The embryonic genome activation (EGA) is genetically activated states that embryos make the materials such as growth factors for using themselves. EGA is various because they have many materials, different site, different stage, also different species. At this time, transcription factors are expressed. Transcription factors bind to specific DNA region, and regulate the gene expression. Thus, we check the expression of transcription factors, we can know that embryo development is very well or not. The development stages of embryos are basically the stages from fertilization to blastocyst. So, we check the embryos oocyte to blastocyst. In our experiments, we focus the early developmental transcription factors such as Cdx2, Oct4, Sox2, Nanog and E-Cadherin. Above antibody factors showed different expression sites, and there were many differentiated parts from other animal species. In addition, we compared the SCNT and parthenogenetic activation (PA) because these are same methods using electrical activation among the embryo production methods. Our results showed not only similar patterns but also different patterns between pig and mouse. Therefore, we have to investigate that different patterns of transcription factors play a role in pigs, and why occur.

Xenotransplantation is proposed as a solution to the problem of organ shortage. However, transplantation of xenogeneic organs induces an antigen-antibody reaction in α-1,3-gal structure that are not present in humans and primates, and thus complement is also activated and organs die within minutes or hours. In this study, we used FasL gene, which is involved in the immune response of NK cell, and US11, which suppresses MHC Class I cell membrane surface expression, to inhibit cell mediated rejection in the interspecific immunity rejection, and also hDAF(CD55) was introduced to confirm the response to C3 complement. These genes were tranfeced into Korean native pig fetal fibroblasts using pCAGGS vector. And cytotoxicity of NK cell and human complement was confirmed in each cell line. The US11 inhibited the cytotoxicity of NK cell and, in addition, the simultaneous expression of US11 and Fas ligand showed excellent suppress to T-lymphocyte cytotoxicity, hDAF showed weak resistance to cytotoxicity of natural killer cell but not in CD8+ CTLs. Cytotoxicity study with human complement showed that hDAF was effective for reducing complement reaction. In this studies have demonstrated that each gene is effective in reducing immune rejection.

Embryo development is very important in reproductive physiology of domestic animal experiments. Therefore, in the above experiment, we want to provide a lot of important information with regard to fertilization breeding by looking at the expression of transcription factor by early embryo development. It is known that mice affect early embryonic development of many transcription factors, many experiments are underway. Different types of mammals showed different expression patterns, thus, we used pigs, which are known to be the most similar to humans, to observe the expression of transcription factors in early embryonic development. Transcription factors were observed using CDX2, OCT4 and E-CADHERIN. CDX2 was expressed in 2 cells, OCT4 and E-CADHERIN were expressed in blastocyst. OCT4 was expressed specifically in ICM (inner cell mass) in blastocyst, and E-CADHERIN was expressed in cell wall and junction of blastocyst. These results show that CDX2, OCT4 and E-CADHERIN play an important role in early embryonic development in pigs.

Porcine litter size is a quantitative trait and its heritability is especially low. So it is necessary to identify porcine reproductive gene and protein. The establishment of pregnancy requires performance of a receptive endometrium. We analyzed the endometrial tissue protein of porcine and would find out biomarker proteins related to porcine litter size. We sorted the two groups according to litter size of porcine: a small litter size group (SLSG) (n=2) and a large litter size group (LLSG) (n=2). The porcine endometrial tissue samples were analyzed separately using 2-dimensional electrophoresis (2-DE) within the isoelectric point ranges of 3.0 to 10.0, and then differential proteins were identified using MALDI-TOF analysis. In comparison of SLSG(small litter size group) with LLSG(large litter size group), a total of 9 protein spots differentially expressed on porcine endometrial tissue 2-DE gels, among which 5 spots were up-regulated proteins as retinol dehydrogenase 16-like isoform 1, Acrosin-binding protein, alpha-N-acetylgalactosaminidase. phosphoglycerate kinase 2, Acrosin-binding protein in LLSG. And 4 spots were up-regulated proteins as phosphoglycerate kinase 2, prenylcysteine oxidase in SLSG.

