I. S. Hwang

Mitochondrial and DNA damage in bovine somatic cell nuclear transfer embryos

The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H2O2 and ˙OH radical levels, mitochondrial morphology and membrane potential (ΔΨ), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H2O2 (35.6 ± 1.1 pixels/embryo) and ˙OH radical levels (44.6 ± 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 ± 1.5 and 23.8 ± 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The ΔΨ of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 ± 0.04 vs. 1.21 ± 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 ± 26.4 µm vs. 425.6 ± 25.0 µm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels.

Effect of a fertilization-promoting peptide on the fertilizing ability and glycosidase activity in vitro of frozen-thawed spermatozoa in the pig.

This study has evaluated the effect of fertilization-promoting peptide (FPP) on the fertilizing ability and glycosidase activity in vitro of frozen-thawed boar spermatozoa. Use of chlortetracycline (CTC) fluorescence analysis, as well as various glycosidase analyses and the oocyte penetration test showed that FPP can promote the fertilizing ability and glycosidase activity of frozen-thawed spermatozoa in vitro. There were significantly (P < 0.05) more acrosome-reacted and penetrated in medium with 100 nM FPP than with 0, 50, 200 or 400 nM. The beta-N-acetylglucosaminidase (beta-GlcNAcase) activity was at least two-fold higher than other glycosidase regardless of FPP concentrations. In the same glycosidase, there were no differences in medium with different concentrations of FPP. The percentages of spermatozoa that reached acrosome reaction were affected by different periods (0, 1, 2, 3 or 4 h) of spermatozoa preincubation and were higher in medium with than without FPP. Penetration rates were decreased with preincubation periods of spermatozoa when oocytes were inseminated with spermatozoa preincubated in medium with and without FPP for the different periods. These rates were higher in spermatozoa preincubated with that than without FPP and had a tendency to increase as time of culture periods when the sperm-oocyte were cultured for 4, 8, 12, 16, 20 or 24 h. The activities of alpha-fucosidase, alpha-mannosidase, beta-galactosidase and beta-GlcNAcase were higher in medium with that than without FPP regardless of periods of sperm preincubation and sperm-oocyte culture. These results suggest that FPP may have a positive role in promoting sperm function and glycosidase activity in the pig.

Comparison of the characteristics and multipotential and in vivo cartilage formation capabilities between porcine adipose-derived stem cells and porcine skin-derived stem cell-like cells.

OBJECTIVE To compare the characteristics and multipotential and in vivo cartilage formation capabilities of porcine adipose-derived stem cells (pASCs) with those of porcine skin-derived stem cell-like cells (pSSCs). ANIMALS Three 6-month-old female pigs and four 6-week-old female athymic mice. PROCEDURES Adipose and skin tissue specimens were obtained from each pig following slaughter and digested to obtain pASCs and pSSCs. For each cell type, flow cytometry and reverse transcription PCR assays were performed to characterize the expression of cell surface and mesenchymal stem cell markers, and in vitro cell cultures were performed to determine the adipogenic, osteogenic, and chondrogenic capabilities. Each cell type was then implanted into athymic mice to determine the extent of in vivo cartilage formation after 6 weeks. RESULTS The cell surface and mesenchymal stem cell marker expression patterns, multipotential capability, and extent of in vivo cartilage formation did not differ significantly between pASCs and pSSCs. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that pSSCs may be a viable alternative to pASCs as a source of progenitor cells for tissue engineering in regenerative medicine.

Antioxidant treatment during manipulation procedures prevents mitochondrial and DNA damage and enhances nuclear reprogramming of bovine somatic cell nuclear transfer embryos

We tried to prevent the mitochondrial and DNA damage caused by mechanical stress-associated reactive oxygen species (ROS), and to improve the reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos by antioxidant treatment during the manipulation procedures of SCNT. Bovine recipient oocytes and reconstituted oocytes were treated with antioxidants during manipulation procedures. The H2O2 level, mitochondrial morphology, membrane potential and apoptosis at the one-cell stage, and in vitro development and DNA methylation status of blastocysts were evaluated. Antioxidant treatment during manipulation procedures reduced the H2O2 level of SCNT embryos. Antioxidant-treated SCNT embryos normally formed mitochondrial clumps, similar to IVF embryos, and showed higher mitochondrial membrane potential versus the SCNT control (P<0.05). Apoptosis and DNA fragmentation were reduced by antioxidant treatment. The development rate to the blastocyst stage was higher (P<0.05) in the antioxidant treatment groups (30.5±2.5 to 30.6±1.6%) versus the control (23.0±1.9%). The DNA methylation status of blastocysts in the antioxidant treatment groups was lower (P<0.05) than that of the control and similar to that of IVF embryos. These results indicate that antioxidant treatment during manipulation procedures can prevent cellular damage that may be caused by mechanical stress-associated ROS, and improve nuclear reprogramming.

