Bovine nuclear transfer using fresh cumulus cell nuclei and in vivo- or in vitro-matured cytoplasts

We examined the effects of the source of recipient oocytes and timing of fusion and activation on the development competence of bovine nuclear transferred (NT) embryos derived from fresh cumulus cells isolated immediately after collection by ovum pickup (OPU). As recipient cytoplasts, we used in vivo-matured oocytes collected from hormone-treated heifers by OPU, or in vitro-matured oocytes from slaughterhouse-derived ovaries. NT embryos were chemically activated immediately (simultaneous fusion and activation, FA) or 2 h (delayed activation, DA) after fusion. When in vitro-matured oocytes were used as recipient cytoplasts, the development rate to the blastocyst stage of NT embryos produced by the DA method (23%) tended to be higher than those by the FA method (15%), but the difference was not significant. NT embryos derived from in vivo-matured cytoplasts have a high blastocyst yield (46%). Pregnancy rate at day 35 did not differ with the timing of fusion and activation (FA vs. DA; 50% vs. 44%) or oocyte source (in vivo- vs. in vitro-matured; 50% vs. 44%). Subsequently, the high fetal losses (88% of pregnancies) were observed with in vitro-matured cytoplasts, whereas no abortions were observed in NT fetuses from in vivo-matured cytoplasts. A total of three embryos derived from fresh cumulus cells developed to term. However, all three cloned calves were stillborn. These results indicate that improvement of development competence after NT is possible by using in vivo-matured oocytes as recipient cytoplasts in bovine NT.     

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.     

Somatic cell nuclear transfer in domestic cat oocytes treated with IGF-I for in vitro maturation

Oocyte maturation and somatic cell nuclear transfer (NT) studies conducted in the domestic cat can provide valuable insights that are relevant to the conservation of endangered species of felids. The present investigation focuses on the in vitro maturation (IVM) of domestic cat oocytes stimulated by insulin-like growth factor-I (IGF-I) and their possible use as recipient cytoplasts for somatic cell NT. In Experiment I, the effects of IGF-I on cat oocyte IVM were monitored. Cumulus-oocyte complexes (COCs) were recovered in TALP-HEPES medium following ovarian follicular aspiration and were classified under a stereomicroscope into four grades using criteria based on cumulus cell investment and the uniformity of ooplasm. The COCs were either cultured in Dulbecco's modified Eagle medium (DMEM) alone as a control group or supplemented with 100 ng/ml IGF-I. After culturing for 32-34 h, oocytes were denuded and maturation rate was evaluated by observing the extrusion of the first polar body and staining with aceto-orcein. The percentages of maturation of Grades 1 and 2 oocytes were significantly increased (P<0.05) in IGF-I supplemented medium compared with medium alone (85.8 versus 65.5 and 70.3 versus 51.8, respectively) whereas the maturation rates of Grades 3 and 4 oocytes were not different. The IVM of Grade 1 oocytes was significantly higher (P<0.05) than for all other grades in both control and experimental groups. In Experiment II, the in vitro development of cat NT embryos using cumulus cells, fetal or adult fibroblasts as donor nuclei was investigated. The IVM oocytes in medium containing IGF-I were enucleated and fused with cumulus cells, fetal or adult fibroblasts between passages 2 and 4 of culture. Reconstructed embryos were cultured and monitored every 24h for progression of development through Day 9. There was no significant difference in the percentage of fusion of NT embryos using different donor nuclei whereas the cleavage rates of NT embryos reconstructed with fetal fibroblasts and cumulus cells were significantly higher (P<0.05) than those reconstructed with adult fibroblasts (72.5 and 70.7% versus 54.8%, respectively). Development of NT embryos reconstructed with adult fibroblast to the morula stage was significantly lower (P<0.05) compared with cumulus cell or fetal fibroblast donor cells (25.8% versus 37.9 or 47.5%, respectively). However, no difference was observed in development to the blastocyst stage. These results demonstrated that IGF-I promoted the IVM of domestic cat oocytes. The enucleated IVM oocytes could be used as recipient cytoplasm for fetal and adult somatic cell nuclei resulting in the production of cloned cat embryos.     

