iSCNT Embryos Research Articles

Reprogramming of Human Somatic Cells Using Human and Animal Oocytes

There is renewed interest in using animal oocytes to reprogram human somatic cells. Here we compare the reprogramming of human somatic nuclei using oocytes obtained from animal and human sources. Comparative analysis of gene expression in morula-stage embryos was carried out using single-embryo transcriptome amplification and global gene expression analyses. Genomic DNA fingerprinting and PCR analysis confirmed that the nuclear genome of the cloned embryos originated from the donor somatic cell. Although the human-human, human-bovine, and human-rabbit clones appeared morphologically similar and continued development to the morula stage at approximately the same rate (39, 36, and 36%, respectively), the pattern of reprogramming of the donor genome was dramatically different. In contrast to the interspecies clones, gene expression profiles of the human-human embryos showed that there was extensive reprogramming of the donor nuclei through extensive upregulation, and that the expression pattern was similar in key upregulation in normal control embryos. To account for maternal gene expression, enucleated oocyte transcriptome profiles were subtracted from the corresponding morula-stage embryo profiles. t-Test comparisons (median-normalized data @ fc>4; p<0.005) between human in vitro fertilization (IVF) embryos and human-bovine or human-rabbit interspecies somatic cell transfer (iSCNT) embryos found between 2400 and 2950 genes that were differentially expressed, the majority (60-70%) of which were downregulated, whereas the same comparison between the bovine and rabbit oocyte profiles found no differences at all. In contrast to the iSCNT embryos, expression profiles of human-human clones compared to the age-matched IVF embryos showed that nearly all of the differentially expressed genes were upregulated in the clones. Importantly, the human oocytes significantly upregulated Oct-4, Sox-2, and nanog (22-fold, 6-fold, and 12-fold, respectively), whereas the bovine and rabbit oocytes either showed no difference or a downregulation of these critical pluripotency-associated genes, effectively silencing them. Without appropriate reprogramming, these data call into question the potential use of these discordant animal oocyte sources to generate patient-specific stem cells.

35 DEVELOPMENT OF INTERSPECIES CLONED MOUSE EMBRYOS RECONSTRUCTED WITH PORCINE ENUCLEATED OOCYTES

Interspecies somatic cell nuclear transfer (iSCNT) offers significant opportunities for the derivation of embryonic stem cells (ESCs) that can model diseases. In this study, we investigated the development of murine-porcine iSCNT embryos and compared these to outcomes associated with porcine-porcine SCNT embryos. Enucleation was performed on in vitro-matured porcine oocytes using the Oosight spindle viewer system (CRI, UK). Either murine embryonic fibroblasts or porcine cumulus cells were injected into porcine ooplasm using a piezo-electric micromanipulator. After being exposed to the porcine ooplasm for 1 h, the reconstructed oocytes were activated by a combination of A23187 and 6-DMAP for 3.5 h. For the murine-porcine embryos (n = 9375), we used a serial culture method, namely porcine zygote medium (PZM) for 48 h followed by incubation with KSOM for another 5 days. We found that the majority of murine-porcine embryos (73.58%) arrested at the 4- to 8-cell stage, whilst a very small minority progressed through to blastocyst (0.064%). Porcine-porcine SCNT reconstructed oocytes (n = 142) cultured in PZM achieved a 15% blastocyst development rate. In the murine-porcine embryos, 10/44 embryos had chromosomal DNA loss associated with apoptosis, as determined by immunocytochemistry using the H2A.X antibody. We also found that at later stages, morula to blastocyst, the mouse-to-pig reconstructed embryos did not exhibit murine-specific Oct-4 expression patterns. Furthermore, murine mitochondrial DNA tended to be eliminated rather than preserved by the murine nuclei, which could render any subsequently derived ESCs dysfunctional due to 2 diverse genomes contributing genes to the electron transfer chain.

