The Journal of Reproduction and Development Supplement

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Preimplantation development of the mammalian embryo is regulated by various cytokines, growth factors, and hormones, for which embryos express specific receptors. Particularly interesting is the action of regulatory proteins that trigger the activation of transcription factors termed STATs. The STAT proteins, of which seven have been described so far, are phosphorylated by Janus kinases (Jak), which are activated by cytokine receptors; phosphorylated STATs form dimers, enter the nucleus, and regulate gene transcription. This study is aimed to investigate the expression of Wnt/stat3 and their role on the rat embryogenesis. Early rat 2-cell embryos were collected from females on day 2 of pregnancy and were then cultured in 100µl droplets of mR1ECM in a humidified atmosphere 5% CO2 in air at 37°C. The GSK3 inhibitor were added to the culture medium in various concentrations : 2 and 5 µM. The expression of LIF receptor and gp-130 mRNA was also not detected at the rat embryos. In contrast, expression of STAT3 and Wnt receptor mRNA was detected. Western blot analysis showed that expression of the STAT3, Tyr705-Phosphorylated STAT3 and Oct3/4 were embryos. Immunohistochemical analysis of the embryos showed of the STAT3, Tyr705-Phosphorylated STAT3 were localized in a punctuate pattern around the inner cell mass. We confirmed that the effect of Bio was specific to its inhibition of Wnt/STAT3 signaling through STAT3 and Oct3/4, as the use of the small molecule inhibitor could potentially affect unrelated signal transduction pathway. In conclusion, low concentration of Bio impair development of cultured rat embryos. However, these effects are modified STAT3, Oct3/4 and Wnt signaling.

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This study was performed to investigate the proteomic analysis of plasma protein between pregnant and non-pregnant Hanwoo during early pregnancy stage. Serum samples (1, 3, 4, 5, 8, and 12 weeks after AI) were obtained from 5 pregnant (P) or non-pregnant (NP) Hanwoo, respectively. After Isoelectric focusing (IEF), the equilibrated strips were performed SDS-PAGE. Normalized protein spots were selected for the significant expression variation deviated over two fold in its expression level between two groups. Protein spots were enzymatically digested in-gel and analyzed using an Ettan MALDI-TOF. By submitting the amino acid sequence, the protein can be identified by a homology search using ProteinInfo, or in a BLAST search using the ExPASy Molecular Biology Server. Among 17 spots selected, 5 spots were NP specific and 12 spots were expressed differentially according to pregnant period. According to mass spectrometry, 8 were identified as albumin, haptoglobin, IgG1, or ferrochelatase etc. By the results of CAF-MALDI sequencing for the unidentified 9 spots (1∼14% coverage), the identifications were paraoxonase 1, hemopexin, and fibrinogen, etc. However, 2 spots were still unidentified. Further studies are needed to analyze the function of the proteins associated with pregnancy. This study was funded by National Institute of Animal Science, Rural Development Administration

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Abnormal developmental and fetal loss during postimplantation period is concerns for production of somatic cell nuclear transferred animals. Aberrant DNA methylation is one of the reasons for poor survival of cloned animals. In this study, we investigated the methylation status of Pre-1 and Satellite sequence in fetal (heart and liver) and placental tissues (endometrial and extraembryonic tissue) from pig fetuses produced by natural mating (control) and somatic cell nuclear transfer (SCNT) at day 35 of pregnancy. In two repeats selected, PRE-1 located in euchromatic region and satellite sequence located in heterochromatic regions. The methylation status of satellite sequences was hypermethylated in SCNT fetal, placental, and endometrial tissues compared to control, while PRE-1 region were demethylated in fetal river and endometrial tissue compared to control. Interestingly, there was a presence of significant non-CpG methylation in PRE-1 regions. Altered DNA methylation was observed in the normal and SCNT fetal, placental, and endometrial tissues using bisulfite sequencing. Different tissue-specific methylated region in the Pre-1 and satellite sequence show a variable methylation status in SCNT fetuses compared to natural mating control.

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