40 EFFECT OF CHEMICAL ACTIVATION ON DEVELOPMENT AND APOPTOSIS OF PREIMPLANTATION PORCINE EMBRYOS DERIVED FROM NUCLEAR TRANSFER

Activation is one of key factors for improving developmental ability of pre-implantation nuclear transfer (NT) embryos. This study investigated the effect of chemical activation following fusion/activation on the development and apoptosis of pre-implantation porcine embryos derived from NT. Oocytes were aspirated from ovaries collected from a local abattoir, and then matured in TCM-199 for 42 to 44 h. Donor cells were prepared from a 35-day-old porcine fetus. Matured oocytes were enucleated and donor cells were introduced into the perivitelline space. Fusion/activation was conducted with two electric pulse of 1.2 kV/cm for 30 µs. Fused embryos were divided into four groups. The first one was the control without chemical activation; the other three groups were treated with thimerosal (0.2 mM for 10 min; T) and then with dithiothreitol (8 mM for 30 min; DTT), 6-dimethylaminopurine (2 mM for 3 h; 6-DMAP), or cycloheximide (10 µg/mL for 6 h; CH). Treated embryos were cultured in porcine zygote medium-3 (PZM-3) at 38.5°C under 5% CO2 in air for 6 days. Cleavage and blastocyst rate were determined on Days 3 and 6, respectively. Apoptosis was analyzed with a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay from day 1 to 7. Embryos treated with chemicals following fusion/activation showed significantly higher blastocyst rates compared to control embryos fused/activated by electric pulse alone (12.6% for control vs. 21.1% for DTT, 20.8% for 6-DMAP, 20.6% for CH; P < 0.05). Although total cell number of blastocysts showed no significant difference, the ratio of inner cell mass to trophectoderm was significantly higher (P < 0.05) in embryos with chemical activation than in those without it (11.9 vs. 19.4, 18.1, and 24.1%; P < 0.05). Occurrence of apoptosis was first observed on Day 3, but there was no significant difference among treatments until Day 6. It was significantly increased in embryos with chemical activation on Day 7 compared to control embryos (5.1 vs. 7.1, 7.8, and 7.8%; P < 0.05). These results indicate that chemical activation following fusion/activation could support significantly a higher blastocyst rate for pre-implantation porcine embryos derived from nuclear transfer; however, it can increase occurrence of apoptotic cells at blastocyst stage.

Effect of L-Glutathione Treatment during Somatic Cell Nuclear Transfer Procedures on the Subsequent Embryonic Development and DNA Methylation Status of Cloned Bovine Embryos

We investigate the effect of L-glutathione (GSH), an antioxidant, treatment during the somatic cell nuclear transfer (SCNT) procedures on the in vitro development and DNA methylation status of bovine SCNT embryos. Bovine in vitro matured (IVM) oocytes were enucleated and electrofused with a donor cell, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine. The recipient oocytes or reconstituted oocytes were treated with 50 μM GSH during these SCNT procedures from enucleation to activation treatment. The SCNT embryos were cultured for 7 days to evaluate the in vitro development, apoptosis and DNA methylation in blastocysts. The apoptosis was measured by TUNEL assay and caspase-3 activity assay. Methylated DNA of SCNT embryos at the blastocyst stages was detected using a 5-methylcytidine (5-MeC) antibody. The developmental rate to the blastocyst stage was significantly higher (P<0.05) in GSH treatment group (32.5±1.2%, 78/235) than that of non-treated control SCNT embryos (22.3±1.8%, 50/224). TUNEL assay revealed that the numbers of apoptotic cells in GSH treatment group (2.3±0.4%) were significantly lower (P<0.05) than that of control (3.8±0.6%). Relative caspase-3 activity of GSH treated group was 0.8±0.06 fold compared to that of control. DNA methylation status of blastocysts in GSH treatment group (13.1±0.5, pixels/ embryo) was significantly lower (P<0.05) than that of control (17.4±0.9, pixels/embryo). These results suggest that antioxidant GSH treatment during SCNT procedures can improve the embryonic development and reduce the apoptosis and DNA methylation level of bovine SCNT embryos, which may enhance the nuclear reprogramming of bovine SCNT embryos.