158 IN VITRO DEVELOPMENT OF WOOD BISON (BISON BISON ATHABASCAE) EMBRYOS BY INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER

Wood bison (Bison bison athabascae) are currently classified as threatened in Canada. Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for embryo production in non-domestic species in which access to gametes is limited. Unlike fertilization, SCNT allows preservation of the entire genome, thus avoiding dilution of valuable alleles, an important factor for the preservation of genetic diversity. The present study compared the developmental competence of iSCNT embryos reconstructed from adult female wood bison ear fibroblasts (bison NT) with development of embryos reconstructed from adult female cattle ear fibroblasts (cattle NT). Domestic cattle (Bos taurus) oocytes were used as recipient ooplasm for both donor cell types. In vitro fertilized (IVF) and parthenogenetic (PA) cattle embryos were used as controls. Fibroblast cultures at passages 3 to 5 confluent for 5 days were used for SCNT. Mature oocytes were enucleated, reconstructed by transfer of donor cells, and fused with an electrical stimulus of 1.5 kV cm–1 for 40 μs in 0.28 m mannitol containing 100 μm CaCl2 and MgCl2. Oocytes for parthenogenesis and following reconstruction were activated for 5 min in 5 μm ionomycin followed by 5 h in 10 μg mL–1 cycloheximide. Embryos produced by IVF, PA, and SCNT were cultured in modified synthetic oviductal fluid medium at 38.5°C in 5% CO2, 5% O2, 90% N2. Cleavage, blastocyst development to day 8, apoptosis (TUNEL assay, Roche Diagnostics, IN, USA), and total cell number were evaluated. Statistical analyses were carried out using one-way ANOVA, followed by Tukey post hoc analysis or the equivalent nonparametrical Kruskal-Wallis test. Cleavage rate was significantly (P &lt; 0.05) higher in the IVF group than in all other groups (86.9 ± 2.9% v. 71.6 ± 4.5% to 78.1 ± 5.1%). Blastocyst rates, expressed as a percentage of cleaved embryos, were similar among all treatment groups (33.4 ± 3.3% to 39.8 ± 5.7%) except for bison NT which had significantly (P &lt; 0.05) lower development to blastocyst (19.2 ± 5.5%). The percentages of TUNEL-positive cells among PA embryos (6.6 ± 1.5%) and bison NT embryos (6.7 ± 2.4%) were significantly (P &lt; 0.05) higher than in IVF embryos (4.2 ± 1.0%), but similar to cattle NT embryos (5.4 ± 1.7%), which did not differ from the IVF group. Total cell number was significantly (P &lt; 0.05) higher in the IVF group than in all other groups (133.2 ± 10.2 v. 91.2 ± 7.8 to 100.1 ± 12.9). These results confirm that in vitro-matured domestic cattle oocytes can serve as suitable recipients of wood bison somatic cells and that iSCNT may provide a possible alternative for embryo production and genetic preservation of endangered cattle species. Both the incidence of apoptotic cells and total cell number did not differ between cattle and bison NT embryos; thus other factors must play a role in the significantly decreased blastocyst development observed in bison NT embryos. This work was supported by Endangered Species Reserve Fund, Toronto Zoo, and the Canada Research Chairs program.

In vitro Development of Interspecies Somatic Cell Nuclear Transfer Embryos Derived from Murine Embryonic Fibroblasts and Bovine Oocytes

Interspecies somatic cell nuclear transfer (iSCNT) is a useful method to preserve endangered species and to study the reprogramming event of a nuclear donor cell by the oocyte. Although several studies of iSCNT using murine cells and bovine oocytes have been reported, the development of murine-bovine iSCNT embryos beyond the 8-cell stage has not been successful. In this paper, we examined the developmental potential of embryos reconstructed with a murine embryonic fibroblast as the nuclear donor and a bovine oocyte as the cytoplasm recipient. The reconstructed embryos were cultured in CZB (murine medium) or CR1aa (bovine medium). In addition, for the development of a murine-bovine iSCNT blastocyst, the antioxidant β-mercaptoethanol (βME) was supplemented to CR1aa medium. Furthermore, to verify the mouse genome activation in murine-bovine iSCNT embryos, RT-PCR analysis of murine Xist was performed. The development of the murine-bovine iSCNT embryos cultured in CR1aa was significantly higher than that in CZB (p<0.05). With respect to the effect of BME on the development of the murine-bovine iSCNT blastocyst, addition of BME produced a significant increase in blastocyst development (p<0.05). Karyotype analysis confirmed that the reconstructed embryos were derived from murine cells (40XX). The Xist gene was gradually increased from the 8-cell stage to the blastocyst stage. This is the first report of blastocyst development of iSCNT embryos derived from murine somatic cells and bovine oocytes. These results demonstrate that bovine cytoplasm can support the development of later stages of a preimplantation embryo from murine-bovine iSCNT.

