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.     

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.     

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.     

Cellular Composition and Viability of Cloned Bovine Embryos Using Exogene-Transfected Somatic Cells

The present study compared the efficiency of transgenic (TG) cloned embryo production by somatic cell nuclear transfer (SCNT) with fetal-derived fibroblast cells (FFCs) which were transfected with pEGFP-N1 to in vitro-fertilized (IVF), parthenogenetic and SCNT counterparts by evaluating the rates of cleavage and blastocyst formation, apoptosis rate at different developmental stages, cell number, ploidy and gene expression in blastocysts. In SCNT and TG embryos, the rates of cleavage and blastocyst formation were significantly lower (p < 0.05) than those of IVF controls, but it did not differ between SCNT and TG embryos. In IVF control, 86.7% embryos displayed diploid chromosomal complements and the rates were significantly (p < 0.05) higher than those of SCNT and TG embryos. Most TG embryos (79%) with FFCs expressed the gene by both PCR and under fluorescence microscopy. The expression of apoptosis by TUNEL was first detected at six to eight cell stages in all embryos of IVF, SCNT and TG groups, but the expression rate at each developmental stages was significantly higher (p < 0.05) in SCNT and TG embryos than in IVF counterparts. The expression rate in inner cell mass (ICM) of TG embryos was significantly higher (p < 0.05) than in SCNT and IVF embryos. These results indicate that the high occurrence of apoptosis observed in SCNT and TG embryos compared with IVF counterparts might influence the developmental competence. Moreover, the SCNT embryos derived using non-transfected donor cells exhibited a lower apoptosis expression in ICM cells than in TG embryos derived using pEGP-N1-transfected donor cells suggesting a possible role of negative gene effect in TG embryos.     

卵母细胞来源对水牛体细胞核移植胚胎发育潜能的影响

[目的] 探索不同来源卵母细胞对体细胞核移植（SCNT）重构胚的发育能力及发育潜能关键蛋白表达水平的影响。[方法] 试验分为活体采卵（OPU）和屠宰场（SLH）卵巢2组,OPU组用超声波活体采卵仪穿刺抽吸10头非泌乳期经产水牛卵巢的卵泡采卵,SLH组从屠宰场卵巢抽吸卵泡采卵。获得的卵母细胞分别进行体外成熟,体外成熟22~24 h后,吹打去除卵丘细胞,挑选具有第一极体的卵母细胞,去核后与水牛耳部成纤维细胞进行SCNT,分别统计SCNT重构胚的融合率、分裂率和囊胚率,用免疫荧光检测2种SCNT重构胚的E-钙黏蛋白（E-cadherin）和转录因子Sox2蛋白的表达水平。[结果] OPU组卵母细胞成熟率及其SCNT重构胚的囊胚率均显著高于SLH组（P<0.05）,但2组SCNT重构胚的融合率和分裂率均无显著差异（P>0.05）;免疫荧光结果显示,E-cadherin蛋白定位于细胞膜上,Sox2蛋白分布在细胞核膜及细胞质中,OPU组SCNT重构胚中E-cadherin和Sox2的表达水平均显著高于SLH组（P<0.05）。[结论] 活体采集的水牛卵母细胞更适合用于SCNT重构胚的构建。     

Effect of sex differences in donor foetal fibroblast on the early development and DNA methylation status of buffalo (Bubalus bubalis) nuclear transfer embryos

Low efficiency of somatic cell nuclear transfer (SCNT) embryos is largely attributable to imperfect reprogramming of the donor nucleus. The differences in epigenetic reprogramming between female and male buffalo cloned embryos remain unclear. We explored the effects of donor cell sex differences on the development of SCNT embryos. We and then compared the expression of DNA methylation (5‐methylcytosine‐5mC and 5‐hydroxymethylcytosine‐5hmC) and the expression level of relevant genes, and histone methylation (H3K9me2 and H3K9me3) level in SCNT‐♀ and SCNT‐♂ preimplantation embryos with in vitro fertilization (IVF) counterparts. In the study, we showed that developmental potential of SCNT‐♀ embryos was greater than that of SCNT‐♂ embryos (p < 0.05). 5mC was mainly expressed in SCNT‐♀ embryos, whereas 5hmC was majorly expressed in SCNT‐♂ embryos (p < 0.05). The levels of DNA methylation (5mC and 5hmC), Dnmt3b, TET1 and TET3 in the SCNT‐♂ embryos were higher than those of SCNT‐♀ embryos (p < 0.05). In addition, there were no significant differences in the expression of H3K9me2 at eight‐stage of the IVF, SCNT‐♀ and SCNT‐♂embryos (p < 0.05). However, H3K9me3 was upregulated in SCNT‐♂ embryos at the eight‐cell stage (p < 0.05). Thus, KDM4B ectopic expression decreased the level of H3K9me3 and significantly improved the developmental rate of two‐cell, eight‐cell and blastocysts of SCNT‐♂ embryos (p < 0.05). Overall, the lower levels of DNA methylation (5mC and 5hmC) and H3K9me3 may introduce the greater developmental potential in buffalo SCNT‐♀ embryos than that of SCNT‐♂ embryos.     

