B. C. Yang

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.

102 PROTEOMIC ANALYSIS OF CONDITIONED MEDIUM SUPPLEMENTED WITH PORCINE FOLLICULAR FLUID

Follicular fluid (FF) contains growth factors, electrolytes, hormones, amino acids, and unknown factors. Supplementation of porcine FF (pFF) to in vitro maturation (IVM) medium was reported to improve the oocyte maturation, monospermic fertilization and embryonic development. This study aimed at investigating whether pFF supplementation affects the characteristics of donor cells for somatic cell nuclear transfer and the proteomic composition of the culture medium. Ear fibroblast cells from an NIH major histocompatibility complex (MHC) inbred miniature pig were cultured with different culture methods: 1) DMEM + 10% FBS (FBS); 2) DMEM + 10% FBS + 10% pFF (pFF). The conditioned medium was collected at 72 h. After isoelectric focusing (IEF), the equilibrated strips were submitted to SDS-PAGE. Normalized protein spots were considered significantly different between the two groups if expression levels varied by two standard deviations. To identify the protein spots, an Ettan MALDI-TOF method was used. Upon submission of the amino acid sequences, proteins were identified by a homology search using ProteinInfo or BLAST search using the ExPASy Molecular Biology Server. The proportion of G0/G1 stage cells in the pFF group was significantly higher than the proportions in the other groups (P < 0.05). Among 42 differentially expressed spots, 36 proteins were identified in the pFF group. Some molecular functions of the spots were: catalytic or methytransferase activity, eukaryotic cell surface binding, or ferric iron binding. It can be concluded that pFF supplementation of culture medium positively affects cell-cycle synchronization and cell metabolism. Further studies are needed to analyse the function of these important cellular proteins. This work received grant support from the Agenda Program (No. PJ006688) and (No. PJ007189), Rural Development Administration, Republic of Korea.

58 DEVELOPMENT OF α1,3-GALACTOSYLTRANSFERASE KNOCK-OUT CLONED PIG EMBRYOS

This study was performed to increase the developmental rate of cloned embryos with the 1,3-Galactosyltransferase (GalT) gene knocked out (KO). Ovaries were collected from local slaughterhouse and immature oocytes were cultured in TCM-199 + 0.1% PVA + FSH + LH (0.5 μg mL-1) + EGF (10 ng mL-1) + 10% porcine follicular fluid (pFF) at 38.5°C in 5% CO2 humidified chamber for 40 h (1-step) or 20 h (with hormone) +20 h (without hormone; 2-step). After IVM, the oocytes with 1st polar body were enucleated and transferred the GalT KO donor cell originated from miniature pig. The embryos transferred with normal mini-pig ear fibroblast cell were used as control. The reconstructed embryos were fused with 2 electric pulses (DC) of 1.2 kV cm-1 for 30 μs. For the development of cloned embryos, the embryos were cultured in PZM-3 under 5% CO2 in air at 38.5°C for 6 days. The embryos were transferred to a surrogate (Landrace) at an earlier stage of the estrus cycle, and pregnancy diagnosis was determined at 28 days after embryo transfer using ultrasonography. Differences among treatment means were determined by a chi-square test. A probability of P < 0.05 was considered statistically significant. The maturation rate was significantly higher in the 2-step method (89.8 ± 2.75) compared with single maturation method (79.6 ± 8.95; P < 0.05). The blastocyst development of cloned embryos reconstructed with GalT KO donor cell (28.4 ± 2.14) was not different from cloned embryos by normal donor cell (27.4 ± 0.01). The cell number of GalT blastocyst (36.1 ± 11.1) was not different statistically from control (26.9 ± 9.3). The apoptosis rate was also not different in both groups (2.9 to 4.9%). Five surrogates were pregnant and the GalT KO fetuses were still ongoing pregnancy at 45 days after embryo transfer. This work received grant support from the Agenda Program (No. 200901FHT010305146 and No. 200901FHT010305535), Rural Development Administration, Republic of Korea.

258 CLASSIFICATION AND IDENTIFICATION OF THE CLONED HANWOO CALVES DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER USING MS GENOTYPES AND mtDNA POLYMORPHISMS

The calves produced by somatic cell nuclear transfer (SCNT) had identical genetic background except for cytoplasmic transmission molecules, which originated from recipient oocytes. To identify the SCNT calves generated from 3 nucleus donors, genotypes for 16 microsatellite (MS) markers and mitochondrial DNA (mtDNA) polymorphisms were analyzed. Using MS genotypes, the parentage test results were not only classified as the single donor-derived calves but mtDNA sequence variations might also discriminated all SCNT calves within the donor-derived families. Comparing MS genotypes, AI-derived progenies were easily discriminated from each other. However, those genotypes could not supply the useful information for identifying the SCNT calves produced from each donor. Informative sequence variations were detected in several regions including D-loop, 12 S rDNA, and ND5 genes. About 19 nucleotide substitutions found within D-loop allowed individual identification for most SCNT-derived progeny except for 5 individuals. However, further investigation on 12 S rDNA and ND5 genes provided the useful polymorphic information for those 5 individuals. Although the experiment had been carried out to produce SCNT calves without previous investigation of mtDNA polymorphism, polymorphic mtDNA sequences provided interesting information that discriminated individuals, even those from the same donor cells. In addition, we could distinguish the 2nd generations produced by AI combinations using SCNT donors and SCNT progeny as the dams and/or the sires when combined molecular data obtained from MS genotypes and mtDNA polymorphisms were derived. These results suggested that mtDNA polymorphisms might supply the critical information for identification and traceability for SCNT-derived calves when combined with MS data. This work received grant support from the Agenda Program (No. 200901FHT010305191), Rural Development Administration, Republic of Korea.

271 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN A 35-DAY-OLD CLONED PIG FETUS USING THE ANNEALING CONTROL PRIMER SYSTEM

Somatic cell nuclear transfer in pig has limitations due to the high incidence of fetal failure after embryo transfer to recipients. Reasons for the inefficient cloning are assumed to be due to abnormal and poorly developed placenta. Thus, this study was designed to determine possible genetic causes of neonatal deaths and other related abnormalities. Genes expressed specifically or prominently on Day 35 were identified in cloned pig placenta utilizing PCR technology regulated by annealing control primers (ACPs). The RNA was isolated using Trizol reagent. By utilizing 120 ACPs, 53 expressed sequence tags (ESTs) of genes that are differentially expressed in cloned pig placenta compared with normal placenta were cloned and sequenced. The cloned genes or ESTs exhibited significant sequence similarities to known genes or ESTs of other species. Ten of the total known genes, i.e., pregnancy associated glycoprotein, H19, 60S ribosomal protein L12, 20-beta hydroxysteroid dehydrogenase, beta galactosidase precursor, aldehyde reductase, glypican 3 precursor, heme oxygenase 2, granulin precursor, and placenta-expressed transcript protein, were selected and their specific expression levels were confirmed by real-time RT-PCR in the normal and cloned pig placentas in triplicate using beta-actin for determining relative expression. The 60S ribosomal protein L12 and heme oxygenase 2 were highly expressed in the cloned pig placenta, whereas pregnancy associated glycoprotein, H19, 20-beta hydroxysteroid dehydrogenase, beta galactosidase precursor, aldehyde reductase, glypican 3 precursor, granulin precursor, and placenta-expressed transcript protein were low or void. Our data suggest that the ACP system effectively identified tissue-specific genes in cloned pig placenta. Furthermore, identified genes would assist in developing insight into the genetic basis of fetal failure and help in resolving low pregnancy rate in the production of cloned pigs.

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.

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.