Development Of Nuclear Transfer Embryos Research Articles

Incomplete activation of Alyref and Gabpb1 leads to preimplantation arrest in cloned mouse embryos.

Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Alyref was needed for the proper formation of inner cell mass by regulating Nanog, whereas Gabpb1 deficiency led to apoptosis. The supplement of Alyref and Gabpb1 mRNA supported efficient preimplantation development of cloned embryos. Alyref and Gabpb1 were silenced in NT embryos partially because of the repressed expression of Klf16 by H3K9me3. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes, and the incomplete activation of such genes results in preimplantation arrest of cloned embryos.

HDAC6 Is Involved in the Histone Deacetylation of In Vitro Maturation Oocytes and the Reprogramming of Nuclear Transplantation in Pig.

It remained unknown whether HDAC6 affected the histone deacetylation of in vitro maturation oocytes and the reprogramming of nuclear transplantation in pig. Our results indicated that HDAC6 specific inhibition did not affect overall HDAC activity and meiosis process, which increased histone H3K9/K14 and H4K8 acetylation of porcine in vitro maturation oocytes and pseudo-pronucleus embryos. HDAC6 inhibition also significantly enhanced the cleavage and blastocyst of nuclear transfer embryos (0.81 ± 0.12 vs. 0.68 ± 0.12 and 0.46 ± 0.19; 0.73 ± 0.13 vs. 0.63 ± 0.18 and 0.40 ± 0.16, P<0.05). The inhibition of HDAC6 significantly enhanced histone H3K9/K14 and H4K8 acetylation, and upregulated the OCT4 and CDX2 expressions (1.83 ± 0.16 vs. 1.00 ± 0.00 %; 2.07 ± 0.09 vs. 1.00 ± 0.00; P<0.05) in porcine SCNT blastocysts. Interestingly, HDAC6 inhibition significantly increased the pseudo-pronucleus volume during somatic cell reprogramming. Thus, HDAC6 was required for porcine histone deacetylation during the in vitro maturation and pseudo-pronucleus stages. HDAC6 inhibition improved the in vitro development of nuclear transfer embryos. HDAC6 may restrict the reprogramming of somatic nuclear transfer by regulating pseudo-pronucleus expansion. We need further research to confirm this in the future.

1 Effect of cytokine-supplemented maturation medium on bovine somatic cell nuclear transfer embryo development

Invitro maturation is an important process in the invitro production of embryos. It has been shown recently that 3 cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1), increase the efficiency of IVM, blastocyst production, and invivo development following somatic cell nuclear transfer (SCNT) in pigs (Yuan et al. 2017 PNAS 114, E5796-E5804). This study was designed to assess the effect of these cytokines on IVM in bovine oocytes, their consecutive development to blastocyst, and pregnancy rate when used in SCNT. Cumulus–oocyte complexes (COCs) were retrieved from abattoir-derived ovaries, matured for 21h in either standard maturation medium [TCM-199 (Gibco/Life Technologies), 10% fetal bovine serum, 0.5µgmL−1 FSH, 5µgmL−1 LH, 100UmL−1 penicillin/streptomycin] or maturation medium supplemented with 20ngmL−1 human LIF, 20ngmL−1 IGF1, and 40ngmL−1 FGF2. After IVM, the first polar body and metaphase plate were removed from MII oocytes. Fibroblast cells were injected in the perivitelline space and fused with enucleated oocytes in 0.28M sorbitol fusion medium (0.1mM calcium, 0.5mM magnesium, 0.5mM HEPES, 1gmL−1 bovine serum albumin) by a single pulse of 1.75 kV/cm for 22ms. Reconstructed embryos were activated by exposure to 5μM ionomycin for 5min, followed by 4h of incubation in 2mM 6-(dimethylamino)purine and 10μgmL−1 cycloheximide. Cleavage and blastocyst rates were assessed at Day 2 and Day 7, respectively. Blastocysts were transferred to oestrus synchronized recipients and initial pregnancy rates assessed at Day 40 after embryo transfer. Statistical analysis was performed using one-way ANOVA or chi-squared test. Data are presented as mean±s.e.m. The MII rate was significantly higher in maturation medium supplemented with cytokines compared with control medium (80.2±2.33%, n=885 vs. 66.8±1.82%, n=822; P&amp;lt;0.05, 7 replicates, one-way ANOVA). No statistical difference was found in the cleavage rate of SCNT embryos between treatment and control groups (94.2±1.34%, n=259 vs. 90.9±1.22%, n=208; P&amp;gt;0.05, 8 replicates, one-way ANOVA), respectively. However, a significant increase in blastocyst rate was observed in the treatment group compared with the control group (40.6±5.1%, n=446 vs. 24.3±2.9%, n=300; P&amp;lt;0.05, 8 replicates, one-way ANOVA). SCNT embryos derived from the treatment group also resulted in a significant increase in initial pregnancy rates (50.3±20.9%, n=48 vs. 29.0±20.6%, n=31; P&amp;lt;0.05, 4 replicates, chi-squared). Full-term pregnancy rates are pending. In conclusion, addition of FGF2, LIF, and IGF1 to maturation medium improves bovine IVM and SCNT blastocyst development and initial pregnancy rates. The effect on full-term pregnancy success is yet to be determined. This research was supported by UAES project (1343) and RFBR (18-29-07089).