Effects of synchronization of donor cell cycle on embryonic development and DNA synthesis in porcine nuclear transfer embryos

The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P<0.05) developmental rate to the blastocyst stage per cleaved embryo (59%) than the G0-SCNT embryos (43%). Moreover, initiation of first DNA synthesis and cleavage occurred significantly earlier in the G1-SCNT embryos than in the G0-SCNT embryos. Delay of initiation of first DNA synthesis in the SCNT embryos by aphidicolin resulted in decreased developmental rates to the blastocyst stage without any effect on cleavage rates. Our data demonstrates that synchronized-G1 phase cells can be used as donor cells for SCNT embryos and that earlier initiation of first DNA synthesis may be important for subsequent development of SCNT embryos. The SCNT system using G1-synchronized cells, in terms of their highly uniform and viable cell states, can be useful for studying the reprogramming processes and embryonic development of SCNT embryos.     

Epigenetic characteristics of cloned and in vitro-fertilized swamp buffalo (Bubalus bubalis) embryos

Swamp buffalos are becoming endangered due to reproductive inefficiencies. This is of concern because many countries depend heavily on their products. Somatic cell nuclear transfer (SCNT) is a potential strategy for preserving endangered species. To date, SCNT in swamp buffalo has succeeded in the creation of blastocyst embryos. However, development to term of SCNT swamp buffalos is extremely limited, and only 1 live birth has been reported. An abnormal epigenetic mechanism is suspected to be the cause of developmental failure, as is also seen in other species. The DNA methylation and histone acetylation are key players in epigenetic modification and display marked variability during embryonic preimplantation development. Knowledge of epigenetic modifications will aid in solving the developmental problems of SCNT embryos and improving reproductive technology in the swamp buffalo. The objective of this study was to determine the relationship between preimplantation embryonic development and 2 epigenetic patterns, global DNA methylation and histone acetylation, in SCNT and in vitro-fertilized (IVF) swamp buffalo embryos. In addition, we examined the correlations between those 2 mechanisms in the SCNT and IVF swamp buffalo embryos throughout the developmental stages using double immunostaining and quantification of the emission intensities using confocal microscopy. We discovered an aberrant methylation pattern in early preimplantation-stage swamp buffalo SCNT embryos. In addition, greater variability in the DNA methylation levels among nuclei within SCNT embryos was discovered. Hyperacetylation was also observed in SCNT embryos compared with IVF embryos at the 4- and 8-cell stages (P < 0.05). Dynamic changes and interplay between these 2 epigenetic mechanisms could be crucial for embryonic development during the early preimplantation period. The aberrancies uncovered here may contribute to the low efficiency of SCNT.     

Expression of Interferon-tau mRNA in Bovine Embryos Derived from Different Procedures

Interferon-tau (IFN-τ) is a secreted conceptus protein which plays a critical role in the establishment of ruminant pregnancy by its antiluteolytic and antiviral effects. In the present study, we hypothesized that IFN-τ expression was temporally and spatially regulated in different pre-implantation embryos and the levels of IFN-τ expression were different among bovine embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). By using in situ hybridization with Digoxingenin (DIG)-labelled IFN-τ cDNA as a probe, we detected IFN-τ mRNA in bovine embryos from days 3 to 9 in culture. However, the timing of the initiation of IFN-τ mRNA expression was different among PA, IVF and SCNT embryos. Interferon-τ mRNA was first expressed in 16-cell stage IVF embryos on day 4, in SCNT morula on day 5 and early PA blastocyst on day 6. Semi-quantitative RT-PCR analysis showed that the expression levels of IFN-τ mRNA did not differ significantly among IVF, SCNT and PA embryos on day 7. In addition, freezing and thawing did not have a major impact either on IFN-τ mRNA expression in IVF or in vivo -produced bovine blastocysts.     