Xenonuclear transplantation of buffalo (Bubalus bubalis) fetal and adult somatic cell nuclei into bovine (Bos indicus) oocyte cytoplasm and their subsequent development

Bovine oocyte cytoplasm has been shown to support the development of nuclei from other species up to the blastocyst stage. Somatic cell nuclei from buffalo fetal fibroblasts have been successfully reprogrammed after transfer to enucleated bovine oocytes, resulting in the production of cloned buffalo blastocysts. The aim of this study was to compare the in vitro development of fetal and adult buffalo cloned embryos after the fusion of a buffalo fetal fibroblast, cumulus or oviductal cell with bovine oocyte cytoplasm. The fusion of oviductal cells with enucleated bovine oocytes was higher than that of fetal fibroblasts or cumulus cells (83% versus 77 or 73%, respectively). There was a significantly higher cleavage rate (P < 0.05) for fused nuclear transferred embryos produced by fetal fibroblasts and oviductal cells than for cumulus cells (84 or 78% versus 68%, respectively). Blastocyst development in the nuclear transferred embryos produced by fetal fibroblasts was higher (P < 0.05) than those produced either by cumulus or oviductal cells. Chromosome analysis of cloned blastocysts confirmed the embryo was derived from buffalo donor nuclei. This study demonstrates that nuclei from buffalo fetal cells could be successfully reprogrammed to develop to the blastocyst stage at a rate higher than nuclei from adult cells.     

Development of embryos reconstructed by interspecies nuclear transfer of adult fibroblasts between buffalo (Bubalus bubalis) and cattle (Bos indicus)

The objective of this study was to explore the feasibility of employing adult fibroblasts as donor cells in interspecies nuclear transfer (NT) between buffaloes and cattle. Buffalo and bovine oocytes matured in vitro for 22 h were enucleated by micromanipulation using the Spindle View system. An ear fibroblast, pretreated with 0.1 microg/mL aphidicolin for 24 h, followed by culture for 2-9 days in Dulbecco's Modified Eagle's Media+0.5% fetal bovine serum, was introduced into the cytoplast by microinjection. Reconstructed oocytes were activated by exposure to 5 microM ionomycin for 5 min and 2 mM 6-dimethylaminopurine for 3 h. When buffalo adult fibroblasts were used as donor cells, there were no differences (P < 0.75) in the cleavage rate (66.2% versus 64.0%) between bovine and buffalo recipient oocytes, but more embryos derived from bovine cytoplasts developed to blastocysts than from buffalo cytoplasts (13.3% versus 3.0%, P < 0.05). When bovine adult fibroblasts were used as donor nuclei, both cleavage rate (45.3%) and blastocyst yield (4.5%) of NT embryos derived from buffalo cytoplasts were lower than those of NT embryos derived from bovine cytoplasts (65.5 and 11.9%, P < 0.05). The proportion of parthenogenetic buffalo (29.1%) or bovine (35.6%) oocytes developing to blastocysts was higher than those of NT embryos (P < 0.01). Interspecies NT embryos were derived from the donor cells and 55.0-61.9% of them possessed a normal diploid karyotype. In conclusion, embryos reconstructed by interspecies NT of adult fibroblasts between buffaloes and cattle developed to blastocysts, but bovine cytoplasts may direct embryonic development more effectively than buffalo cytoplasts, regardless of donor cell species.     

in vitro development of porcine enucleated oocytes reconstructed by the transfer of porcine fetal fibroblasts and cumulus cells