Meta-analysis of melatonin treatment and porcine somatic cell nuclear transfer embryo development.

Porcine somatic cell nuclear transfer (SCNT) plays an important role in many areas of research. However, the low efficiency of SCNT in porcine embryos limits its applications. Porcine embryos contain high concentrations of lipid, which makes them vulnerable to oxidative stress. Some studies have used melatonin to reduce reactive oxygen species damage. At present there are many reports concerning the effect of exogenous melatonin on porcine SCNT. Some studies suggest that the addition of melatonin can increase the number of blastocyst cells, while others indicate that melatonin can reduce the number of blastocyst cells. Therefore, a meta-analysis was carried out to resolve the contradiction. In this study, a total of 63 articles from the past 30 years were analyzed, and six papers were finally selected. Through the analysis, it was found that the blastocyst rate was increased by adding exogenous melatonin. Melatonin had no effect on cleavage rate or the number of blastocyst cells, but did decrease the number of apoptotic cells. This result is crucial for future research on embryo implantation.

Factors Affecting In Vivo Development Of Nuclear Transfer Embryos Using Freeze-Dried Somatic Cells In Cattle

Factors Affecting In Vivo Development Of Nuclear Transfer Embryos Using Freeze-Dried Somatic Cells In Cattle

45 The role of TRIM28 in porcine somatic cell nuclear transfer embryo development

During fertilization, the parental genome undergoes extensive demethylation. Global DNA demethylation is a hallmark of epigenetic reprogramming. Embryos engage non-canonical DNA methylation maintenance mechanisms to ensure inheritance of exceptional germline features. However, the mechanisms ensuring demethylation resistance in light of global reprogramming remain poorly understood. TRIM28 is a maternal-effect factor that controls genomic imprinting during early embryonic reprogramming. In this study, cytoplasmic injections of siRNA were performed into oocytes matured in vitro for 26h to interfere with the expression of TRIM28 in oocytes. The injected oocytes were continually matured in vitro until 42h and used to construct somatic cell nuclear transfer (SCNT) embryos. During 2-cell to blastocyst stages, the expression of development-related genes (NANOG, POU5F1, CDX2, BAX, and BCL2), maternal imprinting genes (IGF2, DIO3, PLAGL1, and DLK1), paternal imprinting genes (H19 and PEG3), TRIM28-recruitment complex-associated genes (ZFP57, PGC7, SETDB1, and DNMT), and epigenetic chromatin modification enzymes were detected by quantitative PCR in the constructed TRIM28-interfered SCNT embryos. The DNA methylation levels in the promoter regions of the imprinted genes (H19 and IGF2) and chromatin repeats (PRE-1 and SATELLITE) were analysed by sodium bisulfite genomic sequencing. The results showed that the TRIM28-interfered SCNT embryos had significantly lower cleavage and blastocyst rates (53.9±3.4% and 12.1±4.3%, respectively) than those in control SCNT embryos (64.8±2.7% and 18.8±1.9%, respectively). The expression levels of development-related genes (NANOG and POU5F1) and TRIM28-recruited transcriptional repression complex-associated genes (PGC7, ZFP57, and DNMT1) in the 4-cell stage were significantly reduced (P&amp;lt;0.05). The imprinted genes were significantly up-regulated (P&amp;lt;0.05) from the 2-cell to blastocyst stage in constructed TRIM28-interfered