Effects of cumulus cells on in vitro maturation of oocytes and development of cloned embryos in the pig

The purpose of this study was to investigate the role of porcine cumulus cells (CC) in oocyte maturation and somatic cell nuclear transfer (SCNT) embryo development in vitro. Denuded pig oocytes were co-cultured with CC or routinely cultured in maturation medium without a feeder layer. Porcine CC inactivated with mitomycin C or non-inactivated were used for the feeder layer in co-culture with porcine SCNT embryos to investigate comparatively the developmental competence of cloned embryos. The DNA damage aspects of apoptosis and expression pattern of genes implicated in apoptosis (Fas/FasL) as well as the mRNA expression of DNA methyltransferase (Dnmt1, Dnmt3a) of porcine SCNT embryos were also evaluated by comet assay or real-time RT-PCR, respectively. The results showed that co-culture with CC improved the extrusion rate of pbI (49.3% vs 31.5%, p<0.05) and survival rate (75.7% vs 53.3%, p<0.05) of denuded oocytes, but had no effects on blastocyst developmental rate or 2-cell-stage survival rate of in vitro fertilization embryos. Co-culture with CC inactivated by mitomycin C improved the blastocyst developmental rate (26.6% vs 13.0%, p<0.05) and decreased the apoptotic incidence (27.6% vs 46.2%, p<0.05) of porcine cloned embryos. Co-culture with inactivated CC reduced Fas and FasL mRNA expression of cloned embryos at the blastocyst stage compared with NT controls (p<0.05), but there were no differences in Dnmt1 and Dnmt3a mRNA expression among groups. Co-culture with inactivated cumulus cell monolayer significantly increased blastocyst formation and decreased the apoptotic incidence in porcine cloned embryos during in vitro development.     

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.     

Extended exposure to trichostatin A after activation alters the expression of genes important for early development in nuclear transfer murine embryos

The low viability of embryos reconstructed by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic modification errors, and reduction of those errors may improve the viability of SCNT embryos. The present study shows the effect of trichostatin A (TSA), a strong inhibitor of histone deacetylase, on the development of murine SCNT embryos. After enucleation and nuclear injection, reconstructed murine oocytes were activated with or without TSA for 6 hr (TSA-6 hr). After activation, TSA treatment was extended to 3 hr (TSA-9 hr), 5 hr (TSA-11 hr) and 18 hr (TSA-24 hr) during culture. As a result, the SCNT embryos in the TSA-11 hr group showed a remarkably higher blastocyst rate (21.1%) when compared with the nontreated embryos (3.4%), while the concentration of TSA did not significantly affect embryonic development. The expressions of histone deacetylase (HDAC1 and HDAC2) and DNA methylation (DNMT3a and DNMT3b) genes decreased in the TSA-11 hr and TSA-24 hr groups, while there was an increase in the expression of histone acetyltransferase (P300 and CBP), pluripotency (OCT4 and NANOG) and embryonic growth/trophectoderm formation (FGF4)-related genes in the same groups. The expression of CDX2, a critical gene for trophectoderm formation was upregulated only in the TSA-24 hr group. Our results show that TSA treatment during the peri- and postactivation period improves the development of reconstructed murine embryos, and this observation may be explained by enhanced epigenetic modification of somatic cells caused by TSA-induced hyperacetylation, demethylation and upregulation of pluripotency and embryonic growth after SCNT.     

In Vitro Development of Bison Embryos Using Interspecies Somatic Cell Nuclear Transfer

Interspecies somatic cell nuclear transfer (interspecies SCNT) has been explored in many domestic and non‐domestic animal species. However, problems arise during the development of these embryos, which may be related to species‐specific differences in nuclear–cytoplasmic communication. The objectives of this study were to investigate the possibility of producing bison embryos in vitro using interspecies SCNT and assess the developmental potential of these embryos. Treatment groups consisted of cattle in vitro fertilization (IVF) and cattle SCNT as controls and wood bison SCNT, plains bison SCNT and wisent SCNT as experimental groups. Cleavage and blastocyst rates were assessed, and blastocyst quality was determined using total cell number, apoptotic incidence and relative quantification of mitochondria‐related genes NRF1, MT‐CYB and TFAM. These results indicate that embryos can be produced by interspecies SCNT in all bison species/subspecies (13.34–33.54% blastocyst rates). Although increased incidence of apoptosis was observed in bison SCNT blastocysts compared to cattle SCNT controls (10.45–12.69 vs 8.76, respectively) that corresponded with significantly lower cell numbers (80–87 cells vs >100 cells, respectively), no major differences were observed in the expression of NRF1, MT‐CYB and TFAM. This study is the first to report the production of bison embryos by interspecies SCNT. Blastocyst development in all three bison species/subspecies was greater than the rates obtained in previous studies by IVF, which supports the potential role of SCNT for in vitro embryo production in this species. Yet, further investigation of developmental competence and the factors influencing blastocyst quality and viability is required.     