Pigs are considered an ideal source of human disease model due to their physiological similarities to humans. However, the low efficiency of in vitro embryo production (IVP) is still a major barrier in the production of pig offspring with gene manipulation. Despite ongoing advances in the associated technologies, the developmental capacity of IVP pig embryos is still lower than that of their in vivo counterparts, as well as IVP embryos of other species (e.g., cattle and mice). The efficiency of IVP can be influenced by many factors that affect various critical steps in the process. The previous relevant reviews have focused on the in vitro maturation system, in vitro culture conditions, in vitro fertilization medium, issues with polyspermy, the utilized technologies, etc. In this review, we concentrate on factors that have not been fully detailed in prior reviews, such as the oocyte morphology, oocyte recovery methods, denuding procedures, first polar body morphology and embryo quality.

Embryonic genome activation (EGA) is a highly complex phenomenon that is controlled at various levels. New studies have ascertained some molecular mechanisms that control EGA in several species; it is apparent that these same mechanisms regulate EGA in all species. Protein phosphorylation, DNA methylation and histone modification regulate transcriptional activities, and mechanisms such as ubiquitination, SUMOylation and microRNAs post-tran-scriptionally regulate development. Each of these regulations is highly dynamic in the early embryo. A better under-standing of these regulatory strategies can provide the possibility to improve the reproductive properties in mammals such as pigs, to develop methods of generating high-quality embryos in vitro, and to find markers for selecting de-velopmentally competent embryos.

Epigenetic status of the genome of a donor nucleus has an important effect on the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). In our previous study has results showed that the donor cells treated with 5-aza-2’- deoxyctidine (5-aza-dC, DNA methylation inhibitors) and Trichostatin A (TSA, histone deacetylase inhibitors) could improve the development of porcine nuclear transfer embryos in vitro. In this study we want to investigate why these two drugs treatment with the donor cell can improve the cloning efficiency, whether they can alter the epigenetic status of the genome of the donor nucleus. This study included 6 groups: control group, the donor cell (porcine fetal fibroblast cell) with no treatment; 2.5 nM 5-aza-dC group, the donor cells treated with 2.5 nM 5-aza-dC for 1h; 5-aza-dC group, the donor cells treated with 5 nM 5-aza-dC for 1h; TSA group, the donor cells treated with 50 nM TSA for 1h; 2.5 nM 5-aza-dC+TSA group, the donor cells treated with 2.5 nM 5-aza-dC for 1h and subsequently treated with 50 nM TSA for another 1h; 5-aza-dC+TSA group, the donor cells treated with 5 nM 5-aza-dC and 50 nM TSA together for 1h. The first experiment detected the DNA methylation status in the different groups. After treatment with these two drugs, the DNA methylation level of the donor cells decreased, however there is no significant difference among the groups. This result indicated that the donor cell treatment with 5-aza-dC and TSA can partially alter the DNA methylation status of the donor cells. The second experiment checked the histone acetylation level of the donor cells treated with these two drugs by western blot. TSA, 2.5 nM 5-aza-dC+TSA, 5 nM 5-aza-aC+TSA, these three groups can significantly improve the hisone acetylation level compared with control and 5-aza-dC groups, there is no significant difference among these three groups. The results of this study suggest that the donor cells treated with 5-aza-dC and TSA can partially decrease DNA methylation and can significantly improve the histone acetylation level of the donor cells, these alterations of the epigenetic modification maybe can improve the clonging efficiency.

In all the studies of mammalian species, chromatin in the germinal vesicle (GV) is initially decondensed with the nucleolus not surrounded by heterochromatin (the NSN configurations). During oocyte growth, the GV chromatin condenses into perinucleolar rings (the SN configurations) or other corresponding configurations with or without the perinucleolar rings, depending on species. During oocyte maturation, the GV chromatin is synchronized in a less condensed state before germinal vesicle breakdown (GVBD) in species that has been minutely studied. As not all the species show the SN configuration and gene transcription always stops at the late stage of oocyte growth, it is suggested that a thorough condensation of GV chromatin is essential for transcriptional repression. Because the GV chromatin status is highly correlated with oocyte competence, oocytes must end the NSN configuration before they gain the full meiotic competence and they must take on the SN or corresponding configurations to stop gene transcription before they acquire the competence for early embryonic development. In this study, we firstly investigated whether the follicle size could determine chromatin configuration in porcine oocyte. For this experiment, follicles was divided into three groups (<1 mm follicle, 1～3 mm follicle and 3～6 follicle). Using DAPI staining, the GV nucleolus and chromatin of porcine oocytes was classified into SN, SN-NSN and NSN configurations. MⅠ and M Ⅱ of three groups's Mature oocytes by staining was confirmed the configuration of chromatin. The maturation rate and parthenogenetic development potential were significant different between the SN and NSN configurations oocytes. These results indicated that chromatin changes in GV oocytes affect the development potential of porcine embryos.