SCNT embryos, except H19 at the 2-cell and blastocyst stage decreased remarkably (P&amp;lt;0.05). The DNA methylation levels of IGF2 decreased 2-fold from the 2-cell to blastocyst stage in TRIM28-interfered SCNT embryos. The PRE-1 and SATELLITE had a remarkably lower (P&amp;lt;0.05) methylation levels in the TRIM28-interfered 2-cell embryos than in control SCNT embryos. The cluster analysis showed some of the chromatin modification enzymes had abnormal expression in the TRIM28-interfered SCNT embryos, especially in the 8-cell stage, where 48 enzymes were significantly decreased (P&amp;lt;0.05). The down-regulation enzymes were mainly clustered in the histone H3K4 methyl transferase and histone acetylase. These results indicate that down-regulation of maternal TRIM28 breaks the steady-state of genomic methylation at a particular locus of the imprinted gene, disrupts the expression of imprinted gene and epigenetic modifications enzymes, and is detrimental to normal development of SCNT embryos. Maternal TRIM28 is needed in maintaining a stable state of genomic methylation and epigenetic modification state during SCNT embryo development.

Intrinsic and extrinsic molecular determinants or modulators for epigenetic remodeling and reprogramming of somatic cell-derived genome in mammalian nuclear-transferred oocytes and resultant embryos

The efficiency of somatic cell cloning in mammals remains disappointingly low. Incomplete and aberrant reprogramming of epigenetic memory of somatic cell nuclei in preimplantation nuclear- transferred (NT) embryos is one of the most important factors that limit the cloning effectiveness. The extent of epigenetic genome-wide alterations, involving histone or DNA methylation and histone deacetylation, that are mediated by histone-lysine methyltransferases (HMTs) or DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) can be modulated/reversed via exogenous inhibitors of these enzymes throughout in vitro culture of nuclear donor cells, nuclear recipient oocytes and/or cloned embryos. The use of the artificial modifiers of epigenomically-conditioned gene expression leads to inhibition of both chromatin condensation and transcriptional silencing the genomic DNA of somatic cells that provide a source of nuclear donors for reconstruction of enucleated oocytes and generation of cloned embryos. The onset of chromatin decondensation and gene transcriptional activity is evoked both through specific/selective inactivating HMTs by BIX-01294 and through non-specific/non-selective blocking the activity of either DNMTs by 5-aza-2'-deoxycytidine, zebularine, S-adenosylhomocysteine or HDACs by trichostatin A, valproic acid, scriptaid, oxamflatin, sodium butyrate, m-carboxycinnamic acid bishydroxamide, panobinostat, abexinostat, quisinostat, dacinostat, belinostat and psammaplin A. Epigenomic modulation of nuclear donor cells, nuclear recipient cells and/or cloned embryos may facilitate and accelerate the reprogrammability for gene expression of donor cell nuclei that have been transplanted into a host ooplasm and subsequently underwent dedifferentiating and re-establishing the epigenetically dependent status of their transcriptional activity during pre- and postimplantation development of NT embryos. Nevertheless, a comprehensive additional work is necessary to determine whether failures in the early-stage reprogramming of somatic cell-inherited genome are magnified downstream in development of cloned conceptuses and neonates.

