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.

An understanding of oocyte gene expression is a necessary for the study of early female gamete development. Recently, oocyte has been used in many techniques such as somatic cell nuclear transfer, intracytoplasmic sperm injection and embryonic stem cell derivation. The purpose of this study was to investigate in the proteomes of pig oocytes and identification of differential proteins between using DIGE technique. In this experiment to overcome of limitation of 2D gel method like a low reproducibility and low sensitivity for proteome analysis of very small quantities, 2D fluorescence difference gel electrophoresis (DIGE), which enables co-detection of up to three samples on the same 2DE gels with CyDyes was used for analysis of oocyte proteins. Proteins within an isoelectric point (pI) range of 3 to 10 and a molecular weight (Mw) range of 20～100 kDa were primarily analyzed in DIGE with 2 replications of each sample. Approximately 1000 spots were detected in 2-D gel. Then, image analysis of DeCyder was performed to detect variations in protein spots between mature oocyte and parthenogenesis embryo. In the comparison of mature oocyte and parthenogenesis embryo, 11 spots were identified to be up-regulated proteins and 2 spots to be down-regulated proteins in parthenogenesis embryo, among which proteins were zona pellucida glycoprotein 4, transferrin receptor, apolipoprotein B, L-3-Hydroxyacyl Coa Dehydrogenase Revisited, cytochrome P450 2C33, similar to Monocarboxylate transporter 2, 2'-5' oligoadenylate synthetase 3, interferon alpha/ beta receptor-1, Chloride channel protein 6, pyruvate carboxylase as well as2'-5' oligoadenylate synthetase 3 using MALDI-TOF-MS. These results suggested that differential proteins were present between mature oocyte and parthenogenesis embryo.

An understanding of oocyte gene expression is a necessary for the study of biological development. Recently, Oocyte has been used in many techniques such as somatic cell nuclear transfer (SCNT), intracytoplasmic sperm injection (ICSI) and embryonic stem cell derivation. However, the molecular mechanism underlying porcine oocyte is still unclear. In this study, we present the description of the porcine oocyte proteome. Proteins within the isoelectric point ranges of 3.0 to 10.0 were analyzed separately using 2‐dimensional electrophoresis (2‐DE). About 450 spots were detected in 2‐ D gel of oocytes, stained with Coomassie blue. Subsequent excision of 227 spots from gels and MALDI‐TOF MS analysis allowed the identification of 85 proteins. Our results indicated the composite profiles of proteins in the porcine oocyte. Tubulin beta chain and meiosis‐specific nuclear structural protein 1 antibody was used to confirm those antibody expression levels in immature, mature and parthenogenetic embryo. Western blot analysis showed that expressions of those proteins increased during mature and parthenogenetic embryo. These protein profiles will make available important guides for the study of oocyte function and assist in functional analysis of the proteins.

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.

X‐box binding protein‐1 (XBP‐1) is an important regulator of a subset of genes active during endoplasmic reticulum (ER) stress. In the present study, we analyzed XBP‐1 level and location to explore the effect of ER stress on oocyte maturation and developmental competency of porcine embryos in an in vitro culture system. First, we examined the localization of XBP‐1 at different meiotic stages of porcine oocytes and at early stages of parthenogenetic embryo development. Fluorescence staining showed that expression of functional XBP‐1 was weak in mature oocytes and at the one‐cell, two‐cell, and eight‐cell stages of embryos, but abundant at the GV oocyte, four‐cell, morula, and blastocyst stages. In addition, RT‐PCR revealed that both spliced XBP‐1 (XBP‐1s ) and unspliced XBP‐1 (XBP‐1u) were expressed at the GV oocyte, four‐cell, morula, and blastocyst stages. Tunicamycin (TM), an ER stress inducer, blocked porcine embryonic development at the four‐cell stage, exhibiting the effect on embryonic genome activation. Next, porcine embryos cultured in the presence of tauroursodeoxycholate (TUDCA), an ER stress inhibitor, were studied. Total cell numbers and the extent of the ICM increased (p<0.05), whereas the rate of nuclear apoptosis decreased (p<0.05). Moreover, expression of the anti‐apoptotic gene Bcl‐2 increased whereas expression of the pro‐apoptotic genes Bcl‐xl and p53 decreased. The results indicated that inhibition of ER stress enhanced porcine oocyte maturation and embryonic development by preventing ER stress‐mediated apoptosis in vitro.

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.

The early diagnosis of bovine pregnancy is an essential component of successful reproductive planning on farms, because lack of bovine pregnancy over the long term results in reproductive failure and low milk yield‐the latter of which is a special concern on dairy farms. This study was designed to identify early pregnancy‐specific whey proteins in bovine, by comparing milk samples collected from cattle during pregnancy (Days 30 and 50) and from non‐pregnant cattle. In this study, differentially expressed proteins in five pregnant and five non‐pregnant Holstein dairy cattle were investigated and compared, using proteomics analysis. The first dimension was applied to a pH 3.0～10.0 strip, by loading a 2‐mg milk protein sample. After the second‐dimension separation was performed, the gels were stained with colloidal Coomassie brilliant blue. The stained gels were scanned and the images were analyzed, to detect variations in protein spots between non‐pregnant and pregnant cattle milk protein spots, using ImageMaster; this was followed by analysis with MALDI TOF‐MS. Analysis of the 2‐DE gel image resulted in a total of approximately 500～600 protein spots, of which 12 spots were differentially expressed, six spots were up‐regulated, and four spots were downregulated; two spots were identified as pregnancy‐specific proteins. These proteins were identified as lactoferrin, NADH dehydrogenase subunit 2, albumin, serum albumin precursor and transferrin. Our results via 2‐D PAGE analysis revealed composite profiles of several milk proteins related to early bovine pregnancy, implying the possible use of these milk proteins in the early detection of bovine pregnancy.