Trichostatin A (TSA) improves the development of rabbit‐rabbit intraspecies cloned embryos, but not rabbit‐human interspecies cloned embryos

The interspecies somatic cell nuclear transfer (iSCNT) technique for therapeutic cloning gives great promise for treatment of many human diseases. However, the incomplete nuclear reprogramming and the low blastocyst rate of iSCNT are still big problems. Herein, we observed the effect of TSA on the development of rabbit-rabbit intraspecies and rabbit-human interspecies cloned embryos. After treatment with TSA for 6 hr during activation, we found that the blastocyst rate of rabbit-rabbit cloned embryos was more than two times higher than that of untreated embryos; however, the blastocyst rate of TSA-treated rabbit-human interspecies cloned embryos decreased. We also found evident time-dependent histone deacetylation-reacetylation changes in rabbit-rabbit cloned embryos, but not in rabbit-human cloned embryos from fusion to 6 hr after activation. Our results suggest that TSA-treatment does not improve blastocyst development of rabbit-human iSCNT embryos and that abnormal histone deacetylation-reacetylation changes in iSCNT embryos may account for their poor blastocyst development.

Interspecies Nuclear Transfer Embryos Reconstructed from Cat Somatic Cells and Bovine Ooplasm

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

61 BOVINE OOPLASM PARTIALLY REMODELS CHIMPANZEE SOMATIC NUCLEI FOLLOWING SOMATIC CELL NUCLEAR TRANSFER

Interspecies somatic cell nuclear transfer (iSCNT) is a tool to address basic questions related to nucleus–cytoplasm interactions between different species. This approach may have practical applications, such as endangered species preservation. To date, live offspring have been obtained only when closely related species were used, such as cow/gaur, cow/buffalo, and domestic cat/wild cat. Several groups have reported preimplantation development of iSCNT embryos using a diverse collection of species; however, further development of these embryos either failed or was not reported. The aim of this study was to investigate developmental events that take place during reprogramming of a somatic nucleus in an enucleated oocyte from a distant species. Our experimental model was cow/chimpanzee iSCNT. We confirmed the donor cell genome origin in SCNT embryos by karyotyping. Embryonic genome activation (EGA) was monitored by Br-UTP incorporation assay and RT-PCR analysis of 6 genes. Chromatin remodeling events such as histone acetylation, histone methylation, and DNA methylation were observed by immunocytochemistry along with examination of Oct4 and Nanog promoter methylation by bisulfite sequencing. As an indicator of embryonic metabolism, ATP levels and apoptotic status of embryonic nuclei were determined by ATP detection and TUNEL assays, respectively. Development of iSCNT embryos did not progress beyond the 8- to 16-cell stage, whereas their cow/cow SCNT counterparts readily developed to the blastocyst stage. Karyotype analysis showed a donor-specific, diploid genomic content in most of the iSCNT nuclei (46 chromosomes). Major EGA takes place at the 16- and 8-cell stages in cow and chimpanzee embryos, respectively. However, our analyses showed a partial EGA pattern in iSCNT embryos at the 8-cell stage, as indicated by lower levels of Br-UTP incorporation and irregular expression of certain embryonic genes compared to that in cow/cow SCNT embryos. GAPDH, Oct4, and Stella transcripts were detected, while Nanog, Glut1, and DSC2 genes failed to be expressed in chimpanzee somatic nuclei, as opposed to robust expression observed for all 6 genes in bovine SCNT embryos. Dynamic chromatin remodeling events as monitored by H3K27 methylation and H4K5 acetylation-specific antibodies were similar in both intra- and interspecies SCNT embryos from 1-cell to 8–16-cell stages. A global demethylation pattern was observed in iSCNT embryos from 1-cell to 8–16-cell stages, which was not significantly different from that of bovine SCNT embryos by immunocytochemistry. However, bisulfite sequencing indicated incomplete demethylaton of Oct4 and Nanog promoters in 8-cell iSCNT embryos, unlike the complete demethylation of the same promoter regions observed in their bovine intraspecific counterparts. ATP levels were significantly higher in bovine SCNT embryos than in iSCNT embryos; TUNEL assay did not reveal any difference in apoptotic status of the nuclei from both types of embryos. Collectively, our results suggest that bovine ooplasm can partially remodel chimpanzee somatic nuclei. However, the remodeling activity was not sufficient to drive embryo development past the 8–16-cell stage.