H3K4 Methylation-Dependent Memory of Somatic Cell Identity Inhibits Reprogramming and Development of Nuclear Transfer Embryos

SummaryVertebrate eggs can induce the nuclear reprogramming of somatic cells to enable production of cloned animals. Nuclear reprogramming is relatively inefficient, and the development of the resultant embryos is frequently compromised, in part due to the inappropriate expression of genes previously active in the donor nucleus. Here, we identify H3K4 methylation as a major epigenetic roadblock that limits transcriptional reprogramming and efficient nuclear transfer (NT). Widespread expression of donor-cell-specific genes was observed in inappropriate cell types in NT embryos, limiting their developmental capacity. The expression of these genes in reprogrammed embryos arises from epigenetic memories of a previously active transcriptional state in donor cells that is characterized by high H3K4 methylation. Reducing H3K4 methylation had little effect on gene expression in donor cells, but it substantially improved transcriptional reprogramming and development of NT embryos. These results show that H3K4 methylation imposes a barrier to efficient nuclear reprogramming and suggest approaches for improving reprogramming strategies.

RE1-silencing Transcription Factor (REST) Is Required for Nuclear Reprogramming by Inhibiting Transforming Growth Factor β Signaling Pathway

Differentiated cells can be reprogrammed by transcription factors, and these factors that are responsible for successful reprogramming need to be further identified. Here, we show that the neuronal repressor RE1-silencing transcription factor (REST) is rich in porcine oocytes and requires for nuclear transfer (NT)-mediated reprogramming through inhibiting TGFβ signaling pathway. REST was dramatically degraded after oocyte activation, but the residual REST was incorporated into the transferred donor nuclei during reprogramming in NT embryos. Inhibition of REST function in oocytes compromised the development of NT embryos but not that of IVF and PA embryos. Bioinformation analysis of putative targets of REST indicated that REST might function on reprogramming in NT embryos by inhibiting TGFβ pathway. Further results showed that the developmental failure of REST-inhibited NT embryos could be rescued by treatment of SB431542, an inhibitor of TGFβ pathway. Thus, REST is a newly discovered transcription factor that is required for NT-mediated nuclear reprogramming.

Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development

Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.

Methyl-CpG-Binding Protein 2 Improves the Development of Mouse Somatic Cell Nuclear Transfer Embryos.

Methyl-CpG-binding domain proteins (MBPs) connect DNA methylation and histone modification, which are the key changes of somatic cell reprogramming. Methyl-CpG-binding protein 2 (MeCP2) was the first discovered MBP that has been extensively studied in the neurodevelopmental disorder Rett syndrome. However, a role for MeCP2 during cellular reprogramming associated with somatic cell nuclear transfer (SCNT) has not been examined. In this study, we discovered that MeCP2 expression was significantly lower in embryos generated by SCNT compared with those generated by intracytoplasmic sperm injection (ICSI). We genetically modified mouse embryonic fibroblasts (MEFs) to overexpress MeCP2 and serve as donor cells for nuclear transfer (NT) to investigate the effects of MeCP2 on preimplantation development of SCNT embryos. The blastocyst rate (35.71%) of MeCP2 overexpressed embryos (NT(+)) was significantly greater than in nontransgenic embryos (NT(-), 24.29%). Furthermore, immunofluorescence experiments revealed that 5-methylcytosine (5mC) was transferred to 5-hydroxymethylcytosine (5hmC) to a greater extent in NT(+) embryos than in NT(-) embryos. Real-time PCR evaluation of gene expression also showed that embryonic development-associated genes, such as Oct4 and Nanog, were significantly higher in the NT(+) group compared to the NT(-) group. Collectively, these results suggested that MeCP2 facilitated Tet3 activity, enhanced expression of pluripotency-related genes, and eventually improved the development of NT embryos. Finally, we performed chromatin immunoprecipitation to identify direct targets of MeCP2 and constructed a protein interaction network to elucidate several putative MeCP2 targets.

