• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.47-47
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• 2003

The present study was conducted to determine the expression of the antioxidant enzyme(CuZn-SOD, Mn-SOD and GPX and apoptosis gene(caspase-3) for in vitro culture in in vitro maturation and in vitro fertilization(IVM/IVF) embryos in porcine. Porcine embryos derived from IVM/IVF were cultured in NCSU23 medium under 5% $CO_2$ in air at 38.5$^{\circ}C$. The patterns of gene expression for several antioxidant enzyme and apoptosis genes during preimplantion porcine embryo development were examined by the modified semi-quantitative single cell reverse transcriptase- polymerase chain reaction (RT-PCR). Preimplantation porcine embryos produced by IVM/IVF have expressed mRNAs for CuZn-SOD and GPX, whereas transcripts for Mn-SOD have not detected at any developmental stages. Expression of caspase-3 mRNA was detected at 2 cell, 8 cell, 16 cell and morula stages. The fas ligand transcripts were detected in porcine blastocyst. These results suggest that various antioxidant enzymes and apoptosis genes play crucial roles in in vitro culture of porcine IVM/IVF embryos.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.183-187
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• 2009

In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro fertilization (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22h post insemination were treated at different concentration of actinomycin D (0, 5, 50 and 500 ng/ml in NCSU medium). Treated embryos were incubated at $39^{\circ}C$ in 5% $CO_2$, 5% $O_2$ for 8h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2days and BL development rate at 7days after in vitro culture. A significantly lower rate of cleavage was found in the 500 ng/ml group compared to others (500 ng/ml vs. 0, 5, 50 ng/ml; 27.8 % vs. 50.0%, 41.2%, 35.9%), and BL formation rate in 500 ng/ml was lower than that of others (500 ng/ml vs. 0, 5, 50 ng/ml; 8.0% vs. 12.6%, 11.2%, 12.6%). In experiment 2, to evaluate apoptotic cells, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng/ml actinomycin D). A high number of BL derived control group contained at least one apoptotic cell. Actinomycin D treated BLs responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and increase in the incidence of apoptotic cell death. In 500 ng/ml group, the incidence of apoptosis increased at 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is useful tool for the apoptosis study of porcine preimplantation embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.1
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• pp.33-38
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• 2004

This study was aimed at testing the gene expression of antioxidant enzymes and apoptosis genes for in vitro culture in porcine embryos produced by in vitro maturation/in vitro fertilization (IVM/IVF). Pocine preimplantation embryos obtainted from IVM/IVF can be successfully culture in vitro, but they are delayed or stop to develop at specific developmental stage. Many factors such as reactive oxygen species and apoptosis in an IVM/IVF system followed by in vitro culture influence the rate of production of viable blastocysts. Porcine embryos derived from IVM/IVF were cultured in the atmosphere of 5% $CO_2$ and 20% $O_2$ at $38.5^{\circ}C$ in NCSU23 medium. The patterns of gene expression for antioxidant enzymes and apoptosis genes during in vitro culture in pocine IVM/IVF embryos were examined by the modified semi-quantitative single cell reverse transcriptase-polymerase chain reaction (RT-PCR). Porcine embryos produced by in vitro procedures were expressed mRNAs for CuZn-SOD, GAPDH and GPX, whereas transcripts for Mn-SOD and catalase were not detected at any developmental stages. Expression of caspase-3 mRNA was detected at 2 cell, 8 cell 16 cell and blastocyst, but p53 mRNA was not detected at any stages. The fas transcripts was only detected in blastocyst stage. These results suggest that various antioxidant enzymes and apoptosis genes play crucial roles in vitro culture of porcine IVM/IVF embryos.

• Reproductive and Developmental Biology
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• v.33 no.1
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• pp.7-11
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• 2009

This study was performed to confirm the microtubule assemblies and methylation patterns of porcine IVF and parthenogenetic embryos. Cumulus-oocyte complexes were collected and matured in vitro for 42 hr. Oocytes were fertilized by prepared fresh sperm or activated parthenogenetically by exposure to electric stimulation and 6-dimethylaminopurine. Porcine IVF and parthenogenetic embryos were cultured in vitro for 6 days. Embryos were stained by immunofluorescence staining method to observe the dynamic of nucleus and microtubules in the first mitotic phase and the methylation patterns in different developmental stages. After then, samples were confirmed and analyzed through a laser-scanning confocal microscope. IVF embryos had a centrosome originated from sperms, which was shown a $\gamma$-tubulin spot. However, $\gamma$-tubulin spot was not observed in parthenogenetic embryos. A lower methylation level was observed in IVF embryos compared to parthenogenetic ones at the morula and blastocyst stages. In conclusion, it is considered that microtubule assemblies and genetic regulation mechanism differ between parthenogenetic and IVF embryos.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.83-90
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• 2007