In the pig little information is available on cytoplasmic events during the reprogramming of oocytes reconstructed with somatic nuclei. The present study was conducted to determine the developmental potential of porcine cumulus cells (CC) and fetal fibroblasts (FF) after they were transferred into enucleated oocytes. Non-quiescent FF were fused to the enucleated oocytes using electrical pulse, whereas CC were directly injected into the oocytes. Transferred nuclei from both CC and FF underwent premature chromosome condensation (PCC), nuclear swelling and pronucleus formation. The remodeled oocytes developed to the mitotic and 2-cell stage at 18 to 24 h after nuclear transfer. The pattern of nuclear remodeling was similar regardless of the sources of karyoplasts or nuclear transfer methods. However, using FF, 24% of nuclear transferred embryos developed to the morula or blastocyst stage, whereas only 8% of those using CC developed to the morula or blastocyst stage. These results suggest that porcine oocyte cytoplasm can successfully reprogram somatic cell nuclei and support the development of nuclear transferred embryos to the blastocyst stage.     

Development to the blastocyst stage of porcine somatic cell nuclear transfer embryos reconstructed by the fusion of cumulus cells and cytoplasts prepared by gradient centrifugation

The present study was designated to examine the possibility of producing somatic cell nuclear transfer (SCNT) embryos in pigs using oocyte cytoplasm fragments (OCFs), prepared by centrifugations, as recipient cytoplasts. In Experiment 1, in vitro matured oocytes were centrifuged at 13,000 x g for 3, 6, and 9 min to stratify the cytoplasm, and then the oocytes were freed from zona pellucida and recentrifuged at 5,000 x g for 4 sec in Percoll gradient solution to produce OCFs as the source of recipient cytoplasts. It was found that a long duration of the first centrifugation tends to produce large-sized OCFs after the second centrifugation. In Experiment 2, two or three cytoplasts without chromosomes were aggregated, and then they were fused with a cumulus cell to produce SCNT embryos. The results showed that 66.4 +/- 9.4% of the reconstructed embryos underwent premature chromosome condensation at 1 h after activation, and 85.2 +/- 7.1% and 61.6 +/- 7.0% of them had pseudopronuclei at 10 and 24 h after activation, respectively. In Experiment 3, when SCNT embryos reconstructed by the fusion of three cytoplasts and one cumulus cell, a significantly higher (p < 0.05) rate of reconstructed embryos developed to the blastocyst stage (10.6 +/- 1.8%) than that of reconstructed with two cytoplasts and one cumulus cell (5.2 +/- 1.5%). These results indicate that cytoplasts obtained by two centrifugations can support the remodeling of a transferred somatic nucleus, resulting in the development of the reconstructed porcine embryos to the blastocyst stage.     

Cell synchronization for the purposes of nuclear transfer in the bovine

We have examined the reprogramming ability of donor fibroblast nuclei in various phases of the cell cycle, upon transfer to cytoplasts, using a bovine nuclear transfer (NT) model. Bovine fetal fibroblasts were cultured in reduced serum and conditioned medium to induce quiescence (G0) and treated with nocodazole to induce M phase arrest. Unsynchronized actively dividing cells (control) were mainly in G1. Cells synchronized in G0, M, and G1 phase were transferred to enucleated bovine MII oocytes by direct injection using the Piezo-Drill microinjector. NT oocytes were artificially activated following injection. Cells at the M phase were also transferred to enucleated oocytes after artificial activation. Cells induced into quiescence by serum starvation and unsynchronized donor cells produced the highest rates of development to the morula/blastocyst stage (20% and 18%, respectively). Development to blastocyst was significantly higher in parthenogenetic controls compared to NT embryos. The transfer of M phase nuclei to MII cytoplasts was not associated with high development to the blastocyst stage. Nevertheless, determining the viability of these embryos requires transfer to recipient animals and assessment of in vivo development.     

