characterize the Suv39h function in early development of Debao porcine SCNT embryos. The global level of H3K9me3 and the expression profiles of Suv39h1/2 in porcine early embryos were analysed by immunohistochemistry and qRT-PCR methods, respectively. Their roles in cell proliferation and histone modification of Debao porcine foetal fibroblast cells (PFFs), and developmental competence of porcine SCNT embryos were investigated by shRNA technology. The methylation levels of H3K9me3 and the expression patterns of Suv39h1 and Suv39h2 were similar (p < .05), and both of them displayed higher levels in Debao porcine SCNT embryos compared with that in PA embryos. The global levels of H3K9me3 and the expressions of G9a, HDAC1 and DNMT1 were decreased by combined inhibition of Suv39h1 and Suv39h2 (p < .05), while the expression of HAT1 was increased (p < .05). Downregulation of Suv39h1/2 also promoted cell proliferation and resulted in a significant increase in the expression of CyclinA2, CyclinB and PCNA in PFFs (p < .05). Furthermore, the use of donor somatic nuclei which depleted H3K9me3 by inhibiting Suv39h1/2 expression markedly increased the cleavage rate, the blastocyst rate and the total cell number of blastocysts of Debao porcine SCNT embryos (p < .05). Altogether, the above results indicate that H3K9me3 levels and Suv39h1/2 expressions display similar patterns in porcine early embryo, and low levels of them are critical to cell proliferation of PFFs and early development of SCNT embryos.     

Comparative evaluation of three different formulas for predicting the parturition date of German Shepherds following somatic cell nuclear transfer

Several studies have reported methods to estimate the parturition date of dogs using ultrasonographic measurements. However, these prediction models were mainly determined using ultrasonographic measurements of naturally pregnant small- and medium-sized dogs, and no such studies have been performed using dogs carrying cloned fetuses produced via somatic cell nuclear transfer. The present study evaluated the abilities of three reference formulas (Luvoni and Grioni, Milani et al., and Groppetti et al.), all of which were developed using data from naturally occurring pregnancies, to accurately predict the parturition date in surrogates carrying cloned German Shepherd (GS) fetuses. All three formulas were based on the use of inner chorionic cavity diameter (ICC) measurements, obtained via ultrasonography. For evaluation, a total of 54 ICC measurements were collected from 14 pregnant bitches carrying cloned GS fetuses. We found that the clinical accuracy of the breed-specific Groppetti et al. formula was highest among those of the three formulas tested, with 87% and 100% of the estimated parturition dates (calculated based on the ICC measurements) being within 1 and 2 days, respectively, of the actual delivery date. By contrast, the Luvoni and Grioni formula showed relatively low accuracy, and the Milani et al. formula showed higher accuracy than that reported previously for natural pregnancies.     

Normal DNA methylation status in sperm from a somatic cell cloned bull and their fertilized embryos

Epigenetic reprogramming confers totipotency even during somatic cell nuclear transfer (SCNT), which has been used to clone various animal species. However, as even apparently healthy cloned animals sometimes have aberrant epigenetic status, the harmful effects of these defects could be passed onto their offspring. This is one of the biggest obstacles for the application of cloned animals for livestock production. Here, we investigated the DNA methylation status of four developmentally regulated genes (PEG3, XIST, OCT4, and NANOG) in sperms from a cloned and a non‐cloned bull, and blastocysts obtained by in vitro fertilization using those sperms and SCNT. We found no differences in the methylation status of the above genes between cloned and non‐cloned bull sperms. Moreover, the methylation status was also similar in blastocysts obtained with cloned and non‐cloned bull sperms. In contrast, the methylation status was compromised in the SCNT blastocysts. These results indicate that sperm from cloned bulls would be adequately reprogrammed during spermatogenesis and, thus, could be used to produce epigenetically normal embryos. This study highlights the normality of cloned bull offspring and supports the application of cloned cattle for calf production.     

Effects of recipient oocyte source,number of transferred embryos and season on somatic cell nuclear transfer efficiency in sheep

To improve the efficiency of somatic cell nuclear transfer (SCNT) in sheep, we investigated the effects of recipient oocyte source, number of transferred embryos and season on the pregnancy and live lamb rates for sheep somatic cell nuclear transfer embryos. Follicle‐stimulating hormone (FSH)‐stimulated ovaries produced significantly more oocytes both in total and of suitable quality for maturation culture than those without FSH treatment (from slaughterhouse). However, their in vitro maturation rates were similar. Embryos were reconstructed using adult fibroblast cells into enucleated MII oocytes. The pregnancy and term rates were significantly higher in the FSH‐stimulated group than in the slaughterhouse one. Oocytes from FSH‐stimulated ovaries were enucleated as recipient cytoplasm for nuclear transfer in the following experiments. The transfer of 7–9 and 11–13 embryos produced significantly higher pregnancy rates than that of six embryos. However, the former groups exhibited similar live lamb rates. FSH‐stimulated ovaries produced significantly more oocytes in November and December (winter) than in May to July (summer), but the associated maturation rate did not increase. Pregnancy and term rates were significantly higher when transfer occurred in winter than in summer. In conclusion, FSH treatment produced significant benefit regarding the number and quality of collected oocytes and also for the pregnancy and live lamb rates for reconstructed embryos. However, the transfer of an appropriate number of embryos (7–13) and at an appropriate season (winter) increased pregnancy and term rates.     

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non‐cloned or cloned dams using semen from non‐cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non‐cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non‐cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.