The expression of β-galactosidase during long-term cultured goat skin fibroblasts and the effect of donor cell passage on in vitro development of nuclear transfer embryos.

The present study aimed to detect the expression of β-galactosidase during long-term cultured goat skin fibroblasts and investigate the effects of donor goat age, sex, and cell passage on senescence and the effects of donor cell passage on in vitro development of nuclear transfer embryos. The results showed that, in the same cell passage, more β-galactosidase-positive cells were detected in cells from older donors than younger donors. Irrespective of the donor age, the number of positive cells was higher in later passages from passages 20 to 50. In the same passage from 20 to 50, the β-galactosidase-positive rate was higher in cells from 5-yr female goat than 5-yr male goat. Using fibroblasts from male goats at various passages as donor cells, reconstructed embryos had similar fusion and cleavage rates, but the blastocyst rate was higher for cells at passages 10 and 20 than passage 30. In conclusion, donor goat age and cell passage had significant effects on the β-galactosidase-positive rate; also, cells from 5-yr female goat had a higher β-galactosidase-positive rate than those from 5-yr male goat, and the donor cell passage affected the developmental potential of nuclear transfer embryos.

Ovine induced pluripotent stem cells are resistant to reprogramming after nuclear transfer.

Induced pluripotent stem cells (iPSCs) share similar characteristics of indefinite in vitro growth with embryonic stem cells (ESCs) and may therefore serve as a useful tool for the targeted genetic modification of farm animals via nuclear transfer (NT). Derivation of stable ESC lines from farm animals has not been possible, therefore, it is important to determine whether iPSCs can be used as substitutes for ESCs in generating genetically modified cloned farm animals. We generated ovine iPSCs by conventional retroviral transduction using the four Yamanaka factors. These cells were basic fibroblast growth factor (bFGF)- and activin A-dependent, showed persistent expression of the transgenes, acquired chromosomal abnormalities, and failed to activate endogenous NANOG. Nonetheless, iPSCs could differentiate into the three somatic germ layers in vitro. Because cloning of farm animals is best achieved with diploid cells (G1/G0), we synchronized the iPSCs in G1 prior to NT. Despite the cell cycle synchronization, preimplantation development of iPSC-NT embryos was lower than with somatic cells (2% vs. 10% blastocysts, p<0.01). Furthermore, analysis of the blastocysts produced demonstrated persistent expression of the transgenes, aberrant expression of endogenous SOX2, and a failure to activate NANOG consistently. In contrast, gene expression in blastocysts produced with the parental fetal fibroblasts was similar to those generated by in vitro fertilization. Taken together, our data suggest that the persistent expression of the exogenous factors and the acquisition of chromosomal abnormalities are incompatible with normal development of NT embryos produced with iPSCs.

Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

To improve nutrient content of goat milk, we describe the construction of a vector (pBLAC) containing a hybrid goat β-lactoglobulin (BLG) promoter/cytomegalovirus (CMV) enhancer. We also describe the generation of transgenic goats expressing rhLA by somatic cell nuclear transfer (SCNT). Of 334 one-cell stage embryos derived from three transgenic cell lines and 99 embryos derived from non-transgenic (NT) cells surgically transferred to the oviducts of 37 recipients, two recipients delivered two kids (2%) from the non-transfected line and five recipients delivered six kids (1.8%) from transgenic cell lines, three of which died within 2 days. Compared to the NT donor cells, transfection of donor cells does not negatively affect the development of nuclear transfer embryos into viable transgenic offspring. However, the clone efficiency in cell line number 1 was lower than that in numbers 2 and 3, and in the NT lines (0.9% versus 1.9% 2.4% and 2%; P < 0.05). Two transgenic cloned goats expressed rhLA in the milk at 0.1–0.9 mg/mL. The mammary gland-specific expression vector pBLAC with hybrid BLG/CMV can drive the hLA gene to express in vitro and in vivo. These data establish the basis for use of a hybrid promoter/enhancer strategy to produce rhLA transgenic goats.