Insulin-like growth factor II (IGF2) and H19 genes are mutually imprinted genes which may be responsible for abnormalities in the cloned fetuses and offspring. This study was performed to identify putative differentially methylated regions (DMRs) of porcine H19 locus and to explore its genomic imprinting in in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) embryos. Based on mice genomic data, we identified DMRs on H19 and found porcine H19 DMRs that included three CTCF binding sites. Methylation patterns in IVF and SCNT embryos at the 2-, 4-, $8{\sim}16$-cells and blastocyst stages were analyzed by BS (Bisulfite Sequencing)-PCR. The CpGs in CTCF1 was significantly unmethylated in the 2-cell stage IVF embryos. However, the 4- (29.1%) and $8{\sim}16$-cell (68.2%) and blastocyst (48.2%) stages showed higher methylation levels (p<0.01). On the other hand, SCNT embryos were unmethylayted ($0{\sim}2%$) at all stages of development. The CpGs in CTCF2 showed almost unmethylation levels at the 2-,4- and $8{\sim}16$-cell and blastocyst stages of development in both IVF ($0{\sim}14.1%$) and SCNT ($0{\sim}6.4%$) embryos. At all stages of development, CTCF3 was unmethylated in IVF ($0{\sim}17.3%$) and SCNT ($0{\sim}1.2%$) embryos except at the blastocyst stage (54.5%) of IVF embryos. In conclusion, porcine SCNT embryos showed an aberrant methylation pattern comprised to IVF embryos. Therefore, we suggest that the aberrant methylation pattern of H19 loci may be a reason for increased abnormal fetus after embryo transfer of porcine SCNT embryos.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.91-96
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• 2007

In vitro development of porcine embryo is affected by culture condition. One possible factor is osmolarity of culture medium. 1his study examined whether high osmolarity of culture medium at the early culture stage improves development of preimplantation porcine in vitro fertilization (IVF) and nuclear transfer (NT) embryos. NT and IVF embryos were divided into three groups and the basic medium was PZM-3 ($250{\sim}270$ mOsmol, control group). The control group of embryos was cultured in PZM-3 for whole culture period. Other two groups of embryos were cultured in a modified PZM-3 with 0.05 M sorbitol or 0.05 M sucrose ($300{\sim}320$ mOsmol, sorbitol or sucrose group) for the first 2 days, and then cultured in PZM-3 for further culture. NT embryos cultured in sucrose group showed a significantly higher developmental rate to the blastocyst stage with a decreased apoptosis rate compared to the sorbitol (p<0.05). For IVF, sucrose group showed a significantly increased the blastocyst formation rate with a decreased apoptosis rate compared to the control (p<0.05). This study represents that the high osmolarity in the early embryo culture stage can enhance the in vitro development of porcine NT and IVF embryos to the blastocyst stage with reduced apoptosis of cells.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.289-297
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• 2000

This study was conducted to investigate whether various protein sources and co-culture affect in vitro development of porcine zygotes derived from In vitro maturation/fertilization (IVM/IVF). These results obtained in these experiments are summarized as follows 1. When porcine oocytes matured and fertilized In vitro were cultured in NCSU 23 medium supplemented with various BSA concentrations (0.4, 0.8 and 3.2%), In vitro developmental rates of porcine zygotes to blastocyst stage were 22.9, 18.4 and 14.6%, respectively. High concentration of BSA (3.2%) showed a smaller nuclei number (36.1$\pm$11.8) of blastocysts than 0.4 and 0.8% BSA groups (53.2$\pm$27.4 and 61.2$\pm$22.5, respectively) (P<0.05). This result indicates that high concentration of BSA is detrimental on preimplantation development of IVF-derived porcine embryos. 2. No differences were detected in the developmental rate and mean nuclei number of porcine embryos between 10 and 20% FBS concentrations in culture medium. 3. IVF-derived porcine embryos co-cultured with mouse or porcine embryonic fibroblast cells showed a lower development to the blastocyst stage than those without co-culture system. Consequently, the present study suggests that high concentration of BSA as a protein source in culture medium suppresses development potential of porcine embryos produced In vitro. In addition, co-culture with somatic cells is not effective on in vitro development of IVF-derived porcine embryos to blastocyst stage.

• Reproductive and Developmental Biology
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• v.31 no.1
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• pp.49-53
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• 2007