Development of Interspecies Cloned Monkey Embryos Reconstructed with Bovine Enucleated Oocytes

This study was carried out to determine whether culture media reconstructed with bovine enucleated oocytes and the expression pattern of Oct-4 could support dedifferentiaton of monkey fibroblasts in interspecies cloned monkey embryos. In this study, monkey and bovine skin fibroblasts were used as donor cells for reconstruction with bovine enucleated oocytes. The reconstructed monkey interspecies somatic cell nuclear transfer (iSCNT) embryos were then cultured under six different culture conditions with modifications of the embryo culture media and normal bovine and monkey specifications. The Oct-4 expression patterns of the embryos were examined at the two-cell to blastocyst stages using immunocytochemistry. The monkey iSCNT embryos showed similar cleavage rates to those of bovine SCNT and bovine parthenogenetic activation (PA). However, the monkey iSCNT embryos were not able to develop beyond the 16-cell stage under any of the culture conditions. In monkey and bovine SCNT embryos, Oct-4 could be detected from the two-cell to blastocyst stage, and in bovine PA embryos, Oct-4 was detectable from the morula to blastocyst stage. These results suggested that bovine ooplasm could support dedifferentiation of monkey somatic cell nuclei but could not support embryo development to either the compact morula or blastocyst stage. In conclusion, we found that the culture conditions that tend to enhance monkey iSCNT embryo development and the expression pattern of Oct-4 in cloned embryos (monkey iSCNT and bovine SCNT) are different than in bovine PA embryos.

Expression of enhanced green fluorescent protein in porcine‐ and bovine‐cloned embryos following interspecies somatic cell nuclear transfer of fibroblasts transfected by retrovirus vector

Interspecies somatic cell nuclear transfer (iSCNT) has emerged as an important tool for studying nucleo-cytoplasmic interactions and cloning of animals whose oocytes are difficult to obtain. This study was designed to explore the feasibility of employing transgenic fibroblasts as donor cells for iSCNT. The study examined the chromatin morphology, in vitro development, and expression of an enhanced green fluorescent protein (EGFP) gene in porcine- and bovine-cloned embryos produced by iSCNT of fetal fibroblast transfected with a pLNbeta-EGFP retroviral vector. Parthenogenetic and transfected or nontransfected intraspecies SCNT embryos were used as controls for comparison. Analysis of data revealed that xenogenic oocyte was able to reprogram somatic cells of different genus and supports their in vitro development to the blastocyst stage. However, the developmental rates of transgenic iSCNT embryos to the blastocyst stage were significantly lower than those of intraspecies SCNT embryos. The reduction in development rates was however, not due to integration of the transgene as the lower (P < 0.05) development rates of the intraspecies SCNT porcine or bovine embryos did not differ between transgenic and nontransgenic groups. Expression of EGFP was observed in 100% of blastocysts and mosaicism was not observed. Furthermore, after iSCNT of porcine or bovine donor nuclei into xenogenic ooplasm, patterns of nuclear remodeling in reconstructed embryos were similar. In conclusion, our data demonstrated the feasibility of producing transgenic iSCNT embryos. To our knowledge, this is the first report of transgenic cloned embryo production by iSCNT approach. In the future, this may provide a powerful research tool for studying developmental events in domestic animals and provide marked cell lines for other genetic manipulations.

