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.

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic status after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Dai et al., 2010), use of m-carboxycinnamic acid bishydroxamide (CBHA) as a histone deacetylase inhibitor during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA treatment on the in vitro preimplantation development of porcine SCNT embryos and on the acetylated status of histone H3K9 on cloned embryos at the zygote stage. We performed the three groups SCNT: SCNT (NT), CBHA treatment at the porcine fetus fibroblast cells (PFFs) used as donor cells prior to SCNT (CBHA-C) and CBHA treatment at the porcine SCNT embryos during the in vitro early culture after oocyte activation (CBHA-Z). The PFFs were treated with a 15 μM of CBHA (8 h) for the early culture and the porcine cloned embryos were treated with a 100 μM concentration of CBHA during the in vitro early culture (10 h). Cleavage rates and development to the blastocyst stage were assessed. No significant difference was observed the cleavage rate among the groups (82.6%, 76.4% and 82.2%, respectively). However, the development competence to the blastocyst stage was significantly increased in CBHA-Z embryos (22.7%) as compared to SCNT and CBHA-C embryos (8.6% and 4.1%)(p<0.05). Total cell numbers and viable cell numbers at the blastocyst stage of porcine SCNT embryos were increased in CBHA-Z embryos as compared to those in CBHA-C embryos (p<0.05). Signal level of histone acetylation (H3K9ac) at the zygote stage of SCNT was increased in CBHA-Z embryos as compared to SCNT and CBHA-C embryos. The results of the present study suggested that treatment with CBHA during the in vitro early culture (10 h) had significantly increased the developmental competence and histone acetylation level at the zygote stage.

Pig embryonic stem cells (ESC) has been suggested to become important animal model for therapeutic cloning using embryonic stem cells derived by somatic cell nuclear transfer (SCNT). However, the quality of cloned embryo and derivation rate of cloned blastocyst has been presented limits for derivation of cloned embryonic stem cell. In this study, we have tried to overcome these problems by aggregating porcine embryos. Zonafree reconstructed SCNT Embryos were cultured in micro-wells singularly (non-aggregated group) or as aggregates of three (aggregated groups) at the four cell stage. Embryo quality of the cloned embryos and attachment on feeder layer rate significantly increased in the aggregates. The aggregation of pig SCNT embryos at the four-cell stage can be a useful technique for improving the quality of pig cloned blastocyst and improvement in the percentage of attachment on the feeder layer of cloned embryos. * This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea.

This study investigates the endoplasmic reticulum (ER) stress and subsequent apoptosis in duced during somatic cell nuclear transfer (SCNT) process of porcine SCNT embryos. Porcine SCNT and in vitro fertilization (IVF) embryos were sampled at 3 h and 20 h after SCNT or IVF and at the blastocyst stage for mRNA extraction. The x-box binding protein 1 (Xbp1) mRNA and the expressions of ER stress-associated genes were confirmed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. Before commencing SCNT, somatic cells treated with tunicamycin (TM), an ER stress inducer, confirmed the splicing of Xbp1 mRNA and increased expressions of ER stress-associated genes. In all the embryonic stages, the SCNT embryos, when compared with the IVF embryos, showed slightly increased expression of spliced Xbp1 (Xbp1s) mRNA and significantly increased expression of ER stress-associated genes (p<0.05). In all stages, apoptotic gene expression was slightly higher in the SCNT embryos, but not significantly different from that of the IVF embryos except for the Bax/Bcl2L1 ratio in the 1-cell stage (p<0.05). The result of this study indicates that excessive ER stress can be induced by the SCNT process, which induce apoptosis of SCNT embryos.