Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 μg/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.     

The influence of nuclear content on developmental competence of gaur x cattle hybrid in vitro fertilized and somatic cell nuclear transfer embryos

In nondomestic and endangered species, the use of domestic animal oocytes as recipients for exotic donor nuclei causes the normal pattern of cytoplasmic inheritance to be disrupted, resulting in the production of nuclear-cytoplasmic hybrids. Evidence suggests that conflict between nuclear and cytoplasmic control elements leads to a disruption of normal cellular processes, including metabolic function and cell division. This study investigated the effects of nuclear-cytoplasmic interactions on the developmental potential of interspecies embryos produced by in vitro fertilization and somatic cell nuclear transfer: cattle x cattle, gaur x cattle, hybrid x cattle. Cattle control and hybrid embryos were examined for development to the blastocyst stage and blastocyst quality, as determined by cell number and allocation, apoptosis incidence, and expression patterns of mitochondria-related genes. These analyses demonstrated that a 100% gaur nucleus within a domestic cattle cytoplasmic environment was not properly capable of directing embryo development in the later preimplantation stages. Poor blastocyst development accompanied by developmental delay, decreased cell numbers, and aberrant apoptotic and related gene expression profiles, all signs of disrupted cellular processes associated with mitochondrial function, were observed. Developmental potential was improved when at least a portion of the nuclear genome corresponded to the inherited cytoplasm, indicating that recognition of cytoplasmic components by the nucleus is crucial for proper cellular function and embryo development. A better understanding of the influence of the cytoplasmic environment on embryonic processes is necessary before interspecies somatic cell nuclear transfer can be considered a viable alternative for endangered species conservation.     

Developmental competence of somatic cell nuclear transfer embryos reconstructed from oocytes matured in vitro with follicle shells in miniature pig

The developmental competence of domestic pig oocytes that were transferred to somatic cell nuclei of miniature pig was examined. A co-culture system of oocytes with follicle shells was used for the maturation of domestic pig oocytes in vitro. Co-cultured oocytes progressed to the metaphase II stage of meiosis more quickly and more synchronously than non co-cultured oocytes. Oocytes were enucleated and fused with fibroblast cells of Potbelly miniature pig at 48 h of maturation. The blastocyst formation rate of nuclear transfer (NT) embryos using cocultured oocytes (24%) was significantly higher (p < 0.05) than that of non-co-cultured oocytes (13%). Cleaved embryos at 48 h after nuclear transfer using co-cultured oocytes were transferred to the oviducts of 14 G?ttingen miniature pigs and four Meishan pigs. Estrus of all G?ttingens returned at around 20-31 days of pregnancy. Two of the four Meishans became pregnant. Three and two cloned piglets were born after modest number of embryo transfer (15 and 29 embryos transferred), respectively. These results indicated that oocytes co-cultured with follicle shells have a high developmental competence after nuclear transfer and result in full-term development after embryo transfer.     

利用IVF废弃胚胎为核移植受体构建人体细胞克隆胚胎

目的：探讨利用IVF废弃胚胎构建人体细胞克隆胚胎的发育潜能及其在人治疗性克隆应用的可能性。方法：收集2008年7-12月在广州医学院第三附属医院进行体外受精-胚胎移植周期中的多精受精胚胎和MII期体外受精失败卵母细胞,运用显微操作技术构建人体细胞克隆胚胎,观察胚胎发育情况。结果：多精受精胚胎为核移植受体的克隆胚胎能够发育到8-细胞期,受精失败MII期卵母细胞为核移植受体的克隆胚胎能够激活,但不能够卵裂。两种IVF废弃的胚胎构建的人体细胞克隆胚胎在去核成功率,注核成功率上无显著差异（P＆gt;0.05）,但卵裂率和8细胞率上具有显著差异（P＆lt;0.05）。结论：多精受精胚胎比MII期体外受精失败卵母细胞更适合作为人核移植受体细胞。     