193 DIFFERENTIAL EFFECTS OF CULTURE ON APOPTOTIC GENE EXPRESSION IN THE PRE-IMPLANTATION CLONED EMBRYOS OF MINIATURE PIG

Manipulations of early embryos require that the embryos be placed in vitro. The ability to reproduce in vivo conditions in vitro would greatly facilitate studies on the development of early embryos. A variety of different conditions have been described that result in development of pig embryos from the 1-cell stage to the blastocyst stage in vitro. There is a species-specific cell stage at which the early embryo is very sensitive to in vitro conditions, which generally corresponds to the stage at which the embryo begins producing significant amounts of RNA. The present study was conducted to investigate the relative amounts of apoptotic gene expression in miniature pig NT embryos under culture conditions of different osmolarity. Oocytes were cultured in TCM-199 for 40–44 h at 38.5°C under 5% CO2 in air. Miniature pig ear fibroblast cells were cultured to reach confluency, and the culture was continued for an additional 5–6 days. The NaCl group of embryos was cultured in PZM-3 supplemented with 138 mM NaCl in total concentration (280–320 mOsmol) for the first 2 days, and then cultured in PZM-3 (250–270 mOsmol) for a further 4 days. The control group of embryos was cultured in the PZM-3 for the entire period of in vitro culture. Total RNA samples were prepared from 2 blastocysts using the Roche 1st strand cDNA synthesis kit. Bax and Bcl-xl gene expression of blastocysts was analyzed by real-time RT-PCR. Developemntal rates were analyzed by a GLM procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Relative gene expression was compared by Students t-test. Blastocyst formation rate in the NaCl group was not different from that in the control group (25.4% and 23.2%, respectively), but the apoptosis rate was significantly lower (P < 0.05) in the NaCl group (1.6%) than in the control (7.1%). The relative abundance of Bax mRNA expression was significantly higher (P < 0.05) in the control group (n = 32) than in the NaCl group (n = 33). However, the relative abundance of Bcl-xl mRNA was significantly higher (P < 0.05) in NaCl group. The relative abundance of Bax/Bcl-xl was significantly higher in the control group than in the NaCl group (P < 0.05). These results indicate that the hypertonic culture condition at the early embryonic stage of miniature pig NT embryos could reduce the frequency of apoptosis through regulating Bax and Bcl-xl gene expression.

336 DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS PRODUCED USING SPERM CYTOSOLIC FACTOR (SCF) AT FUSION-ACTIVATION

At fertilization, the sperm activates the developmental program of the oocyte by inducing an elevation in the intracellular free Ca2+ concentration ([Ca2+]i). One possible explanation is that at sperm–oocyte fusion the fertilizing spermatozoon introduces a factor into the cytoplasm of the oocyte which opens the Ca2+ release channels from the intracellular stores through a yet unidentified mechanism. This study investigated the development of porcine nuclear transfer embryos fused and activated in the presence of sperm cytosolic factor (SCF) isolated from porcine sperm. Ovaries were collected at a local slaughterhouse and transported to the laboratory within 2 h at 35–39°C, and rinsed in 0.9% NaCl. Fetal fibroblast cells were prepared from a 35-day-old porcine fetus for use as donor cells. For parthenogenesis, matured oocytes were activated using 2 DC pulses of 1.2 kV cm-1 for 30 µs in fusion medium (0.1 mM CaCl2) supplemented with 100, 200, or 300 µg mL-1 SCF. For NT, matured oocytes were enucleated, reconstructed, and fused. Reconstructed embryos were divided into 2 groups. The embryos in one group were fused with fusion medium (1.0 mM CaCl2), and the embryos in the other group were fused with fusion medium (0.1 mM CaCl2) supplemented with 100 µg mL-1 SCF. After fusion, the embryos were cultured in PZM-3 under 5% CO2 in air at 38.5°C. A TUNEL assay was used to assess the presence of apoptotic cells (In Situ Cell Death Detection Kit, TMR red; Roche, Mannheim, Germany). Data were subjected to a generalized linear model procedure (PROC-GLM) of the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). Oocytes activated parthenogenetically in the presence of SCF showed significantly higher developmental rate to the blastocyst stage compared to that of controls (21.3–27.6% vs. 12.5%; P < 0.05). For NT, there was no difference between treatments in developmental rate to the blastocyst stage (18.2% vs. 17.1%). However, the apoptosis rate was slightly lower in blastocysts produced in the presence of SCF than that in controls. These results indicate that the presence of SCF in fusion medium can support a higher quality of porcine nuclear transfer embryos.