Development of bovine–ovine interspecies cloned embryos and mitochondria segregation in blastomeres during preimplantation

The objective of the study was to investigate interspecies somatic cell nuclear transfer (iSCNT) embryonic potential and mitochondrial DNA (mtDNA) segregation during preimplantation development. We generated bovine–ovine reconstructed embryos via iSCNT using bovine oocytes as recipient cytoplasm and ovine fetal fibroblast as donor cells. Chromosome composition, the total cell number of blastocyst and embryonic morphology were analyzed. In addition, mtDNA copy numbers both from donor cell and recipient cytoplasm were assessed by real-time PCR in individual blastocysts and blastomeres from 1- to 16-cell stage embryos. The results indicated the following: (1) cell nuclei of ovine fetal fibroblasts can dedifferentiate in enucleated bovine ooplasm, and the reconstructed embryos can develop to blastocysts. (2) 66% of iSCNT embryos had the same number of chromosome as that of donor cell, and the total cell number of iSCNT blastocysts was comparable to that of sheep parthenogenetic blastocysts. (3) RT-PCR analysis in individual blastomeres revealed that the ratio of donor cell mtDNA: recipient cytoplasm mtDNA remained constant (1%) from the one- to eight-cell stage. However, the ratio decreased from 0.6% at the 16-cell stage to 0.1% at the blastocyst stage. (4) Both donor cell- and recipient cytoplasm-derived mitochondria distributed unequally in blastomeres with progression of cell mitotic division. Considerable unequal mitochondrial segregation occurred between blastomeres from the same iSCNT embryos.

In vitro development of reconstructed ibex ( Capra ibex) embryos by nuclear transfer using goat ( Capra hircus) oocytes

The ibex ( Capra ibex) is currently listed as a priority protected animal by the Chinese government. Due to the limited availability of ibex oocytes, the objective of this study was to explore the feasibility of using domestic goat ( Capra hircus) cytoplasts to reprogram nuclei from ibex donor fibroblasts in interspecies nuclear transfer (iSCNT). An effort was thus made to produce ibex embryos via iSCNT. The trial was performed using three treatment groups. The embryo cleavage rates on day 3 of in vitro culture and the blastocyst development rates on day 7 were recorded. In Treatment A, the female ibex fibroblasts were used as donor cells and of the fused cell-cytoplast couplets, 68% cleaved and 11% reached the blastocyst stage. In Treatment B, domestic adult dairy doe fibroblasts were used as donor cells and as comparison of homologous nuclear transfer (NT). Of fused cell-cytoplast couplets, 76% cleaved and 31% reached the blastocyst stage. In Treatment C, the development of parthenogenetic goat oocytes was used as the control and 90% of the embryos cleaved and 43% reached the blastocyst stage. No significant difference was found between treatment groups A and B in embryo cleavage rate, but a significant difference ( P < 0.01) was recorded between treatment groups A and B in the blastocyst production rate. There was a significant difference ( P < 0.01) between Treatment C and Treatment A/B in embryo cleavage rate and blastocyst production rate. The results indicated that although both cleavage rate (68%) and blastocyst yield (11%) of iSCNT embryos derived from female ibex fibroblasts (Treatment A) were lower than those of nuclear transfer embryos (Treatment B) and parthenogenetic development embryos (Treatment C). The domestic goat ( C. hircus) cytoplasts supported the mitotic cleavage of ibex karyoplasts and were capable of reprogramming the nucleus to achieve a blastocyst stage embryo in exotic Capra which has the potential of alleviating species on the endangered list.