Telophase-stage host ooplasts support complete reprogramming of roscovitine-treated somatic cell nuclei in cattle

Nuclear-cytoplasmic incompatibilities are known to play a significant role in the developmental outcome of embryos produced by nuclear transfer, particularly when metaphase arrested oocytes are used as hosts for interphase donor nuclei. To further our understanding of how cell cycle coordination affects somatic cell cloning, somatic cells at different stages of the cell cycle were fused to host oocytes either before (metaphase II, M-II) or after (telophase II, T-II) activation. To obtain cells at different stages of the cell cycle, fetal fibroblast (FF) and granulosa cells (GC) were treated with roscovitine, an inhibitor of cyclin-dependent kinases (CDKs) resulting in a large percentage of cells in S/G(2)-phase. In contrast to the M-II group, which did better with confluent cells, embryos reconstructed with T-II cytoplasts resulted in higher rates of blastocyst formation when fused to cells recovered at 16-24 h after passage. Embryos reconstructed with FF treated with roscovitine and T-II cytoplasts (Rosc/T-II) resulted in similar blastocyst rate compared to those produced with confluent cells and M-II cytoplasts (Conf/M-II). Transfer of blastocysts to surrogate heifers resulted pregnancies and birth of healthy calves from Rosc/T-II and Conf/M-II reconstructed embryos. These results indicate that, when combined with nuclear donor cells at specific cell cycle stages, M-II and T-II bovine oocytes are similarly effective in supporting the reprogramming of somatic cell nuclei.     

Production of cloned bovine embryos by somatic cell transfer into enucleated zona-free oocytes

Cloned bovine embryos were produced at the blastocyst stage. Prior to enucleation, oocytes were freed from the zona pellucida. Fibroblasts isolated from the bovine fetus were used as nuclear donors. Pairs of fetal fibroblasts and enucleated oocytes (cytoplasts) were glued in phytohemagglutinin solution under a binocular microscope. The subsequent electrofusion of 39 fetal fibroblast-cytoplast pairs yielded 36 reconstructed one-cell embryos (92.3%). After culturing in synthetic oviduct fluid for 7.5 days, seven cloned embryos developed to the blastocyst stage (19.4%) and six blastocysts were considered fit for transplantation. The applied technique of bovine embryo growth allowed 31.1% zona-free oocytes parthenogenetically activated by to reach the blastocyst stage.     

In vitro production and initiation of pregnancies in inter-genus nuclear transfer embryos derived from leopard cat (Prionailurus bengalensis) nuclei fused with domestic cat (Felis silverstris catus) enucleated oocytes

The leopard cat (Prionailurus bengalensis), a member of the felidae family, is currently listed as threatened by the Ministry of Environment in South Korea. In exotic or endangered species, the lack of oocytes and recipients precludes the use of traditional somatic cell nuclear transfer, and an approach such as inter-genus nuclear transfer may be the only alternative for producing embryos and offspring. In the present study, we used the leopard cat as a somatic cell donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of leopard cat fibroblast cell nuclei with domestic cat cytoplasts. A total of 412 enucleated domestic cat oocytes were reconstructed with either male (Group A) or female (Group B) adult leopard cat fibroblasts. There was no significant difference in fusion rate (60.4% versus 56.9%) between Groups A and B. Of the cultured embryos, the cleavage and blastocyst developmental rate were not significantly different between Groups A and B (69.5% versus 60.8%; 7.2% versus 7.8%, P > 0.05). In Group A, in vivo developmental studies at 30-45 days postimplantation demonstrated 4.8% (21/435) of reconstructed embryos (n = 435) had entered into the uterine lining of recipients, while 1.4% (6/435) formed fetuses. However, all of the reconstructed embryos failed to develop to term (65 days). Microsatellite analyses confirmed that the nuclear genome of the cloned fetus were leopard cat in origin.     

Heterogeneous nuclear transfer embryos reconstructed by bovine oocytes and camel (Camelus bactrianus) skin fibroblasts and their subsequent development

Summary This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplasts to investigate the reprogramming of camel somatic cell nuclei in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Serum-starved skin fibroblast cells, obtained from adult camel, were electrically fused into enucleated bovine metaphase II (MII) oocytes that were matured in vitro. The fused eggs were activated by Inomycin with 2 mM/ml 6-dimethylaminopurine. The activated reconstructed embryos were cocultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum for 168 h. Results showed that 53% of the injected oocytes were successfully fused, 34% of the fused eggs underwent the first egg cleavage, and 100% of them developed to four- or 16-cell embryo stages. The first completed cleavage of xenonuclear transfer camel embryos occurred between 22 and 48 h following activation. This study demonstrated that the reconstructed embryos underwent the first embryonic division and that the reprogramming of camel fibroblast nuclei can be initiated in enucleated bovine MII oocytes.     

Oocyte age and nuclear donor cell type affect the technical efficiency of somatic cloning in rabbits

The present study in rabbits compared, in the first experiment, the effect of two commonly used oocyte ages, 13 h and 17 h after ovulation induction treatment, on the technical efficiency of somatic nuclear transfer steps, using fresh cumulus cells as nuclear donors. Recently ovulated metaphase II oocytes (13 h) showed higher fusion (13 h: 83% vs 17 h: 67%, p < 0.05) and in vitro development rates than in vivo slightly aged metaphase II oocytes (morula, 13 h: 74% vs 17 h: 25%, p < 0.05; blastocyst, 13 h: 16% vs 17 h: 8%; p < 0.05). In contrast, activation rate was higher in the 17 h group (13 h: 45% vs 17 h: 67%; p < 0.05). In a second experiment, using recently ovulated oocytes (13 h) as recipients, two donor cell types (from primary cultures of either cumulus cells or fetal fibroblasts) were tested to evaluate their effects on the efficiencies of the different technical steps of somatic nuclear transfer procedure. A better fusion ratewas obtained when fetal fibroblasts were used as nuclear donors (cumulus cells: 45% vs fetal fibroblasts: 67%, p < 0.05). No statistically significant differences were detected in cleavage rate regardless of the cell type used (cumulus cells: 44% vs fetal fibroblasts: 60%, p > 0.05). However, in vitro development to morula (cumulus cells: 41% vs fetal fibroblasts: 14%, p < 0.05) and to blastocyst stage (cumulus cells: 27% vs fetal fibroblasts: 3%, p < 0.05) were different between cell types.     

Production of calves by transfer of nuclei from cultured somatic cells obtained from Japanese black bulls

We investigated the possibility of producing calves from transferable bovine embryos obtained by nuclear transfer using somatic cell-derived cell lines. Muscle cells obtained from 2 Japanese Black bulls were dispersed in Hank's solution supplemented with collagenase Type-I. The separated muscle cells were cultured in Dulbecco's Modified Eagle's medium (D-MEM) supplemented with 10% fetal bovine serum (FBS) at 39 degrees C in an atmosphere of 5% CO2 in air. Cells were passaged at least 4 times, and for 5 d prior to nuclear transfer they (donor cells: karyoplasts) were cultured in D-MEM supplemented with 0.5% FBS (to induce quiescence) or 10% FBS. Recipient oocytes were produced by in vitro culture of bovine oocytes that were obtained at a slaughterhouse and then enucleated in modified phosphate buffered saline supplemented with cytochalasin B. Embryos were reconstructed by 3 protocols using karyoplasts cultured in the medium with 0.5% FBS. 1) Group A: recipient oocytes (cytoplasts; n = 157) were treated with Ca ionophore A 23187, ethanol and cycloheximide, and then a karyoplast was fused to an activated cytoplast. 2) Group B: karyoplasts were transferred to cytoplasts (n = 117), and the couplets were treated with electric stimulation and then Ca ionophore A 23187 and cycloheximide. 3) Group C: cytoplasts (n = 104) were cultured for a further 12 h before fusion, and then the couplets were treated with electric stimulation and cycloheximide. 4) Group D: in addition to the above 3 groups, karyoplasts cultured in the medium with 10% FBS were transferred to recipient cytoplasts (n = 137) and treated as in Protocol 2. Reconstructed embryos were cultured in modified CR1aa for 8 d, and the development of embryos was assessed. In total 73 blastocysts were obtained, and the frequency of development to the blastocyst stage in Group A (2.5%) was lower than that of Groups B, C and D (20.5, 18.3 and 19.0%, respectively; P < 0.01). Of these the sex of 21 blastocysts was determined by rapid Y-chromosome detection assay, and all were male, suggesting that nuclear replacement had been achieved successfully. When 26 blastocysts were transferred to 20 recipient cows, 8 of them became pregnant; 4 cows subsequently aborted about 60 d after embryo transfer while the remaining 4 cows calved. These results indicate that reconstructed embryos obtained by nuclear transfer of muscle cell-derived cell lines can develop to the blastocyst stage, and some are sufficiently competent to develop to term. Particularly important was the finding that special culture protocols for somatic cells prior to nuclear transfer were not necessary in our system.     

Nuclear and cytoskeletal dynamics during oocyte maturation and development of somatic cell cloned pig embryos injected with membrane disintegrated donor cells

The objectives of this study were to characterize the nuclear and cytoskeletal changes of pig oocytes during in vitro maturation (IVM) and the development of the reconstructed embryos after injection with membrane intact or disintegrated donor cells. Cumulus-oocyte complexes (COCs) were collected from abattoir ovaries by follicle (2-8mm) aspiration. In Experiment 1, COCs were cultured in NCSU-23 medium for 0, 11, 22, 33, and 44 h. Oocytes were fixed at different time points for nuclear and cytoskeletal labeling. Forty-three percent and 75% oocytes progressed to MII stage at 33 and 44 h after IVM culture, respectively. Dynamic shift of spindle and cytoplasmic microtubules was evident. In Experiment 2, matured oocytes were injected with either the whole cumulus cell with or without intact cell membranes after enucleation. The reconstructed oocytes were fixed at 0, 2, or 4 h after cell injection for nuclear and cytoskeletal evaluation. When an intact cumulus cell was injected, the injected cell remained intact within 4h after injection. When a cell with disintegrated membrane was injected, 59-63% (n=146) of the injected cell underwent premature chromosome condensation (PCC). In Experiment 3, the reconstructed pig oocytes received membrane-disintegrated cumulus cells or fetal fibroblasts were cultured in PZM medium. The blastocyst rate of the fibroblast-injected embryos was 10%, which was lower than the non-cloned parthenotes (33%, P<0.05) but higher than the cumulus cell-injected embryos (2.7%). These results suggest that pig oocytes are subjected to nuclear and cytoskeletal reorganization during maturation. Pig oocytes injected with membrane-disintegrated fibroblast cells support better blastocyst development of the cloned embryos.     

Heterogeneous nuclear-transferred-embryos reconstructed with camel (Camelus bactrianus) skin fibroblasts and enucleated ovine oocytes and their development H-M

This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and ovine oocytes as recipient cytoplasts for investigating the developmental potential of the reconstructed embryos. Serum-starved adult camel skin fibroblast cells were used as donor somatic cells. Ovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electrofusion. The fused oocytes were activated by 5mM/ml inomycin with 2mM/ml 6-dimethylaminopurine (6-DMAP). The activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum (FCS) for 168h. A total of 300 enucleated ovine oocytes were available for xenonuclear embryo reconstruction. The results showed that 71% of the nuclear transfer couplets were successfully fused, 55% of the fused oocytes cleaved within 48h after activation, 82% of the cleaved oocytes developed to 2-16-cell embryo stages and 18% of the cleaved nuclear transfer zygotes developed to the morula stage. This study demonstrated that the xenonuclear transfer camel embryos can undergo the first embryonic division and subsequent development to morula stage in vitro.     