Constant transmission of mitochondrial DNA in intergeneric cloned embryos reconstructed from swamp buffalo fibroblasts and bovine ooplasm

Although interspecies/intergeneric somatic cell nuclear transfer (iSCNT) has been proposed as a tool to produce offspring of endangered species, conflict between donor nucleus and recipient cytoplasm in iSCNT embryos has been identified as an impediment to implementation for agricultural production. To investigate the nuclear–mitochondrial interactions on the developmental potential of iSCNT embryos, we analyzed the mtDNA copy numbers in iSCNT embryos reconstructed with water buffalo (swamp type) fibroblasts and bovine enucleated oocytes (buffalo iSCNT). As controls, SCNT embryos were derived from bovine fibroblasts (bovine SCNT). Buffalo iSCNT and bovine SCNT embryos showed similar rates of cleavage and development to the 8‐cell stage (P > 0.05). However, buffalo iSCNT embryos did not develop beyond the 16‐cell stage. Both bovine and buffalo mtDNA content in buffalo iSCNT embryos was stable throughout the nuclear transfer process, and arrested at the 8‐ to 16‐cell stage (P > 0.05). In bovine SCNT embryos that developed to the blastocyst stage, mtDNA copy number was increased (P < 0.05). In conclusion, both the donor cell and recipient cytoplast mtDNAs of buffalo iSCNT embryos were identified and maintained through the iSCNT process until the 8–16‐cell stage. In addition, the copy number of mtDNA per embryo was a useful monitor to investigate nuclear–mitochondrial interactions.     

卵母细胞核因子促进核移植四倍体胚胎早期发育研究

研究旨在探讨猪卵母细胞核因子在重编程过程中发挥的作用。将体细胞引入未去核的MⅡ期卵母细胞中,构建体细胞核与卵母细胞核共存的核移植四倍体胚胎。通过分析核移植四倍体胚胎的早期发育情况探讨卵母细胞核因子对核移植四倍体胚胎早期发育的影响。结果显示,核移植四倍体胚胎、孤雌二倍体胚胎及孤雌单倍体胚胎这3组胚胎的卵裂率极显著高于核移植二倍体胚胎（P<0.01）,且核移植四倍体囊胚率及总细胞数也极显著高于核移植二倍体囊胚（P<0.01）。与通过标准核移植程序构建的核移植二倍体胚胎相比,核移植四倍体胚胎具有更强的发育能力。本研究建立了一个体细胞核与完整卵母细胞核因子物质共存的四倍体胚胎模型,有助于研究供体核与卵母细胞核之间的联系,为研究核因子在重编程过程中发挥的作用提供了平台。     

Study on Oocyte Nuclear Factor Promotes the Early Development of Tetraploid Somatic Cell Nuclear Transfer Embryos

The study was aimed to investigate the role of porcine oocyte nuclear factors during reprogramming. Somatic cell nuclei was introduced into intact MⅡ oocytes to establish tetraploid somatic cell nuclear transfer (SCNT) embryos containing both somatic nuclei and oocyte nuclei. And then the influence of the oocyte nucleus on tetraploid SCNT embryo development was examined by assessing characteristics including cleavage rate and blastocyst rate. The results showed that the cleavage rate of tetraploid SCNT embryos,diploid parthenogenetic embryos and haploid parthenogenetic embryos was extremely significantly higher than that of standard diploid SCNT embryos (P<0.01). The blastocyst rate and the total number of cells in tetraploid SCNT embryos were extremely significantly higher than that of standard diploid SCNT embryos (P<0.01).Overall,tetraploid SCNT embryos had a higher developmental competence than standard diploid SCNT embryos. In conclusion, the embryonic model was established in which a fetal fibroblast nucleus and an oocyte M Ⅱ plate coexist. Tetraploid SCNT represented a new research platform that was potentially useful for examining interactions between donor nuclei and oocyte nuclei. This platform should facilitate further understanding of the roles played by nuclear factors during reprogramming.