56 DEVELOPMENT OF INTERSPECIES CLONED EMBRYOS USING SOMATIC CELLS FROM VARIOUS SPECIES AND BOVINE CYTOPLASTS

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleus–cytoplasm interaction and it provides a possible alternative to cloning animals whose oocytes are limited. In Experiment 1 of the present study, we investigated the developmental potential of iSCNT embryos created from monkey, pig, and goat donor cells and bovine cytoplasts. Bovine ovaries were obtained at a local slaughterhouse and the cumulus-oocyte complexes (COCs) aspirated. COCs were matured in vitro in TCM-199 supplemented with 10 IU mL–1 pregnant mare serum gonadotropin (PMSG), 10 IU mL–1 hCG, and 10 ng mL–1 epidermal growth factor (EGF) at 38.5�C and 5% CO2 in air for 20–22 h. At the end of IVM, half of the COCs were inseminated using frozen semen (1 � 106 sperm mL–1) and the remainder were used for iSCNT after the cumulus cells were removed with 0.1% hyaluronidase in TCM-199. The procedure of iSCNT and establishment of donor cells were according to Koo et al. (2002 Biol. Reprod. 67, 487–492). After IVF and iSCNT, presumptive zygotes were cultured in CR1-aa medium supplement with 0.3% BSA. After 3 days, cleaved embryos were transferred to CR1-aa medium supplemented with 10% FBS and cultured for an additional 4 days. In Experiment 2, we investigated the developmental ability of reconstructed embryos produced from monkey cells and bovine cytoplasts using various IVC media, such as IVC-1/2 (InVitroCare, Frederick, MD, USA), G-1/2 (Vitrolife, Inc., Englewood, CO, USA) and complete medium (CM; Irvine Scientific, Santa Clara, CA, USA). All experiments were repeated more than three times and data were analyzed with t-test of one-way ANOVA using the SAS 8.01 program (SAS Institute, Inc., Cary, NC, USA). Cleavage and developmental rate of blastocysts were expressed as mean � SEM. In Experiment 1, we investigated the development ability among IVF, SCNT (bovine-bovine), and iSCNT (monkey-bovine, pig-bovine, and goatbovine) embryos cultured in CR1-aa medium. Our results showed that the cleavage rate of IVF (73.6 � 1.8%, 86/117) embryos was not significantly different compared to SCNT (84.6 � 2.7%, 38/45), and iSCNT (89.3 � 2.7%, 100/110, monkey; 89.3 � 3.3%, 45/49, pig; and 86.0 � 2.3%, 87/95, goat). Although cloned embryos reconstructed with monkey cells did not develop to the blastocyst stage, iSCNT embryos derived from pig and goat cells did (3.3 � 3.0%, 2/49, and 7.9 � 1.7%, 7/95, respectively). However, these blastocyst formation rates were significantly lower compared to those of IVF and SCNT bovine embryos (32.5 � 2.9%, 38/117, and 26.7 � 2.8%, 12/88, respectively; P &lt; 0.05). The success of iSCNT was confirmed by PCR of mitochondrial DNA, porcine PKA region, and SRY region. In Experiment 2, we investigated the developmental potential of cloned embryos produced by monkey cells using various IVC media (IVC-1/2, G-1/2, and CM). The cleavage rate of iSCNT embryos was not significantly different among these media (86.9 � 2.7%, 78.1 � 2.1%, and 82.3 � 1.8%, respectively). However, we did not observe blastocyst formation using these media. Therefore, we suggest that the cytoplasts of bovine oocytes can support blastocyst development of cloned embryos with pig and goat cells, but they were not suitable for monkey cells. In conclusion, our results suggest that species-specific differences are apparent in the production of iSCNT embryos.

Bovine Ooplasm Partially Remodels Primate Somatic Nuclei following Somatic Cell Nuclear Transfer

Interspecies somatic cell nuclear transfer (iSCNT) has the potential to become a useful tool to address basic questions about the nucleus-cytoplasm interactions between species. It has also been proposed as an alternative for the preservation of endangered species and to derive autologous embryonic stem cells. Using chimpanzee/ bovine iSCNT as our experimental model we studied the early epigenetic events that take place soon after cell fusion until embryonic genome activation (EGA). Our analysis suggested partial EGA in iSCNT embryos at the eight-cell stage, as indicated by Br-UTP incorporation and expression of chimpanzee embryonic genes. Oct4, Stella, Crabp1, CCNE2, CXCL6, PTGER4, H2AFZ, c-MYC, KLF4, and GAPDH transcripts were expressed, while Nanog, Glut1, DSC2, USF2, Adrbk1, and Lin28 failed to be activated. Although development of iSCNT embryos did not progress beyond the 8- to 16-cell stage, chromatin remodeling events, monitored by H3K27 methylation, H4K5 acetylation, and global DNA methylation, were similar in both intra- and interspecies SCNT embryos. However, bisulfite sequencing indicated incomplete demethylation of Oct4 and Nanog promoters in eight-cell iSCNT embryos. ATP production levels were significantly higher in bovine SCNT embryos than in iSCNT embryos, TUNEL assays did not reveal any difference in the apoptotic status of the nuclei from both types of embryos. Collectively, our results suggest that bovine ooplasm can partially remodel chimpanzee somatic nuclei, and provides insight into some of the current barriers iSCNT must overcome if further embryonic development is to be expected.