ドナー細胞におけるRNA polymerase IIの局在が核移植胚の発生に与える影響

ドナー細胞におけるRNA polymerase IIの局在が核移植胚の発生に与える影響

XenopusEgg Extract Treatment Reduced Global DNA Methylation of Donor Cells and Enhanced Somatic Cell Nuclear Transfer Embryo Development in Pigs

The efficiency to produce offspring by somatic cell nuclear transfer (SCNT) is low. It has been showed that treatment of donor cells with Xenopus oocyte extract increased live births in ovine and handmade cloned embryo development in pigs. Scriptaid treatment after oocyte activation is another approach to improve SCNT efficiency. The present study was carried out to investigate (a) the effects of treatment of donor cells with Xenopus egg extract on donor cell DNA methylation at days 0 and 4 with two digitonin permeabilization concentrations (10 and 15 μg/mL), (b) the effects of treatment of donor cells with Xenopus egg extract on early development of cloned embryos, and (c) the effects of combined treatments, treating donor cells with extract before nuclear transfer and treatment of cloned embryos with scriptaid after oocyte activation, on embryo development. Compared to the control, a decrease of DNA methylation in donor cells was observed at 2.5 h after extract treatment. However, this effect was not observed after the cells were cultured for four more days. More embryos developed into blastocysts in the Xenopus egg extract-treated group than in the control (13.4±1.9% vs. 9.1±1.9%, p=0.01). Furthermore, scriptaid treatment of cloned embryos further increased the frequency of development to blastocyst, compared to the control reconstructed with the same extract-treated cells (22.5±0.9% vs. 15.3±0.9%, p<0.01). In addition, egg extract treatments increased the cell number in the blastocysts. This study demonstrated that Xenopus egg extract treatment reduced donor cell DNA methylation and enhanced the SCNT embryo development. Moreover, the combined treatments of donor cells with egg extract before nuclear transfer and of cloned embryos with scriptaid could improve cloned embryo development additively.

23 DEVELOPMENTAL POTENTIAL OF CLONED TRANSGENIC PORCINE EMBRYOS PRODUCED BY SERIAL NUCLEAR TRANSFER CAN BE IMPROVED BY TREATMENT WITH HISTONE DEACETYLASE INHIBITORS

Recently, we generated cloned transgenic pigs by nuclear transfer (NT) using fetal fibroblasts transfected with a LEA29Y gene specifically expressed in pancreatic β-cells (INS-LEA). Transfer of 216 NT embryos into 3 recipients resulted in the birth of 9 piglets. Furthermore, we examined serial NT with donor cells of the INS-LEA cloned pigs as a means of propagating the genotype of these valuable animals. Surprisingly, no piglets were obtained after transfer of 512 NT embryos into 5 recipients, which might be due to epigenetic alterations that presumably occurred during post-implantation development of the first round cloned embryos or during nuclear reprogramming in the second round of NT. In this study we tested whether in vitro development of re-cloned embryos can be improved by their treatment with histone deacetylase inhibitors (HDACi), scriptaid and suberoylanilide hydroxamic acid (SAHA). As nuclear donors, ear fibroblast cells derived from the INS-LEA cloned pig were used. Nuclear transfer was performed using in vitro-matured oocytes as previously reported (Kurome et al. 2006, Transgenic Res. 15, 229–240). After activation, reconstructed embryos were treated immediately by scriptaid (500 nM) and SAHA (10 μM) for 16 and 10 h, respectively. Development of NT embryos was assessed by cleavage and blastocyst formation during culture for 7 days. The cell number of blastocysts was also counted after fixation and staining. There was no significant difference in the cleavage rate between treated and non-treated by both HDACi, whereas treatment of NT embryos with scriptaid or SAHA significantly enhanced their development to blastocyst compared with non-treated NT embryos (22.2%, 43/194 and 22.7%, 34/150 vs 7.7%, 15/195 and 12.3%, 18/146, respectively; P &lt; 0.05). Notably, blastocyst rates obtained after treatment of re-cloned embryos with HDACi were similar to those in the first round of NT (21.2%, 33/156). Treatment of NT embryos with HDACi did not increase mean cell number of blastocysts compared with non-treated embryos. The results of our study show that in vitro developmental competence of embryos produced by serial NT can be improved by both HDACi used, scriptaid as well as SAHA, which has not been reported before in pig cloning. To determine the post-implantation developmental potential, re-cloned embryos treated with HDACi will be transferred to surrogate gilts. This work is supported by the DFG (FOR535, FOR793), the Bayerische Forschungsstiftung and Mukoviszidose e.V.