Astaxanthin is a kind of carotenoid compounds, having a antioxidant and anti-inflammatory activities. The antioxidative mechanism by which carotenoid scavenge free radicals has been clearly elucidated, but has not tried for the development of mammalian preimplantation embryo. This study was conducted to investigate the antioxidative effect of astaxanthin on in vitro development of porcine in vitro fertilized embryos. Porcine embryos derrived from in vitro fertilization (IVF) were cultured in 5% $CO_{2} in air at $38.5^{\circ}C$ in PZM-3 medium supplemented with different dosages of astaxanthin ($0,\;1,\;5\;and\;10{\mu}M$) and taurine (0, 1, 2.5 and 5 mM) as a positive control, and execute to compare the effects of various antioxidants such as taurine, melatonin and asculatin on in vitro development. The proportions of embryos developed to the blastocyst stage were increased when $1\;and\;5\;{\mu}M$ of astaxanthin (26.6 and 23.4%, respectively) and 1 and 2.5 mM taurine (25.8 and 26.4%, Respectively) were supplemented, compared to controls (p<0.05). Also, various antioxidant-treated groups were significantly higher rates of blastocysts (astaxanthin, 27.4%; taurine, 29.1%; melatonin, 26.8%; aesculetin, 27.9%, respectively) than control (18.8%). There was no difference in mean cell number of blastocysts between antioxidants and control. This result indicates that astaxanthin has an antioxidant feature when porcine IVF embryos were cultured in vitro.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.201-210
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• 1996

This experiment was carried out to evaluate the effect of early mouse embryonic development in vitro by co-culture with bovine and porcine oviductal epithelial cells (BOEC and POEC). The 2-cell embryos were collected from the oviducts of the superovulated and mated cultured in D-PBS /15% FCS at 48 hours after hCG injection. The in vitro developmental rate of blastocyst formation in the embryos were examined under the fllowing treatments; 1) TCM 199 added 15% HCS, 2) Ham's F-10 added 15% HCS, 3) MediCult IVF medium, 4) TCM 199 added 15% HCS + BOEC, 5) TCM 199 added 15% HCS + POEC, 6) Ham's F40 added 15% HCS + BOEC, 7) Ham's F-10 added 15% HCS + POEC,8) MediCult IVF medium + BOEC, 9) MediCult IVF medium + POEC. For a comparative study of in vitro development for 96 hours after hCG injection, were cultured with oviductal epithelial cell and media only. The obtained results were 2-cell embryos developed to the blastocyst stage in TCM 199, Ham's F-10 and MediCult IVF medium at the rates of 84.4,83.2 and 81.6%. respectively. The higher developmental rates(91~97%) of blastocyst formation was appeared when the embryos were co-cultured with a monolayer of bovine or porcine oviductal epithelial cells in TCM 199 or Ham's F-10 and MediCult IVF media. No significant difference in developmental rates was shown between bovine and porcine oviductal epithelial cells but significant difference in co-culture system in comparison between media only system and co-cultures. In conclusions, oviductal epithelial cells, BOEC and POEC, when co-culture with mouse early embryos improved the rates of development, blastocyst and hatching. Therefore, it is suggested that co-culture system using oviductal epithelial cells improve early embryonic developtnent in mouse.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.59-66
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• 2014

Transcription activator like effector nucleases (TALENs) are artificial restriction enzymes generated by fusing a TALE DNA binding domain to a DNA cleavage domain which remove and introduce specific genes to produce transgenic animals. To investigate the efficient laboratory techniques for the injection of TALEN mRNA, pEGFP-N1 commercial plasmid were microinjected into porcine parthenogenetic and in vitro fertilization (IVF). In Experiment 1, to investigate injection time, compared 4 different time durations (2 hr, 4 hrs, 6 hrs & 8 hrs) after post activation of parthenogenetic embryos and after 6 hrs of co-incubation with sperms in IVF embryos. There were significant difference (P<0.05) in development to the blastocysts (4.4, 8.9, 3.9, 0.6%), GFP expression in blastocysts (1.3, 5.7, 2.3, 0.0%) which injected after post activation of 4 hrs compared with other 3 groups. IVF embryos after 2 hrs and 4 hrs injected were expressed GFP significantly higher than rest of two groups (P<0.05). In Experiment 2, compared development of 2 different concentrations ($20ng/{\mu}l$ and $50ng/{\mu}l$) of EGFP injection. There were significant difference (P<0.05) between two treatments which has higher cleavage (58.8 vs 41.9%), blastocysts development rate (13.0 vs 11.1%) and GFP expressed blastocysts (5.7 vs 0.0%) in $20ng/{\mu}l$ than the $50ng/{\mu}l$ in parthenogenetic embryos. In IVF embryos, only $20ng/{\mu}l$ injected embryos were expressed GFP (4.2%) after 7 days of incubation and 77.3 vs 64.7% of cleavage, 26.4 vs 23.5% development to blastocysts. In Experiment 3, three different volumes (5, 10 and 20 pl) were microinjected into porcine embryos to determine the most appropriate volume. Out of 3 groups, significantly higher development rates of cleavage (68.3, 58.0, 29.3%), blastocysts (11.7, 12.7, 0.5%) and GFP expressed blastocysts (2.9, 7.8, 0.0%) were shown in the 10 pl group (P<0.05). In conclusion, these results imply that $20ng/{\mu}l$ concentration, 10 pl of volume and injection at 4 hrs after post activation for parthenogenetic and 2~4 hrs after IVF, $20ng/{\mu}l$ concentration and 10 pl volume for IVF embryos were more effective microinjection conditions.