62 EXPRESSION AND DISTRIBUTION OF Oct-4 IN INTERSPECIES-CLONED LONG-TAILED MONKEY (MACACA FASCICULARIS) EMBRYOS

The transcription factor Oct-4 is one of the important factors for early embryonic development and differentiation in several animal species such as monkey (Mitalipov et al. 2003 Biol. Reprod. 69, 1785–1792) and bovine (Kurosaka et al. 2004 Biol. Reprod. 71, 1578–1582). However, Oct-4 expression and distribution in interspecies somatic cell nuclear transfer (iSCNT) embryo have not yet been studied. Therefore, this study was undertaken to evaluate the Oct-4 distribution in monkey iSCNT embryos at the 2-cell (2C), 4-cell (4C), 8-cell (8C), 16-cell (16C)/morula, hatching (Day 7), and hatched blastocyst stages (Day 8), compared with bovine SCNT embryos. The skin fibroblasts from adult monkey (Macaca fascicularis) and bovine were used as donor cells, and bovine enucleated oocytes were used as recipient cytoplasts. SCNT procedures were performed as previously reported (Laowtammathron et al. 2005 Theriogenology 64, 1185–1196). The result was that monkey iSCNT embryos have cleavage (93.8%; 76/81) and development rates up to the 8-cell stage (76.5%; 62/81) similar to those in bovine SCNT (96.0%; 96/100, and 89.0%; 89/100, respectively). Bovine SCNT developed to morulae (61.5%; 59/96) and blastocysts (41.7%; 40/96) at a higher rate than monkey iSCNT (43.4%; 33/76, and 11.8%; 9/76, respectively). Moreover, bovine SCNT blastocysts showed inner cell mass (ICM) and trophectoderm (TE), but monkey iSCNT embryos formed a blastocoel and ICM and TE could not be clearly seen. Five embryos from each group were used to study Oct-4 expression and distribution by immunocytochemistry. The embryos were incubated with 1 : 1000 anti-mouse Oct-4 polyclonal antibodies (Chemicon International, Inc., Temecula, CA, USA), then rinsed in PBS, and incubated with 1 : 200 FITC-labeled antimouse IgG (Sigma-Aldrich Corp., St Louis, MO, USA), followed by 2 �g mL-1 422-6-Diamidino-2-phenylindole (DAPI) (Sigma); the embryos were then mounted and examined by fluorescence microscopy. The results showed Oct-4 staining in every nucleus of bovine and monkey iSCNT embryos at the 2C, 4C, 8C, and 16C/morula stages but was varied in blastocysts. The Oct-4 staining was reduced in TE cells when half of the embryonic cells hatched out of the zona pellucida (60.0%; 3/5). The Oct-4 staining continued to decrease in the TE of hatched blastocysts (Day 8) but did not affect the ICM. The total cell number of bovine SCNTs at blastocyst stage were 140.9 � 13.2 and, of these, 13.5% (19/140) showed apoptotic nuclei morphologies, whereas the cell numbers of monkey iSCNT early blastocysts had only 15.6 � 2.3 cells and, of these, 19.2% (3/15) showed apoptotic nuclei. It can be concluded from this study that monkey iSCNT embryos show Oct-4 staining in every intact nucleus.

Interspecies nuclear transfer reveals that demethylation of specific repetitive sequences is determined by recipient ooplasm but not by donor intrinsic property in cloned embryos

DNA methylation/demethylation of donor genomes in recipient ooplasm after nuclear transfer occurs in a species-specific way. In cloned rabbit and bovine embryos, repetitive sequences maintain the donor-type methylation status, but typical demethylation of repetitive sequences takes place in cloned porcine embryos. To clarify whether the demethylation is controlled by donor nucleus intrinsic property or by recipient ooplasm, we used interspecies somatic cell nuclear transfer (iSCNT) model to examine the methylation status of repetitive sequences in pig-to-rabbit and rabbit-to-pig interspecies embryos. We found that no demethylation of pig repetitive sequences was observed in pig-to-rabbit iSCNT embryos, while the examined rabbit repetitive sequence Rsat IIE was demethylated in rabbit-to-pig iSCNT embryos. These results indicate that demethylation of donor repetitive sequences is determined by ooplasm but not by donor intrinsic property and that ooplasm from different species have different capabilities to demethylate genes.