332 Embryonic development after somatic cell nuclear transfer of porcine oocytes meiotically inhibited with roscovitine.

Numerous factors affect on the developmental competence of cloned embryos, and one of the factors might be the disturbed synchronization of nuclear and cytoplasm maturation. Roscovitine, a purine known to specifically inhibit M-phase promoting factor (MPF) kinase activity by blocking the ATP in numerous cell systems, has been successfully used in maintaining porcine oocytes at GV stage without affecting their developmental potential. However, developmental ability of roscovitine treated porcine oocytes after nuclear transfer has not been evaluated. The purpose of this study was to examine the development of nuclear transferred porcine embryos after meiotic inhibition with roscovitine (ROS). Cumulus-oocyte complexes (COCs) were collected from antral follicles of slaughtered prepubertal gilts. COCs were cultured in pre-maturation medium (TCM-199 containing 50 ¼M Roscovitine) for 24 h, and then further cultured in conventional maturation medium for 44 h. A control group was cultured in the maturation medium for 44 h. Matured oocytes were enucleated and a porcine fetus cell was inserted into each enucleated oocyte. Couplets were simultaneously fused and activated with electric pulse of two 1.2 kV/cm for 30 ¼s. Nuclear transferred (NT) embryos were cultured in PZM-1 medium for 6 days (five replicates). Apoptotic cell death was analyzed by using a TUNEL assay and total cell number was examined by Hoechest 33342 counterstaining. At 3 h after fusion, NT embryos were fixed for microfilament staining. Data were analyzed by ANOVA and Students t-test. The rates of fusion, cleavage, and blastocyst formation of the ROS-treated group (85, 68, and 18%, respectively) after nuclear transfer did not differ from control (78, 76, and 16%, respectively). The cell number in blastocysts of the ROS-treated group (30.8 ± 10.6) was significantly lower than that of the control (42.3 ± 13.7) (P < 0.01), but the mean proportion of apoptotic cells was not different between the two groups (6.9 ± 7.1 and 4.8 ± 4.9% for control and ROS group, respectively). Recovery of microfilaments after fusion was delayed in NT embryos derived from ROS-treated oocytes. This study demonstrated that porcine oocytes pre-cultured for 24 h in presence of roscovitine can be developed to blastocysts after somatic cell nuclear transfer. This could provide flexibility for studying porcine oocyte development and embryo cloning.

163 SUCROSE ADDITION AT EARLY CULTURE STAGE IMPROVES DEVELOPMENT OF PRE-IMPLANTATION PORCINE NT AND IVF EMBRYOS

Apoptosis is a form of cell death leading to fragmentation of the DNA, shrinkage of the cytoplasm, membrane changes, and cell death without lysis or damage to neighboring cells. It might contribute to the low developmental rate of in vitro-produced (IVP) embryos, but apoptosis in porcine embryos is still unclear. This study investigated the effect of sucrose in the culture medium on the development of porcine NT and IVP embryos. Oocytes were aspirated from the follicles in ovaries collected from a local abattoir, and then matured in TCM-199 for 40-44 h. Fresh semen was diluted and equilibrated at 16°C. A final concentration of motile spermatozoa was adjusted to 5 × 105 cells/mL in fertilization medium. Fetal fibroblast cells were prepared from a 35-day-old porcine fetus and used as donor cells. Embryos were cultured in PZM-3 supplemented with 0.05 M sucrose for 2 days, and then cultured in the PZM-3 without sucrose for 4 days at 38.5°C under 5% CO2 in air. Apoptotic cell death was analyzed by using a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. All data were subjected to a generalized linear model procedure (PROC-GLM) of the statistical analysis system (SAS; SAS Institute, Inc., Cary, NC, USA). NT and IVF embryos cultured in the medium supplemented with sucrose showed a significantly higher blastocyst formation rate than those cultured with no addition (28.8 vs. 20% and 32.3 vs. 17.9%; P < 0.05, respectively). For apoptosis, both NT and IVF embryos cultured in the medium with sucrose showed significantly lower frequency of apoptosis compared to embryos cultured in the medium without sucrose (3.4 vs. 6.3% and 0.6 vs. 1.8%; P < 0.05, respectively). Finally, the number of nuclei in NT blastocysts cultured in the medium with sucrose was higher than that of NT blastocysts cultured without sucrose (30.8 vs. 25.5; P < 0.05, respectively). However, the number of nuclei in the IVF blastocysts was not significantly different between groups. These results indicate that sucrose addition may increase the development of porcine NT and IVF embryos to the blastocyst stage and decrease the rate of apoptotic cells.