High in vitro development after somatic cell nuclear transfer and trichostatin A treatment of reconstructed porcine embryos

Abnormal epigenetic modification is supposed to be one of factors accounting for inefficient reprogramming of the donor cell nuclei in ooplasm after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone deacetylase, potentially enhancing cloning efficiency. The aim of our present study was to establish the optimal TSA treatment in order to improve the development of handmade cloned (HMC) porcine embryos and examine the effect of TSA on their development. The blastocyst percentage of HMC embryos treated with 37.5nM TSA for 22-24h after activation increased up to 80% (control group-54%; P<0.05). TSA mediated increase in histone acetylation was proved by immunofluorescence analysis of acH3K9 and acH4K16. 2-cell stage embryos derived from TSA treatment displayed significant increase in histone acetylation compared to control embryos, whereas no significant differences were observed at blastocyst stage. During time-lapse monitoring, no difference was observed in the kinetics of 2-cell stage embryos. Compact morula (CM) stage was reached 15h later in TSA treated embryos compared to the control. Blastocysts (Day 5 and 6) from HMC embryos treated with TSA were transferred to 2 recipients resulting in one pregnancy and birth of one live and five dead piglets. Our data demonstrate that TSA treatment after HMC in pigs may affect reprogramming of the somatic genome resulting in higher in vitro embryo development, and enable full-term in vivo development.     

Effect of cytochalasins B and D on the developmental competence of somatic cell nuclear transfer embryos in miniature pigs

In many animals, cytochalasins have generally been used as cytoskeletal inhibitors for the diploid complement retention of somatic cell nuclear transfer (SCNT) embryos. However, limited information is available on the effects of cytochalasins on the in vitro development of SCNT embryos. Hence, we compared the effects of cytochalasin B (CB) and cytochalasin D (CD) on pseudo-polar body (pPB) extrusion, cortical actin filament (F-actin) distribution in porcine parthenogenetic oocytes and in vitro development of SCNT embryos that were reconstructed using foetal fibroblasts in the G0/G1 phase derived from miniature pigs. CB (7.5 microg/ml) and CD (2.5 microg/ml) treatments effectively inhibited pPB extrusion in SCNT embryos. CB (2.5 microg/ml) treatment could not inhibit pPB extrusion and insufficiently destabilized F-actin immediately following artificial activation. In parthenogenetic oocytes treated with 2.5 microg/ml CD, normal reorganization and uniform distribution of cortical F-actin at the cytoplasmic membrane were observed at 8 h after artificial activation; this finding was similar to that of control oocytes. In contrast, parthenogenetic oocytes treated with 7.5 microg/ml CB showed non-uniform distribution of F-actin at 8 h after artificial activation. On day 5 after in vitro cultivation, the blastocyst formation rate of SCNT embryos treated with 2.5 microg/ml CD was significantly higher than that of SCNT embryos treated with 2.5 and 7.5 microg/ml CB (p < 0.05). Hence, the present findings suggest that CD is more effective than CB as the cytoskeletal inhibitor for the production of SCNT embryos in miniature pigs.     

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.     

In vitro development of mouse somatic nuclear transfer embryos: effects of donor cell passages and electrofusion

In this study, C57BL/6 adult male mouse ear fibroblast cells and Kunming mouse M2 oocytes were used as donors and recipients, respectively, to investigate the effect of passage number on donor cells and electrofusion times on the in vitro development of nuclear transfer (NT) embryos. The results demonstrated firstly that when the ear fibroblast cells from either 2-4, 5-7 or 8-10 passages were used as donors, respectively, to produce NT embryos, the number of passages undergone by the donor cells had no significant effect on the in vitro development of NT embryos. The developmental rates for morula/blastocyst were 15.2, 13.3 and 14.0%, respectively, which were not significantly difference (p>0.05). Secondly, when the NT embryos were electrofused, there was no significant difference between the fusion ratio for the first electrofusion and the second electrofusion (p>0.05). The developmental rates of the 2-cell and 4-cell stages that had undergone only one electrofusion, however, were significantly higher than those that had had two electrofusions (65.7% compared with 18.4% and 36.4% compared with 6.1%; p<0.01), furthermore the NT embryos with two electrofusions could not develop beyond the 4-cell stage. This study suggests that this protocol might be an alternative method for mouse somatic cloning, even though electrofusion can exert negative effects on the development of NT embryos.     