In vitro culture and mtDNA fate of ibex–rabbit nuclear transfer embryos

Rabbit oocyte can be used as the recipient in interspecies somatic cell nuclear transfer (iSCNT). This work was undertaken in order to study the developmental competence of Capra ibex somatic cells reprogrammed by rabbit oocytes and the fate of mitochondria in iSCNT embryos. Metaphase II (MII) oocytes from superovulated rabbit were used as nuclear recipients. The nuclear donors were Capra ibex somatic cells with different proliferative status: population doubling time (PDL) = 15 +/- 2 (group 1), 35 +/- 2 (group 2), 55 +/- 2 (group 3) and 70 +/- 2 (group 4). Oocytes reconstructed with electrical pulses (2.1kV/cm, 10 micros, 2 times) were activated (1.4kV, 20 micros, 2 times) and then cultured in Medium199 containing 10% fetal bovine serum at 38.5 degrees C, 5% CO2 in air. In groups 1, 2, 3 and 4, the fusion rates were 35.83%, 66.03%, 65.40% and 35.35%, respectively. Similar cleavage rates were observed among the four groups. However, the developmental potential to morula/blastocyst from early nuclear donor embryos (16.42%/10.45%) was significantly higher (p < 0.05) than in terminal donor embryos (9.52%/3.81%). Polymerase chain reaction analysis of the mitochondrial (mt) DNA cytb gene demonstrated that mtDNAs from ibex and rabbit could be detected at various developmental stages before implantation. In conclusion, our results provide some original information about rescuing Capra ibex using the iSCNT technique. These results indicate that: (1) enucleated rabbit oocytes make Capra ibex fibroblast nuclei reprogramme; (2) the proliferative status of donor cells affects the efficiency of iSCNT; and (3) rabbit ooplasm rescues the donor-derived mtDNAs, resulting in mtDNA heteroplasmy before implantation.

Combined activation treatments of recipient cytoplasts prior to nuclear transfer improves development of cloned embryos

The ibex (Capra ibex) is currently listed as a priority protected animal by the Chinese government. Due to the limited availability of ibex oocytes, the objective of this study was to explore the feasibility of using domestic goat (Capra hircus) cytoplasts to reprogram nuclei from ibex donor fibroblasts in interspecies nuclear transfer (iSCNT). An effort was thus made to produce ibex embryos via iSCNT. The trial was performed using three treatment groups. The embryo cleavage rates on day 3 of in vitro culture and the blastocyst development rates on day 7 were recorded. In Treatment A, the female ibex fibroblasts were used as donor cells and of the fused cell-cytoplast couplets, 68% cleaved and 11% reached the blastocyst stage. In Treatment B, domestic adult dairy doe fibroblasts were used as donor cells and as comparison of homologous nuclear transfer (NT). Of fused cell-cytoplast couplets, 76% cleaved and 31% reached the blastocyst stage. In Treatment C, the development of parthenogenetic goat oocytes was used as the control and 90% of the embryos cleaved and 43% reached the blastocyst stage. No significant difference was found between treatment groups A and B in embryo cleavage rate, but a significant difference (P < 0.01) was recorded between treatment groups A and B in the blastocyst production rate. There was a significant difference (P < 0.01) between Treatment C and Treatment A/B in embryo cleavage rate and blastocyst production rate. The results indicated that although both cleavage rate (68%) and blastocyst yield (11%) of iSCNT embryos derived from female ibex fibroblasts (Treatment A) were lower than those of nuclear transfer embryos (Treatment B) and parthenogenetic development embryos (Treatment C). The domestic goat (C. hircus) cytoplasts supported the mitotic cleavage of ibex karyoplasts and were capable of reprogramming the nucleus to achieve a blastocyst stage embryo in exotic Capra which has the potential of alleviating species on the endangered list.