In vitro development of nuclear transfer embryos derived from porcine embryonic germ cells and their descendent neural precursor cells

Undifferentiated stem cells may support a greater development of cloned embryos compared with differentiated cell types due to their ease of reprogramming during the nuclear transfer (NT) process. Hence, stem cells may be more suitable as nuclear donor cells for NT procedures than are somatic cells. Embryonic germ (EG) cells are undifferentiated stem cells that are isolated from cultured primordial germ cells (PGC) and can differentiate into several cell types. In this study, the in vitro development of NT embryos using porcine EG cells and their derivative neural precursor (NP) cells was investigated, thus eliminating any variation in genetic differences. The rates of fusion did not differ between NT embryos from EG and NP cells; however, the rate of cleavage in NT embryos derived from EG cells was significantly higher (p < 0.05) than that from NP cells (141/247 [57.1%] vs. 105/228 [46.1%]). Similarly, the rate of blastocyst development was significantly higher (P < 0.05) in NT using EG cells than the rate using NP cells (43/247 [17.4%] vs. 18/228 [7.9%]). The results obtained from the present study in pigs demonstrate a reduced capability for nuclear donor cells to be reprogrammed following the differentiation of porcine EG cells. Undifferentiated EG cells may be more amenable to reprogramming after reconstruction compared with differentiated somatic cells.

Nuclear transfer procedures in the ovine can induce early embryo fragmentation and compromise cloned embryo development

Cytoplasmic fragmentations are frequently observed in early mammalian embryos, and especially in the human. In our research we have observed subtle clues that the occurrence of fragmentation was most likely a result of somatic cell nuclear transfer (NT) protocols, and in particular, the in vitro culture system. In this study we examined various putative factors that might induce early embryo fragmentation in the ovine. The results indicate that nuclear transfer protocols such as the fusion parameter, activation treatment, and especially the choice of culture medium affected embryo cleavage rates and resulted in a higher incidence of fragmented embryos. Upon using the same fusion parameter, activation parameters that were based upon amino acids containing synthetic oviduct fluids (SOFaa) culture system resulted in significantly lower fragmentation rates than when utilizing a Charles Rosenkrans 1 (CR1aa) culture system. Fragmented embryos typically exhibited irregular numbers of blastomeres with the majority of blastomeres devoid of chromatin. Factors such as fusion DC pulse, activation treatment and culture system led to higher fragmentation and also affected in vitro and in vivo embryo development. The SOFaa based culture system produced a higher number of quality NT embryos resulting in higher pregnancy rates and the birth of live lambs as compared to the CR1aa based system ( P < 0.05). We conclude that early embryo fragmentation in the ovine is caused by suboptimal cloning protocols, and NT embryo development is especially affected by the culture system used.

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos

Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti-DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer.