Development and calcium level changes in pre-implantation porcine nuclear transfer embryos activated with 6-DMAP after fusion

This study investigated the effect of treatment with 6-dimethylaminopurine (6-DMAP) following fusion on in vitro development of porcine nuclear transfer (NT) embryos. Frozen thawed ear skin cells were transferred into the perivitelline space of enucleated oocytes. Reconstructed oocytes were fused and activated with electric pulse in 0.3 M mannitol supplemented with either 0.1 or 1.0 mM CaCl(2). In each calcium concentration, activated oocytes were divided into three groups. Two groups of them were exposed to either ionomycin (I + 6-DMAP or 6-DMAP alone. In experiment 2, fused NT embryos in 0.3 M mannitol containing 1.0 mM CaCl(2) were exposed to 6-DMAP either immediately or 20 min after fusion/activation. For 0.1 mM CaCl(2), oocytes activated with either I + 6-DMAP or 6-DMAP alone showed a higher (P < 0.05) developmental rate to the blastocyst stage than those activated with an electric pulse alone (26.7 and 22.5 vs. 12.5%). For 1.0 mM CaCl(2), oocytes activated with either I + 6-DMAP or 6-DMAP alone showed significantly higher (P < 0.05) developmental rate to the blastocyst stage (35.6 and 28.3 vs. 19.8%). Developmental rate to the blastocyst stage was (P < 0.05) increased in NT embryos activated with 6-DMAP 20 min after fusion. 6-DMAP made a higher and wider Ca(2+) transient compared to that induced by electric pulses (Fig. 3). The fluctuation lasted during the time that oocytes were cultured in 6-DMAP. Regardless of Ca(2+) concentration in fusion medium, activation with 6-DMAP following electric pulses supported more development of porcine NT embryos. Activation of NT embryos with 6-DMAP after fusion in the presence of 1.0 mM CaCl(2) could support better developmental rate to the blastocyst stage.     

Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein

This study was performed to develop a system for porcine somatic cell nuclear transfer (SCNT) and to produce human erythropoietin (hEPO)-transgenic cloned piglets. Porcine fetal fibroblasts were transfected with an expression plasmid (phEPO-GFP). In Experiment 1, the effect of transfection of phEPO-GFP transgene on development of porcine SCNT embryos was investigated. Three fetal fibroblast cell lines (two male and one female) with or without transfected with phEPO-GFP trasngene were used as donor cells for SCNT. Lower fusion rates were observed in two lines of transfected cells as compared to those of the control cells. In Experiment 2, the effect was examined of elevated Ca2+ concentration in the fusion/activation medium on development of transfected SCNT embryos. The rates of fusion and blastocyst formation were significantly increased by supplementing 1.0 mM of CaCl2 (versus 0.1 mM) into the fusion/activation medium. In Experiment 3, the effect was studied of a chemical treatment (cytochalasin B) after electric fusion/activation (F/A) on porcine transgenic SCNT embryo development. The electric F/A + cytochalasin B treatment increased total cell number in blastocysts as compared to that of electric F/A treatment alone. In Experiment 4, transgenic cloned embryos were transferred to surrogate mothers and a total of six cloned piglets were born. Transgenic cloned piglets were confirmed by polymerase chain reaction and Southern blot analysis. From a single surrogate mother, female and male transgenic cloned piglets were produced by transferring pooled SCNT embryos derived from female and male transfected donor cells. In conclusion, a system for porcine SCNT was developed and led to the successful production of hEPO transgenic cloned piglets.     

Effects of fusion/activation methods on development of embryos produced by nuclear transfer of porcine fetal fibroblast

The present studies were carried out to investigate the effects of intensity of dc pulse, number of dc pulse and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. In experiment 1, different fusion/activation intensity (two dc pulses of 0.4, 0.8, 1.2, 1.6 and 2.0 kV/cm for 30 micros, respectively) was carried out to investigate development of embryos. In experiment 2, the reconstructed oocytes were fused and activated with one, two or three dc pulses of 1.2 kV/cm for 30 micros. In experiment 3, reconstructed oocytes were equilibrated in TCM-199 medium for 0-6 h, respectively, and fused/activated with one dc pulse of 1.2 kV/cm for 30 micros. The reconstructed embryos were cultured in PZM-3 medium containing 0.3% BSA. When oocytes were fused with donor cell by two dc pulses of 0.4 kV/cm for 30 micros, the rates of cleavage and blastocyst formation were significantly lower (32.9% and 2.5%) than those of fused by 0.8 kV/cm (59.0% and 17.4%) or 1.2 kV/cm (63.3% and 18.4%), respectively. One dc pulse of 1.2 kV/cm for 30 micros was enough to fuse and activate embryos to develop to blastocyst (24.8%). Equilibration for 2-3 h in TCM-199 before fusion/activation was beneficial for improving the developmental ability of embryos produced by nuclear transfer (25.6-23.3% at blastocysts).     

Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts

The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P < 0.05). When electrofusion was induced 27 h after the onset of oocyte maturation, the cleavage rate (78.0%) was higher than that of electrofusion induced at 28 h (67.2%, P < 0.05), and the blastocyst yield (18.1%) was higher (P < 0.05) than that of electrofusion induced at 25 or 26 h (7.4 and 8.5%, respectively). A higher proportion of NT embryos activated at 3 h after electrofusion developed to the blastocyst stage (18.6%) in comparison with NT embryos activated at 1 h (6.0%), 2 h (8.3%), or 4 h (10.6%) after fusion (P < 0.05). No recipient was pregnant 60 d after transfer of blastocysts developed from NT embryos activated at 1 h (0/8), 2 h (0/10), or 4 h (0/9) after fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation.     

Roscovitine is a novel agent that can be used for the activation of porcine oocytes reconstructed with adult cutaneous or fetal fibroblast cell nuclei

The present study was undertaken to investigate the preimplantation developmental competence of cloned pig embryos that were derived from fibroblast cell nuclei by different methods for the activation of reconstructed oocytes. In subgroups IA and IB, nuclear-transferred (NT) oocytes derived from either adult cutaneous or fetal fibroblast cells that had been classified as nonapoptotic by intra vitam analysis for programmed cell death using the YO-PRO-1 DNA fluorochrome underwent sequential physical (i.e., electrical) and chemical activation (SE-CA). This novel method of SE-CA, which was developed and optimized in our laboratory, involves treatment of reconstituted oocytes with direct current pulses and subsequent exposure to 7.5 μM calcium ionomycin, followed by incubation with 30 μM R-roscovitine (R-RSCV), 0.7 mM 6-dimethylaminopurine and 3.5 μg/mL cycloheximide. In subgroups IIA and IIB, NT oocytes were subjected to the standard method of simultaneous fusion and activation mediated by direct current pulses. The proportion of cloned embryos in subgroup IA that reached the morula and blastocyst stages was 145/248 (58.5%) and 78/248 (31.5%), respectively. The proportions of cloned embryos in subgroup IB that reached the morula and blastocyst stages were 186/264 (70.5%) and 112/264 (42.4%), respectively. In turn, subgroup IIA yielded proportions at the morula and blastocyst stages of 110/234 (47.0%) and 49/234 (20.9%), respectively. Subgroup IIB yielded proportions at the morula and blastocyst stages of 144/243 (59.3%) and 74/243 (30.5%), respectively. In summary, the SE-CA of NT oocytes reconstructed from either type of nonapoptotic/nonnecrotic (i.e., YO-PRO-1-negative) fibroblast cell resulted in porcine cloned embryos with considerably better in vitro developmental outcomes than those of cloned embryos generated using the simultaneous fusion and activation approach. To our knowledge, this is the first report of the successful stimulation of porcine NT oocytes using electric pulses followed by an additional activation with a higher dose (1.5 times) of calcium ionomycin and subsequent exposure to a combination of 30 μM R-RSCV and lower concentrations (by 3 times) of 6-dimethylaminopurine and cycloheximide. Moreover, we report here the first use of R-RSCV, a novel meiosis-promoting factor-related p34^cdc2 kinase inhibitor, in the oocyte activation protocol for the somatic cell cloning of pigs.     

Improvement in development of porcine embryos reconstituted with cells from blastocyst-derived cell lines and enucleated oocytes by optimization of reconstruction methods

The present study was conducted to establish the most suitable system for producing porcine reconstructed embryos by transferring cells from blastocyst-derived cell lines into enucleated oocytes. When the cells were fused to preactivated metaphase II oocytes, or the cells and arrested metaphase II oocytes were fused in medium without CaCl(2) and MgSO(4), the percentages (43-53%) of fused embryos were significantly lower than those (72-79%) produced by fusing the cells to arrested metaphase II oocytes in medium containing CaCl(2) and MgSO(4). High productive efficiency (7%) of blastocysts was obtained when reconstituted embryos produced by the last method were activated again at 3 hours after fusion (F/A --> Activation). Pronuclear formation was observed in 80-91% of the reconstructed embryos produced by F/A --> Activation, with no significant differences between different culture periods in the medium containing cytochalasin B. When cultured in the medium containing cytochalasin B for 0-1 h, almost all (83-85%) the embryos had one pronucleus and one polar body. However, the number of embryos with two pronuclei and no polar bodies was increased significantly by culturing in the medium containing cytochalasin B for 2-4 h. The cleavage rate (34-48%) of reconstructed embryos was not affected by the presence of cytochalasin B for 2 h after activation. However, the percentage of embryos that developed to the blastocyst stage was significantly higher in the presence (23%) than absence (5%) of cytochalasin B. The results indicate that F/A --> Activation and cytochalasin B treatment are effective for the production of porcine embryos reconstituted with cells from blastocyst-derived cell lines and enucleated oocytes.     

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.     

Differential development of rabbit embryos derived from parthenogenesis and nuclear transfer

Parthenogenetic development (PA) is often used as a model to investigate activation protocols for nuclear transfer (NT) embryos. The objective of this study was to compare the development, as well as the dynamics of the nuclear materials and microtubules of PA and NT embryos following similar activation treatment. Our results demonstrate that, during parthenogenesis, activation through either electrical pulses or chemical stimulation alone resulted in low cleavage rates and compromised development. A combination of two sets of electrical pulses and a 2-h-exposure to chemical activation medium (5 microg/ml cycloheximide (CHX) and 2 mM 6-dimethylaminopurine (6-DMAP) in KSOM+0.1% BSA) could effectively activate rabbit oocytes, and resulted in a 99% (n = 73) cleavage rate with greater than 60% (n = 73) developing to blastocysts at day 4. However, the same activation protocol following NT resulted in only 65-72% of oocytes cleaved (depending on donor cell type), with less than 20% developing to the blastocyst stage. The differences observed between NT and PA embryos subjected to the same activation protocol were also evident in terms of the time required for their development to the blastocyst stage, as well as the cell numbers present in blastocysts at day 6. Furthermore, laser confocal microscopy revealed that pronuclear formation in the NT embryos was delayed by comparison to that in the parthenotes. In conclusion, our study suggests that an effective protocol for parthenogenesis cannot promise a comparable outcome for NT embryos.     

Development of nuclear transfer and parthenogenetic rabbit embryos activated with inositol 1,4,5-trisphosphate

The present study was carried out to evaluate the effects of different activation protocols, enucleation methods, and culture media on the development of parthenogenetic and nuclear transfer (NT) rabbit embryos. Electroporation of 25 mM inositol 1,4, 5-trisphosphate (IP3) in calcium- and magnesium-free PBS immediately induced a single intracellular calcium transient in 6 out of 14 metaphase II-stage rabbit oocytes evaluated during a 10-min recording period. The percentage of oocytes treated with IP3 followed by 6-dimethylaminopurine (IP3 + DMAP) that cleaved (83.9%) and reached the blastocyst stage (50%) was significantly higher (p < 0.05) than those activated with multiple pulses (61.6% and 30.1%, respectively) or treated with ionomycin + DMAP (52.9% and 5.7%, respectively). Development of IP3 + DMAP-activated rabbit oocytes and in vivo-fertilized zygotes in different culture media was studied. Development of activated oocytes to the blastocyst stage in Earle's balanced salt solution (EBSS) supplemented with MEM nonessential amino acids, basal medium Eagle amino acids, 1 mM L-glutamine, 0.4 mM sodium pyruvate, and 10% fetal bovine serum (FBS) (EBSS-complete) (40.6%) was significantly higher (p < 0.05) than those that developed in either Dulbecco's Modified Eagle's medium (DMEM)/RPMI + 10% FBS (15.5%) or CR1aa + 10% FBS (4%) medium. In addition, 100% of in vivo-fertilized rabbit zygotes developed to the blastocyst stage in EBSS-complete. A third set of experiments was carried out to study the efficiency of blind versus stained (Hoechst 33342) enucleation of oocytes. Twenty-nine of 48 blind enucleated and IP3 + DMAP-activated oocytes cleaved (60.4%), and 15 (31.2%) subsequently reached the blastocyst stage, whereas 9 of 52 oocytes enucleated using epifluorescence (17.3%) cleaved, and none of these reached the blastocyst stage. When the above parameters that yielded the highest blastocysts were combined in an NT experiment using adult rabbit fibroblast nuclei, 72.2% (39 of 54) of the fused nuclear transplant embryos cleaved and 29.6% (16 of 54) reached the blastocyst stage.     

Effect of electrofusion pulse in either electrolyte or nonelectrolyte fusion medium on subsequent murine embryonic development.

This study was designed to determine what effect electropulse parameters would have on rate of fusion, lysis, and embryo viability when embryos were subjected to electrofusion treatment in nonelectrolyte or electrolyte pulse media. Previous experiments have shown electrolyte medium (i.e., phosphate-buffered saline; PBS) to have a positive effect on electric pulse-induced murine oocyte activation. In addition, these results also indicated that pulse media containing 0.9 mM Ca2+ induced a dramatic increase in the rate of murine oocyte activation compared with oocytes pulsed in media containing 0.0 or 0.05 mM Ca2+. Pronuclear or two-cell-stage embryos were obtained from superovulated prepubertal randomly bred Swiss (albino) female mice. Embryos were randomly assigned to three nonelectrolyte and three electrolyte treatment media. Nonelectrolyte media consisted of 0.3 M mannitol (T1), 0.3 M mannitol + 0.05 mM CaCl2 (T2), and 0.3 M mannitol + 0.9 mM CaCl2 (T3). Electrolyte media consisted of Ca(2+)-free PBS (T4), PBS containing 0.05 mM CaCl2 (T5), and PBS containing 0.9 mM CaCl2 (T6). Three experiments were carried out; the objective of the first was to determine the rate of fusion and rate of lysis in murine two-cell embryos placed in the two types of (0.3 M mannitol, T1-T3; and PBS, T4-T6) fusion media and subjected to a fusion procedure (3 V, 5 sec AC alignment pulse, followed by a 1.56 kV.cm-1, 99 microsec DC fusion pulse). Control two-cell embryos were placed in T1 for 2 min and did not receive a fusion pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental potential of porcine nuclear transfer embryos derived from transgenic fetal fibroblasts infected with the gene for the green fluorescent protein: comparison of different fusion/activation conditions.

The in vitro developmental potential of porcine nuclear transfer (NT) embryos was evaluated. Oocytes were matured for 42-44 h, and metaphase II-oocytes were enucleated. Fetal fibroblasts infected with the enhanced green fluorescent protein (EGFP) gene were serum-starved for 3-5 days. A single cell was injected into the perivitelline space of the enucleated oocytes. The reconstructed oocytes were allocated to different fusion and activation conditions. In experiment 1, two different fusion/activation conditions were compared: two pulses of 1.2 kV/cm for 30 microsec (group A), or one pulse of 1.6 kV/cm for 30 microsec followed in 30 min by one pulse of 1.2 kV/cm for 30 microsec (group B). Parthenogenetic controls were created by using the group A parameter. The fusion rate in group A (mean +/- SEM, 68.4% +/- 3.9%) was higher (P < 0.05) than in group B (59.4% +/- 2.3%). The rates of cleavage (50.1% +/- 4.6% to 62.8% +/- 5.5%) were not different among control and treatment groups. However, the rate of parthenogenetic control embryos developing to the blastocyst stage (18.1% +/- 3.1%) was higher (P < 0.05) than the rate of NT embryos (5.9% +/- 1.7% and 4.9% +/- 2.5%). In experiment 2, we compared two pulses of 1.2 kV/cm (group C) versus two pulses of 1.3 kV/cm (group D). For two control groups, the same pulses as those given to group C or D, respectively, were supplied. The fusion rate in group D (70.6% +/- 4.2%) was higher (P < 0.05) than in group C (58.9% +/- 2.7%). The cleavage rates were not different among control and treatment groups (58.1% +/- 8.1% to 73.6% +/- 6.0%). However, the rate of embryos developing to the blastocyst stage in group D (3.5% +/- 1.7%) was lower (P < 0.05) than in controls and group C (11.4% +/- 2.0% to 16.4% +/- 1.1%). In experiment 3, we examined whether the presence of cytochalasin B (CB) during donor cell injection affects the development of NT embryos. The fusion rate of oocytes in the group with CB (78.4% +/- 1.4%) was higher (P < 0.05) than in the group without CB (70.9% +/- 0.2%). The cleavage rate of the control group (85.5% +/- 4.9%) was higher (P < 0.05) than those of the treatment groups (61.6% +/- 2.7% and 63.9% +/- 4.3%). However, the rates of embryos developing to the blastocyst stage (8.1% +/- 2.5% to 19.1% +/- 6.0%) and the mean cell number of blastocysts (29.4 +/- 5.2 to 45.7 +/- 6.4) were not different among control and treatment groups. Green fluorescence was observed at all stages in NT embryos. These results indicate that two pulses of 1.2 kV/cm are enough for fusion/activation of NT embryos to develop to the blastocyst stage, and that the presence of CB during donor cell injection is not necessary for early development of NT embryos.     

Production of transgenic porcine blastocysts by nuclear transfer

In this study the in vitro development of porcine nuclear transfer (NT) embryos was investigated. Transgenic fetal fibroblast cells that were frozen after 5 days of serum starvation were injected immediately after thawing into enucleated metaphase II (MII) oocytes. Reconstructed embryos were activated by incubation in 200 microM thimerosal followed by a 30-min treatment of 8 mM DTT. The embryos were subsequently cultured in NCSU23, supplemented with 4 mg/ml BSA for 7 days. The actual cleavage rate (embryos showing > or =2 nuclei) in 6 replicates was 33% (ranging from 15% to 50%). Three blastocysts with cell numbers of 14, 15, and 18 were obtained. The blastocyst rate was significantly lower for NT embryos as opposed to parthenogenetically activated embryos (1% vs. 5%; P<0.05). The neomycin-resistance gene was amplified by PCR in all three NT embryos, indicating their origin from the injected transgenic fibroblasts. Efforts are now being directed in improvements in the nuclear transfer technology, whereby viable fetuses or offspring can be produced from these NT-embryos.     

Genetic reprogramming of lactate dehydrogenase, citrate synthase, and phosphofructokinase mRNA in bovine nuclear transfer embryos produced using bovine fibroblast cell nuclei

Adult animal cloning has progressed to allow the production of offspring cloned from adult cells, however many cloned calves die prenatally or shortly after birth. This study examined the expression of three important metabolic enzymes, lactate dehydrogenase (LDH), citrate synthase, and phosphofructokinase (PFK), to determine if their detection in nuclear transfer (NT) embryos mimics that determined for in vitro produced embryos. A day 40 nuclear transfer produced fetus derived from an adult cell line was collected and fetal fibroblast cultures were established and maintained. Reconstructed NT embryos were then produced from this cell line, and RT-PCR was used to evaluate mRNA reprogramming. All three mRNAs encoding these enzymes were detected in the regenerated fetal fibroblast cell line. Detection patterns were first determined for IVF produced embryos (1-cell, 2-cell, 6-8 cell, morula, and blastocyst stages) to compare with their detection in NT embryos. PFK has three subunits: PFK-L, PFK-M, and PFK-P. PFK-L and PFK-P were not detected in bovine oocytes. PFK subunits were not detected in 6-8 cell embryos but were detected in blastocysts. Results from NT embryo RT-PCR demonstrated that PFK was not detected in 8-cell NT embryos but was detected in NT blastocysts indicating that proper nuclear reprogramming had occurred. Citrate synthase was detected in oocytes and throughout development to the blastocyst stage in both bovine IVF and NT embryos. LDH-A and LDH-B were detected in bovine oocytes and in all stages of IVF and NT embryos examined up to the blastocyst stage. A third subunit, LDH-C was not detected at the blastocyst stage in IVF or NT embryos but was detected in all earlier stages and in mature oocytes. In addition, LDH-C mRNA was detected in gonad isolated from the NT and an in vivo produced control fetus. These results indicate that the three metabolic enzymes maintain normal expression patterns and therefore must be properly reprogrammed following nuclear transfer.     

Parthenogenetic and nuclear transfer rabbit embryo development and apoptosis after activation treatments

Previous studies mainly evaluated the effect of culture conditions on preimplantation embryo apoptosis. In order to inhibit apoptosis of nuclear transfer (NT) embryos, putative apoptosis inhibitors were used to treat donor cells. However, little is known about the effect of activation treatments on embryo apoptosis. We firstly investigated the effect of various parthenogenetic activation (PA) treatments on embryo development, blastocyst cell number, and apoptosis, and then one of these activation treatments proved to be most efficient was selected for activation rabbit NT embryos. The activation by electrical pulses and 30 min later, electroporation with 25 muM D-myoinositol 1,4,5-trisphosphate (IP3) in Ca(2+)- and Mg(2+)-free PBS, then exposure to 2.0 mM 6-dimethylaminopurine (6-DMAP) for 3 hr effectively activated rabbit oocytes, and resulted in significantly a higher blastocyst development rate (72.7%) and total cell number (175 +/- 14.1), and markedly lower apoptosis level of blastocyst (4.3 +/- 0.5) than all the other groups. When the same activation protocol was applied in NT embryo activation, we found that exposure of the embryos to 6-DMAP for 3 hr could decrease the apoptosis level of blastocyst and increase blastocyst rate and cell number. The results demonstrate that oocyte activation affects not only embryo development and quality but also embryo apoptosis.     

Somatic cell cloning in Buffalo (Bubalus bubalis): effects of interspecies cytoplasmic recipients and activation procedures

Successful nuclear transfer (NT) of somatic cell nuclei from various mammalian species to enucleated bovine oocytes provides a universal cytoplast for NT in endangered or extinct species. Buffalo fetal fibroblasts were isolated from a day 40 fetus and were synchronized in presumptive G(0) by serum deprivation. Buffalo and bovine oocytes from abattoir ovaries were matured in vitro and enucleated at 22 h. In the first experiment, we compared the ability of buffalo and bovine oocyte cytoplasm to support in vitro development of NT embryos produced by buffalo fetal fibroblasts as donor nuclei. There were no significant differences (p > 0.05) between the NT embryos derived from buffalo and bovine oocytes, in fusion (74% versus 71%) and cleavage (77% versus 75%) rates, respectively. No significant differences were also observed in blastocyst development (39% versus 33%) and the mean cell numbers of day 7 cloned blastocysts (88.5 +/- 25.7 versus 51.7 +/- 5.4). In the second experiment, we evaluated the effects of activation with calcium ionophore A23187 on development of NT embryos after electrical fusion. A significantly higher (p < 0.05) percentage of blastocyst development was observed in the NT embryos activated by calcium ionophore and 6-DMAP when compared with 6-DMAP alone (33% versus 17%). The results indicate that the somatic nuclei from buffalo can be reprogrammed after transfer to enucleated bovine oocytes, resulting in the production of cloned buffalo blastocysts similar to those transferred into buffalo oocytes. Calcium ionophore used in conjunction with 6-DMAP effectively induces NT embryo development.     

In vitro development of reconstructed porcine oocytes after somatic cell nuclear transfer

This study was designed to examine the developmental ability of porcine embryos after somatic cell nuclear transfer. Porcine fibroblasts were isolated from fetuses at Day 40 of gestation. In vitro-matured porcine oocytes were enucleated and electrically fused with somatic cells. The reconstructed eggs were activated using electrical stimulus and cultured in vitro for 6 days. Nuclear-transferred (NT) embryos activated at a field strength of 120 V/mm (11.6 +/- 1.6%) showed a higher developmental rate as compared to the 150-V/mm group (6.5 +/- 2.3%) (P: < 0.05), but the mean cell numbers of blastocysts were similar between the two groups. Rates of blastocyst development from NT embryos electrically pulsed at different times (2, 4, and 6 h) after electrofusion were 11.6 +/- 2.9, 6.6 +/- 2.3, and 8.1 +/- 3.3%, respectively. The mean cell numbers of blastocysts developed from NT embryos were gradually decreased (30.4 +/- 10.4 > 24.6 +/- 10.1 > 16.5 +/- 7.4 per blastocyst) as exposure time (2, 4, and 6 h) of nuclei to oocyte cytoplast before activation was prolonged. There was a significant difference in the cell number between the 2- and 6-h groups (P: < 0. 05). Nuclear-transferred embryos (9.4 +/- 0.9%) had a lower developmental rate than in vitro fertilization (IVF)-derived (21.4 +/- 1.9%) or parthenogenetic embryos (22.4 +/- 7.2%) (P: < 0.01). The mean cell number (28.9 +/- 11.4) of NT-derived blastocysts was smaller than that (38.6 +/- 10.4) of IVF-derived blastocysts (P: < 0. 05) and was similar to that (29.9 +/- 12.1) of parthenogenetic embryos. Our results suggest that porcine NT eggs using somatic cells after electrical activation have developmental potential to the blastocyst stage, although with smaller cell numbers compared to IVF embryos.     

Efficient in vitro production of porcine blastocysts by handmade cloning with a combined electrical and chemical activation

The purpose of our work was to establish an efficient protocol for activation of porcine cytoplast-fibroblast constructs produced by the handmade cloning technique. Firstly, we investigated a combined electrical and chemical activation protocol for parthenogenetic development of in vitro matured zona-free oocytes. Oocytes were activated by one 80 micros pulse and subsequently cultured in cytochalasin B and cycloheximide. Developmental rates of blastocysts from activated oocytes were 49+/-1 and 40+/-2%, when using one 80 micros pulse of 0.85 or 1.25 kV/cm, respectively. The activation procedure was further confirmed by a simultaneous re-fusion and activation of bisected oocytes, resulting in a blastocyst rate of 41+/-8%. Secondly, the activation protocol was applied in the handmade cloning technique. In vitro matured zona-free porcine oocytes were bisected and halves containing no chromatin, i.e. the cytoplasts, were selected. Reconstructed embryos were produced by a two-step fusion procedure. At the first step, one cytoplast was fused to one fibroblast by one 80 micros pulse of 1.25 kV/cm. After 1h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously by one 80 micros pulse of 0.85 kV/cm, and subsequently cultured in cytochalasin B and cycloheximide. The development of reconstructed embryos to the blastocyst stage was in average 21+/-4%, and total blastocyst cell counts were in average 48+/-3. Thus, the combined electrical and chemical activation procedure resulted in efficient blastocyst development in the handmade cloning technique.     

Establishment of a porcine cell line from in vitro-produced blastocysts and transfer of the cells into enucleated oocytes

The present study was conducted to establish a porcine cell line from blastocysts produced in vitro and to examine the developmental ability of nuclear transfer embryos reconstituted with the cells and enucleated mature oocytes. When hatched blastocysts were cultured in Dulbecco's modified Eagle's medium with supplements, no colonies of embryo-derived cells were observed. In contrast, 56% of embryos that were attached to feeder layers of STO cells formed colonies in NCSU-23 with supplements. When the colonies were subcultured in the absence of feeder cells, a cell line with an epithelial-like cell morphology was obtained. This cell morphology was stable up to at least passage 30. Although no fused embryos were observed when a pulse of 100 V/mm was applied, the fusion rate increased significantly at 150 V/mm (28%) and 200 V/mm (64%). At 200 V/mm, 39% of fused embryos cleaved, but no embryos developed beyond the 3-cell stage. When cocultured with electro-activated oocytes, percentages of reconstructed embryos cleaved (65%) and developed to the 4-cell stage (23%) were significantly higher than percentages for those (cleavage: 38%; 4-cell stage: 3%) in the absence of activated oocytes. At 7 days after culture, one reconstructed embryo successfully developed to the blastocyst stage in the presence of activated oocytes. When green fluorescent protein-expressing cells and enucleated oocytes were fused and the fused embryos were cultured with electro-activated oocytes, 3 of 102 reconstructed embryos developed to the blastocyst stage. All of the blastocysts were positive for fluorescent green under ultraviolet light. The results of the present study indicate that a porcine cell line can be established from the hatched blastocyst and maintained in vitro for a long period, and that reconstructed embryos obtained by transferring the blastocyst-derived cells into enucleated oocytes have the ability to develop to the blastocyst stage in vitro.     

Establishment of pregnancy after the transfer of nuclear transfer embryos produced from the fusion of argali (Ovis ammon) nuclei into domestic sheep (Ovis aries) enucleated oocytes

Cloning mammalian species from cell lines of adult animals has been demonstrated. Aside from its importance for cloning multiple copies of genetically valuable livestock, cloning now has the potential to salvage endangered or even extinct species. The aim of this study was to investigate the effect of the bovine and domestic (Ovis aries) ovine oocyte cytoplasm on the nucleus of an established cell line from an endangered argali wild sheep (Ovis ammon) after nuclear transplantation. A fibroblast cell line was established from skin biopsies from an adult argali ram from the People's Republic of China. Early karyotype analysis of cells between 3-6 passages revealed a normal diploid chromosome number of 56. The argali karyotype consisted of 2 pairs of biarmed and 25 pairs of acrocentric autosomes, a large acrocentric and minute biarmed Y. Bovine ovaries were collected from a local abattoir, oocytes aspirated, and immediately placed in maturation medium consisting of M-199 containing 10% fetal bovine serum, 100 IU/mL penicillin, 100 microg/mL streptomycin, 0.5 microg/mL follicle-stimulating hormone (FSH), 5.0 microg/mL luetinizing hormone (LH) and 1.0 microg/mL estradiol. Ovine (O. aries) oocytes were collected at surgery 25 hours postonset of estrus from the oviducts of superovulated donor animals. All cultures were carried out at 39 degrees C in a humidified atmosphere of 5% CO2 and air. In vitro matured MII bovine oocytes were enucleated 16-20 hours after onset of maturation and ovine oocytes within 2-3 hours after collection. Enucleation was confirmed using Hoechst 33342 and UV light. The donor argali cells were synchronized in G0-G1 phase by culturing in Dulbecco's modified Eagle's medium (DMEM) plus 0.5% fetal bovine serum for 5-10 days. Fusion of nuclear donor cell to an enucleated oocyte (cytoplast) to produce nuclear transfer (NT) embryos was induced by 2 electric pulses of 1.4 kV/cm for 30 microsc. Fused NT embryos were activated after 24 hours of maturation by exposure to ionomycin (5 microM, 4 minutes) followed by incubation in 6-dimethylaminopurine (0.2 mM, 4 hours) and cultured in microdrops of CR1aa medium. From a total of 166 constructed nuclear donor cell-bovine cytoplasm NT couples, 128 (77%) successfully fused, 100 (78%) developed to 8-16 cell stage, and 2 (1.56%) developed to the blastocyst stage. The presence of argali nuclei in 8-16 cell stage embryo clones was confirmed after observation of Hoechst 33342 stained embryos under UV light and chromosome analysis of metaphase spreads from blastomeres. A total of 127 constructed nuclear donor cell-ovine cytoplasm NT couples were produced, 101 (80%) successfully fused, 81 (80% of fused) developed to the 16- to 32-cell stage. A total of 28 hybrid (argali-sheep) and 21 sheep-sheep NT embryos were transferred into 6 recipients and 4 recipients, respectively. Two of these recipients, 1 carrying argali-sheep and 1 sheep-sheep, were confirmed pregnant at 49 days by ultrasound, but both pregnancies terminated by 59 days. The results of this study demonstrate the possibility of using xenogenic oocytes to produce early-stage embryos and pregnancies from an established fibroblast cell line of an endangered species.     

The effect of donor cell serum starvation and oocyte activation compounds on the development of somatic cell cloned embryos

Two experiments, one comparing nuclear transfer (NT) embryo activation compounds, the other donor cell treatments, were conducted with a goal of identifying factors that improve the in vitro development of cloned bovine embryos. In experiment 1, 539 NT embryos were produced by combining serum starved bovine fetal fibroblasts with enucleated in vitro matured oocytes, activated with ionomycin, then randomly allocated to be incubated for 4 hours in either Butyrolactone-I (BL-I) or 6-dimethylaminopurine (DMAP). There was no significant difference in development to blastocyst or compact morula of fused embryos at Day 6.5 between BL-I and DMAP activated embryos (22.4% vs. 20.2%; p = 0.18). Karyotyping of 20 blastocysts and compact morula from each group determined that 65% of BL1 and 63% of DMAP embryos were diploid with the remainder mixoploid (2n + 4n). In Experiment 2, the development of 389 NT embryos reconstructed from either serum starved or serum fed fetal fibroblasts was assessed. More Day 7 blastocysts and compact morula developed in the serum starved group (34.5% vs. 18.8%; p = 0.008). To verify the viability of BL-I activated embryos, 10 blastocytes from experiment 2 were transferred into 4 recipient cows. Two morphologically normal fetuses, genetically identical to the original fetal cell line, were surgically recovered at day 45 of gestation. In summary, serum starvation of bovine fetal fibroblasts prior to NT significantly improved development to blastocyst. Additionally, we have shown that BL-I is a novel alternative compound for use in combination with ionomycin to activate NT embryos.