Defined media optimization for in vitro culture of bovine somatic cell nuclear transfer (SCNT) embryos

The objective was to establish an efficient defined culture medium for bovine somatic cell nuclear transfer (SCNT) embryos. In this study, modified synthetic oviductal fluid (mSOF) without bovine serum albumin (BSA) was used as the basic culture medium (BCM), whereas the control medium was BCM with BSA. In Experiment 1, adding polyvinyl alcohol (PVA) to BCM supported development of SCNT embryos to blastocyst stage, but blastocyst formation rate and blastocyst cell number were both lower (P < 0.05) compared to the undefined group (6.1 vs. 32.6% and 67.3 ± 3.4 vs. 109.3 ± 4.5, respectively). In Experiment 2, myo-inositol, a combination of insulin, transferrin and selenium (ITS), and epidermal growth factor (EGF) were added separately to PVA-supplemented BCM. The blastocyst formation rate and blastocyst cell number of those three groups were dramatically improved compared with that of PVA-supplemented group in Experiment 1 (18.5, 23.0, 24.1 vs. 6.1% and 82.7 ± 2.0, 84.3 ± 4.2, 95.3 ± 3.8 vs. 67.3 ± 3.4, respectively, P < 0.05), but were still lower compared with that of undefined group (33.7% and 113.8 ± 3.4, P < 0.05). In Experiment 3, when a combination of myo-inositol, ITS and EGF were added to PVA-supplemented BCM, blastocyst formation rate and blastocyst cell number were similar to that of undefined group (30.4 vs. 31.1% and 109.3 ± 4.4 vs. 112.0 ± 3.6, P > 0.05). In Experiment 4, when blastocysts were cryopreserved and subsequently thawed, there were no significant differences between the optimized defined group (Experiment 3) and undefined group in survival rate and 24 and 48 h hatching blastocyst rates. Furthermore, there were no significant differences in expression levels of H19, HSP70 and BAX in blastocysts derived from optimized defined medium and undefined medium, although the relative expression abundance of IGF-2 was significantly decreased in the former. In conclusion, a defined culture medium containing PVA, myo-inositol, ITS, and EGF supported in vitro development of bovine SCNT embryos.     

Ultrastructural analysis reveals striking differences of intercellular contact lengths in bovine embryos produced in vivo, in vitro and by somatic cell nuclear transfer

Cellular coherence and communication, thus cell-to-cell contact is an indispensable premise to sustain the formation of complex, multi-cellular organisms. We have analyzed intercellular contact lengths in NT-cloned bovine embryos compared to the in vivo or in vitro produced counterparts. Therefore, ultrastructural analysis was carried out by transmission electron microscopy (TEM) at the 8-cell and blastocyst stage of development. To obtain embryos generated in vivo, oviducts of superovulated cows were flushed 3 days after insemination, subsequent to slaughter. Standard in vitro maturation (IVM) and -fertilization (IVF) were utilized to obtain in vitro embryos. Cloned embryos by somatic nuclear transfer were produced by the handmade cloning (HMC) procedure. The points of apposition/focal contact points (CPs) between the blastomeres were of the shortest order in cloned embryos (236 +/- 135 nm) and of highest order in the in vivo produced embryos (2,085 +/- 1,540 nm), although no significant differences regarding the blastomere sizes in the various groups of 8-cell embryos could be established. In summary, the CP lengths in case of in vitro and in vivo 8-cell embryos were, on an average, five or nine times longer, respectively, than in the case of the cloned embryos. These differences of CP lengths vanished in embryos reaching the blastocyst stage of embryonic development in all the three groups of embryos. The observed differences of intercellular contact length at distinct stages of embryonic development could be responsible for differences in intercellular communication between the blastomeres at the beginning of cellular differentiation. These may be one reason for the lower developmental competence of cloned (NT) embryos.     