Live offspring is obtained from in vitro production of porcine embryos, but the procedure is still associated with great inefficiencies. In mammalian oocytes, acquisition of meiotic competence coincides with a decrease in general transcriptional activity at the end of the oocyte growth phase. In this study, we investigated the expression and sub-cellular localization of positive transcription elongation factor P-TEFb (CDK9/Cyclin T1), a RNA polymerase II CTD kinase during pig oocyte growth and early embryonic development. Localization and expression of components involved in mRNA and rRNA transcription were assessed by immunocytochemistry in growing and fully-grown oocytes. In addition, meiotic resumption, germinal vesicle breakdown, nuclear transcription and embryonic genome activation (EGA) were analyzed in oocytes and embryos cultured in presence of a potent CDK9 inhibitor, flavopiridol. Our analyses, demonstrated that CDK9 became co- localized partially with phosphorylated Pol II CTD and mRNA splicing complexes. Surprisingly, CDK9 was co-localized with Pol I-specific transcription factor, UBF, and gradually localized in nucleolar peripheries at the final steps of oocyte growth. Later, CDK9 became associated with nucleolar structures at 4-cell stage. Treatment with flavopiridol resulted in arrest in meiotic resumption, germinal vesicle breakdown as well as a decline in global transcription. Flavopiridol also inhibited embryo development beyond EGA. All together, these data suggest that CDK9 has a dual role in both Pol I- and Pol II-dependent transcription in pig oocyte growth and embryonic development.

In all the studies of mammalian species, chromatin in the germinal vesicle (GV) is initially decondensed with the nucleolus not surrounded by heterochromatin (the NSN configurations). During oocyte growth, the GV chromatin condenses into perinucleolar rings (the SN configurations) or other corresponding configurations with or without the perinucleolar rings, depending on species. During oocyte maturation, the GV chromatin is synchronized in a less condensed state before germinal vesicle breakdown (GVBD) in species that has been minutely studied. As not all the species show the SN configuration and gene transcription always stops at the late stage of oocyte growth, it is suggested that a thorough condensation of GV chromatin is essential for transcriptional repression. Because the GV chromatin status is highly correlated with oocyte competence, oocytes must end the NSN configuration before they gain the full meiotic competence and they must take on the SN/corresponding configurations and stop gene transcription before they acquire the competence for early embryonic development. In this study, we firstly investigated whether the layer of cumulus cells and size of oocytes could determine chromatin configurations in porcine oocytes. Using Hoechst3342 staining, the GV nucleolus and chromatin of porcine oocytes was classified into SN and NSN configurations. Next, we examined the changes in GV chromatin configurations during growth and maturation of porcine oocytes. In addition, the maturation and parthenogenetic development abilities in vitro were significant different between the SN and NSN configurations oocytes. These results indicated that chromatin changes in GV oocytes affect the development potential of parthenogenetic embryos.