Improvement of a porcine somatic cell nuclear transfer technique by optimizing donor cell and recipient oocyte preparations

This study was conducted to improve a porcine somatic cell nuclear transfer (SCNT) technique by optimizing donor cell and recipient oocyte preparations. Adult and fetal fibroblasts, and cumulus and oviduct cells were used as donor cells, and in vivo- and in vitro-matured oocytes were employed as recipient oocytes. The percentages of fusion and development to the blastocyst stage, the ratio of blastocysts to 2-cell embryos, and cell number of blastocysts were monitored as experimental parameters. In Experiment 1, donor cells of four different types were transferred to enucleated oocytes matured in vitro, and more (P < 0.05) blastocysts were derived from SCNT of fetal fibroblasts than from that of other cells (15.9% versus 3.1-7.9%). For SCNT using fetal fibroblasts, increasing the number of subcultures up to 15 times did not improve developmental competence to the blastocyst stage (12.2-16.7%). In Experiment 2, fetal fibroblasts were transferred to enucleated oocytes that matured in vivo or in vitro. When parthenogenetic activation of both types of oocytes was conducted as a preliminary control treatment, a significant increase in blastocyst formation was found for in vivo-matured compared with in vitro-matured oocytes (36.4% versus 29.5%). However, no improvement was achieved in SCNT using in vivo-matured oocytes. In conclusion, the type of donor somatic cell is important for improving development after porcine SCNT, and fetal fibroblasts were the most effective among examined cells. A system with good reproducibility has been established using fetal fibroblasts as the donor karyoplast after subculturing 1-10 times, and using both in vivo and in vitro-matured oocytes as the recipient cytoplast.     

Effect of telophase enucleation on bovine somatic nuclear transfer

Telophase enucleation has been proven to be an efficient method for preparing recipient cytoplasts in bovine embryonic nuclear transfer (2, 11). This research was designed to study in vitro development of bovine oocytes containing transferred somatic cell nuclei, reconstructed by using enucleated in vitro-matured oocytes 32 h of age at telophase II stage as recipient cytoplasts, compared with those 24 h of age at metaphase II stage. Two protocols for donor cell injection were adopted, i.e., subzonal injection (SUZI) and intracytoplasmic injection (ICI). Bovine oviduct epithelial cells (BOECs) and bovine cumulus cells (BCCs) from an adult cow were used as nuclear donors for these experiments. In SUZI groups, the fusion rate of donor cells, both BOECs and BCCs, with MII enucleated oocytes were higher than those with TII enucleated oocytes (54% vs. 41% and 53% vs. 39%, respectively; P<0.05), but the development rates to morula plus blastocyst stage in MII groups were lower than those in TII groups (22% vs. 39% and 21% vs. 41%, respectively; P<0.05). In ICI groups, about 26% of enucleated MII oocytes injected with BOECs or BCCs cleaved and only small parts of them developed to blastocyst stage (4% and 3%, respectively; P>0.05). When BOECs or BCCs were intracytoplasmically injected into oocytes enucleated at TII stage, no blastocyst was formed in either donor cell group and no cleavage occurred in BOEC group. Our data demonstrated that telophase enucleation is beneficial to early embryo development when bovine somatic nuclei are transferred by subzonal injection. However, it is harmful when donor cells are directly injected into the cytoplast of the enucleated oocytes.     

Nuclear transfer of synchronized african wild cat somatic cells into enucleated domestic cat oocytes