Cleavage pattern and survivin expression in porcine embryos by somatic cell nuclear transfer

Mammalian embryos produced in vitro show a high rate of early developmental failure. Numerous somatic cell nuclear transfer (SCNT) embryos undergo arrest and show abnormal gene expression in the early developmental stages. The purpose of this study was to analyze porcine SCNT embryo development and investigate the cause of porcine SCNT embryo arrest. The temporal cleavage pattern of porcine SCNT embryos was analyzed first, and the blastocyst origin at early developmental stage was identified. To investigate markers of arrest in the cleavage patterns of preimplantation SCNT embryos, the expression of survivin—the smallest member of the inhibitor of apoptosis (IAP) gene family, which suppresses apoptosis and regulates cell division—was compared between embryos showing normal cleavage and arrested embryos.A total of 511 SCNT embryos were used for cleavage pattern analysis. Twenty-four hours post activation (hpa), embryos were classified into five groups based on the cleavage stage as follows; 1-cell, 2-cell, 4-cell, 8-cell and fragmentation (frag). In addition, 48 hpa embryos were more strictly classified into 15 groups based on the cleavage stage of 24 hpa; 1-1 cell (24 hpa-48 hpa), 1-2 cell, 1-4 cell, 1-8 cell, 1 cell-frag, 2-2 cell, 2-4 cell, 2-8 cell, 2 cell-frag, 4-4 cell, 4-8 cell, 4 cell-frag, 8-8 cell, 8 cell-frag, and frag-frag. These groups were cultured until 7 d post activation, and were evaluated for blastocyst formation. At 24 hpa, the proportion of 2-cell stage was significantly higher (44.5%) than those in the other cleavage stages (1-cell: 13.4%; 4-cell: 17.9%; 8-cell: 10.3%; and frag: 13.9%). At 48 hpa, the proportion of embryos in the 2-4 cell stage was significantly higher (32.4%) than those in the other cleavage stages (2-8 cell: 8.2%; 4-8 cell: 12.1%; and frag-frag: 13.9%). Some embryos arrested at 48 hpa (1-1 cell: 5.8%; 2-2 cell: 2.8%; 4-4 cell: 3.8%; 8-8 cell: 6.5%; and total arrested embryos: 18.9%). Blastocyst formation rates were higher in 2-4 cell cleavage group (20.2%) than in other groups. SCNT embryos in 2-4 cell stage showed stable developmental competence. In addition, we investigated survivin expression in porcine SCNT embryos during the early developmental stages. The levels of survivin mRNA in 2-cell, 4-cell stage SCNT embryos were significantly higher than those of arrested embryos. Survivin protein expression showed a similar pattern to that of survivin mRNA. Normally cleaving embryos showed higher survivin protein expression levels than arrested embryos. These observations suggested that 2-4 cell cleaving embryos at 48 hpa have high developmental competence, and that embryonic arrest, which may be influenced by survivin expression in porcine SCNT embryos.     

Qualitative and quantitative differences of cytoskeleton proteins in embryos produced in vitro, in vivo, and by somatic nuclear transfer