In vitro production of porcine embryos, including in vitro maturation of oocytes followed by in vitro fertilization and in vitro culture, may result in live offspring, but it is still associated with great inefficiencies. In mammalian oocytes, acquisition of meiotic competence coincides with a decrease in general transcriptional activity at the end of the oocyte growth phase. In this study, we investigated the expression and sub‐cellular localization of CDK9, a RNA polymerase II CTD kinase during pig oocyte growth. Localization and expression of components involved in mRNA and rRNA transcription were assessed by immunocytochemistry in growing and fully‐grown oocytes. In addition, meiotic resumption, germinal vesicle breakdown and nuclear transcription were analyzed in oocytes cultured in presence of a potent CDK9 inhibitor, flavopiridol. Our analyses, demonstrated that CDK9 became co‐localized partially with phosphorylated Pol II CTD and mRNA splicing complexes. Surprisingly, CDK9 was co‐localized with Pol Ispecific transcription factor, UBF, and gradually localized in nucleolar peripheries at the final steps of oocyte growth. Treatment with flavopiridol resulted in arrest in meiotic resumption, germinal vesicle breakdown as well as a decline in global transcription. All together, this data suggest that CDK9 has a dual role in both Pol I‐ and Pol II‐dependent transcription in pig oocyte growth.

5‐aza‐2’‐deoxyctidine (5‐aza‐dC) is DNA methylation inhibitor and Trichostatin A (TSA) is histone deacytlase inhibitor, both of them can alter the level of the epigenetic modification of cells. The objective of this study was to investigate the effects of treatment with 5‐aza‐dC and TSA into fetal fibroblasts on the development of porcine nuclear transfer (NT) embryos. In this study, experiments were performed in order to modify epigenetic status in donor cells and evaluate developmental potential of NT embryos. 5‐ aza‐dC or TSA or combining treatment of TSA and 5‐aza‐dC was treated into growing donor cells for 1 h exposure and development of NT embryos was evaluated. Experiment was performed with 3 groups: control group (donor cells without treatment); TSA group (donor cell treated with 50 nM TSA for 1 h); TSA + 5‐aza‐dC group (donor cells were treated with 50 nM TSA and 5 nM 5‐aza‐dC for 1 h); TSA+1/2(5‐aza‐dC) group (donor cells were treated with 50 nM TSA for 1h and subsequently treated with 2.5 nM 5‐aza‐dC for another 1h). When donor cells were individually treated with 5 nM 5‐aza‐dC or 50 nM TSA for 1h, the blastocyst rate of NT embryos increased significantly compared with control group [18.8% vs 13.4% (5 nM 5‐aza‐dC group vs control group), and 26.2% vs 11.8% (50 nM TSA group vs control group), p<0.05]. However, the blastocyst rate in combining treatment group (50 nM TSA + 5 nM 5‐aza‐dC) did not increase compare with control group (12.3% vs 11.8%, p>0.05). When the donor cell were individually treated with 50nM TSA for 1 h firstly and then treated with 2.5 nM 5‐aza‐dC for another 1h, the blastocyst rate was significantly improved compared with control and TSA group (28% vs 10.2% and 23.7%, p<0.05). The present study suggested that donor cells treated with TSA or low concentration of TSA+5‐azadC in short time exposure may enhance the development of porcine NT embryo.

The pig has been considered to serve as an appropriate model of human disease. Therefore, establishment of porcine embryonic stem cell lines is important. The purpose of the present study was to further work in this direction. We produced porcine parthenogenetic embryos, and separately aggregated two of each of two-cell (2×2), four-cell (2×4), and eight-cell (2×8) embryos derived by parthenogenesis. After culture for 4 days, the developmental ability of the aggregates and total blastocyst cell numbers were evaluated. The percentage of blastocysts was significantly higher in both 2×4- and 2×8-aggregated embryos (58.3±1.9% and 37.2±2.8%, respectively) than in the control or 2×2-aggregated embryos (23.6±1.1% and 12.5±2.4%, respectively). Total blastocyst cell numbers were increased in the 2×4- and 2×8-aggregated embryos (by 44±3.0% and 45±3.3%, respectively) compared with those of control or 2×2-aggregated embryos (30.5±2.1% and 30.7±2.6%, respectively; p<0.05). The levels of mRNA encoding Oct-4 were higher in both the 2×4- and 2×8-aggregated embryos than in the control. When blastocysts derived from 2×4- aggregated embryos or intact normal embryos were cultured on mouse embryonic fibroblast feeder cells to obtain porcine stem cells, blastocysts from aggregated embryos formed colonies that were better in shape compared with those derived from intact blastocysts. Together, the data show that aggregation of porcine embryos not only improves blastocyst quality but also serves as an efficient procedure by which porcine embryonic stem cells can become established.