The African wild cat is one of the smallest wild cats and its future is threatened by hybridization with domestic cats. Nuclear transfer, a valuable tool for retaining genetic variability, offers the possibility of species continuation rather than extinction. The aim of this study was to investigate the ability of somatic cell nuclei of the African wild cat (AWC) to dedifferentiate within domestic cat (DSH) cytoplasts and to support early development after nuclear transplantation. In experiment 1, distributions of AWC and DSH fibroblasts in each cell-cycle phase were assessed by flow cytometry using cells cultured to confluency and disaggregated with pronase, trypsin, or mechanical separation. Trypsin (89.0%) and pronase (93.0%) yielded higher proportions of AWC nuclei in the G0/G1 phase than mechanical separation (82.0%). In contrast, mechanical separation yielded higher percentages of DSH nuclei in the G0/G1 phase (86.6%) than pronase (79.7%) or trypsin (74.2%) treatments. In both species, pronase induced less DNA damage than trypsin. In experiment 2, the effects of serum starvation, culture to confluency, and exposure to roscovitine on the distribution of AWC and DSH fibroblasts in various phases of the cell cycle were determined. Flow cytometry analyses revealed that the dynamics of the cell cycle varied as culture conditions were modified. Specifically, a higher percentage of AWC and DSH nuclei were in the G0/G1 phase after cells were serum starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%), or after roscovitine (56% vs. 84%) treatment, respectively. In experiment 3, we evaluated the effects of cell synchronization and oocyte maturation (in vivo vs. in vitro) on the reconstruction and development of AWC-DSH- and DSH-DSH-cloned embryos. The method of cell synchronization did not affect the fusion and cleavage rate because only a slightly higher percentage of fused couplets cleaved when donor nuclei were synchronized by serum starvation (83.0%) than after roscovitine (80.0%) or contact-inhibition (80.0%). The fusion efficiency of in vivo and in vitro matured oocytes used as recipient cytoplasts of AWC donor nuclei (86.6% vs. 85.2%) was similar to the rates obtained with DSH donor nuclei, 83.7% vs. 73.0%, respectively. The only significant effect of source of donor nucleus (AWC vs. DSH) was on the rate of blastocyst formation in vitro. A higher percentage of the embryos derived from AWC nuclei developed to the blastocyst stage than did embryos produced from DSH nuclei, 24.2% vs. 3.3%, respectively (P < 0.05). In experiment 4, the effect of calcium in the fusion medium on induction of oocyte activation and development of AWC-DSH-cloned embryos was determined. The presence of calcium in the fusion medium induced a high incidence of cleavage of DSH oocytes (54.3%), while oocyte cleavage frequency was much lower in the absence of calcium (16.6%). The presence or absence of calcium in the fusion medium did not affect the fusion, cleavage, and blastocyst development of AWC-DSH-cloned embryos. In experiment 5, AWC-DSH-cloned embryos were transferred to the uteri of 11 synchronized domestic cat recipients on Day 6 or 7 after oocyte aspiration. Recipients were assessed by ultrasonography on Day 21 postovulation, but no pregnancies were observed. In the present study, after NT, AWC donor nuclei were able to dedifferentiate in DSH cytoplasts and support high rates of blastocyst development in vitro. Incomplete reprogramming of the differentiated nucleus may be a major constraint to the in vivo developmental potential of the embryos.     

Clonal lines of transgenic fibroblast cells derived from the same fetus result in different development when used for nuclear transfer in pigs

Different factors are believed to influence the outcome of nuclear transfer (NT) experiments. Besides the cell cycle stage of both recipient cytoplast and donor karyoplast, the origin of the donor cells (embryonic, fetal, and adult) is of interest. We compared in vitro development of NT embryos derived from small serum-starved (G0) or small cycling (G1) porcine fetal fibroblast cells. Serum starvation did not have a positive effect on cleavage rate or the percentage of embryos that developed to the morula and blastocyst stages. Next, we investigated the development of porcine NT embryos derived from different transgenic clonal cell lines that had originated from the same fetus. When different clonal lines of fetal fibroblasts were fused to enucleated metaphase II oocytes, differences in fusion rates as well as in development to the morula and blastocyst stages were observed (P < 0.05). When oocytes derived from sow ovaries were used as recipient cytoplasts, significantly better cleavage (P = 0.03) and blastocyst formation (P < 0.014) was obtained when compared with oocytes derived from gilts. Our data indicate that not only different cell lines, but also different clones derived from one primary cell line, result in different development when used for NT. In addition, the use of sow oocytes as a cytoplast source also improves the efficiency of NT experiments.