The cytoskeleton, consisting of complex and dynamic systems of structural filaments, intermediate filaments and microtubules, is not only a structural element but also contributes to many cellular processes such as functional compartments, transportation, mitosis, secretion, formation of cell extensions, and intercellular communication. Suggestions in rat 2-cell embryos that abnormal distributions of cytoskeletal proteins occurred following the initiations of developmental arrest and our former studies showing reduced intercellular contact zones in cloned bovine embryos prompted us to conduct comparative studies on 8-cell stage bovine embryos from nuclear transfer (NT), in vitro, and in vivo production. Immunohistochemistry and Laser-Scanning-Microscopy facilitated detection of cytoskeleton proteins--alpha-tubulin, F-actin, beta-catenin, and the cell adhesion protein cadherin; image and cluster analysis were subsequently used to study the distribution pattern of the proteins, whereas Western blot was carried out for their qualitative and quantitative analysis. The maximum fluorescence intensity of stained alpha-tubulin was observed in the cloned and the in vitro embryos. A significant higher intensity of staining for F-actin was observed in the in vivo and in vitro embryos. In contrast, Western blot revealed no differences of actin, tubulin, and catenin between the three tested groups whereas a lower abundance of cadherin proteins in the cloned embryos was visible. The distribution of actin filaments in cloned embryos was more centric or one-sided and not peripheral whereas the stained spots of catenin were smaller in comparison to in vivo or in vitro produced embryos. These differences recorded in the distribution patterns may be associated with cell physiological processes related to an influenced actin-catenin-cadherin system. In conclusion, reduced intercellular contacts coupled with abnormal distribution of cytoskeletal proteins seem to play an important role in the developmental arrest encountered normally at the 8-cell stage in bovine cloned embryos.     

In vitro and in vivo developmental potential of nuclear transfer embryos using bovine cumulus cells prepared in four different conditions

We examined the effect of culture of donor cells on nuclear transfer efficiency using bovine cumulus cells treated with four different conditions: (1). group A, the cells removed from cumulus-oocyte complexes (COC) after aspiration of ovarian follicles; (2). group B, the cells removed from COC after in vitro maturation; (3). group C, the cells cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) for 3 days after some subculture; and (4). group D, the cells cultured in DMEM with 0.5% FBS for an additional 5 days. Analysis of cell cycle using flow cytometry revealed that the relative proportion of donor cells at G0/G1 phase of cell cycle was 89.7% in group A, 89.5% in group B, 76.0% in group C, and 90.6% in group D. The developmental rates to blastocyst stage in groups C (45.3%) and D (46.4%) were significantly (p < 0.05) higher than in groups A (17.5%) and B (31.9%). After transfer of blastocysts produced in each group, nine of 24 recipients became pregnant on day 30. A total of five live calves were obtained from cumulus cells in all groups (group A [n = 1], group B [n = 1], group C [n = 2], and group D [n = 1]).     

Critical developmental stages for the efficiency of somatic cell nuclear transfer in zebrafish

Somatic cell nuclear transfer (SCNT) has been performed extensively in fish since the 1960s with a generally low efficiency of approximately 1%. Little is known about somatic nuclear reprogramming in fish. Here, we utilized the zebrafish as a model to study reprogramming events of nuclei from tail, liver and kidney cells by SCNT. We produced a total of 4,796 reconstituted embryos and obtained a high survival rate of 58.9-67.4% initially at the 8-cell stage. The survival rate exhibited two steps of dramatic decrease, leading to 8.7-13.9% at the dome stage and to 1.5-2.96% by the shield stage. Concurrently, we observed that SCNT embryos displayed apparently delayed development also at the two stages, namely the dome stage (1:30 ± 0:40) and the shield stage (2:50 ± 0:50), indicating that the dome and shield stage are critical for the SCNT efficiency. Interestingly, we also revealed that an apparent alteration in klf4 and mycb expression occurred at the dome stage in SCNT embryos from all the three donor cell sources. Taken together, these results suggest that the dome stage is critical for the SCNT efficiency, and that alternated gene expression appears to be common to SCNT embryos independently of the donor cell types, suggesting that balanced mycb and klf4 expression at this stage is important for proper reprogramming of somatic nuclei in zebrafish SCNT embryos. Although the significant alteration in klf4 and mycb expression was not identified at the shield stage between ZD and SCNT embryos, the importance of reprogramming processes at the shield stage should not be underestimated in zebrafish SCNT embryos.