Tumor necrosis factor receptor associated factor 4 (TRAF4) is a member of the TNF receptor associated factor family, which involved in cancer metastasis, production of reactive oxygen species, and cell polarity. During mouse embryonic development, TRAF4 was expressed from 8.5 to 13.5 days of post-coitum. Hence TRAF4 at tight junction of cell membrane is related with epithelial to mesenchymal transition, it may be essential in early embryogenesis to start of cell differenciation. However, little is known about characteristics and functions of TRAF4 in pig, specifically preimplantation embryonic development in porcine. This study was performed to investigate an isolation and identification of TRAF4 mRNA sequences in pig as the first step to understand relevant characteristics and functions. To analyze the full-length of TRAF4 mRNA, it was carried out rapid amplification of cDNA ends with total RNA from pK15 cells. Using 5’ and 3’ TRAF4 specific primers, three PCR products were obtained and applied for sequencing analyses. Consequently, 2030 bp nucleotide sequences were analyzed, seeming to putative full-length mRNA of TRAF4. The coding region of porcine TRAF4 was shown to 93 and 90 % homologies of human and mouse, respectively. The sequences were encoded to 470 amino acids of which the sequences were different from 12 and 7 amino acids of human and mouse, respectively. It was found that porcine TRAF4 contained GTWRGS domain at the C-terminal region, known to be in only TRAF4 among other TRAF family members in human. Based on these sequencing results, it can be postulated that the porcine TRAF4 mRNA sequences may provide an important information for an initial study to understand characterizations and functions of TRAF4 during porcine preimplantation embryonic development.

We have recently shown that attachment of active sperm to bovine uterine epithelial cells (BUECs) induces a pro-inflammatory response in BUECs in vitro (MRD 2018), but the molecular mechanism remains unknown. Therefore, we aimed to investigate the role(s) of TLR2/4 signaling pathway in sperm-BUECs inflammatory process. Using immunohistochemistry, we found that TLR2 and TLR4 proteins are localized in the luminal and glandular epithelia of bovine endometrium. The co-culture of BUECs with sperm stimulated the transcription of pro-inflammatory genes and enhanced the phosphorylation of TLR2/4 downstream targets (p38MAPK and JNK) in BUECs. Importantly, the pre-incubation of BUECs with TLR2/4 blocker abrogated sperm-induced inflammation. Additionally, the phosphorylation of p38MAPK was suppressed by the addition of TLR4 antibody, while the phosphorylation of JNK was suppressed by the addition of either TLR2 antagonist or TLR4 antibody. In conclusion, the present in vitro findings suggest that uterine epithelial cells respond to sperm inflammatory effect via TLR2/4 signal pathway.

Studies have shown that epidermal growth factor (EGF) is critical for porcine oocyte maturation and repair the damage of epidermal cells. EGF acts through EGF receptor (EGFR) present in the blastocyst seems to regulate embryonic production of IFNT. In pigs, expression and amounts of biologically active interferon tau (IFNT) and transforming growth factor-β (TGF-β) at the conceptus-maternal interface increase significantly as conceptuses elongate and begin the implantation process. Mature oocytes were collected and subjected to in vitro fertilization and cultured. Hatched pig blastocysts (days 7–10) and matured oocytes were cultured and exposed to EGF (0, 10, 40 and 80 ng/ml) for 24, 48 and 72 h. To understand the mechanisms regulating the interaction between the hatched blastocyst and maternal uterine environment, the production of IFNT and TGF-β were investigated. Protein concentrations of IFNT and TGF-β in the cultured media were determined by commercial enzyme-linked immunosorbent assay (ELISA) kit. Epidermal growth factor 40 ng/ml increased embryonic production of IFNT and TGF-β production by hatched blastocysts. The above results suggest that epidermal growth factor produced by epithelial cell stimulates the production of IFNT by pig trophoblasts. The capacity of conceptus to increase IFNT and TGF-β production in response to EGF stimulation may be important for the establishment of maternal uterine environment. In the study was carried out with the support of "Agenda Program (Project No. PJ01201601)", RDA, Republic of Korea.

The aim of this study was to investigate the improvement in developmental rate of porcine nuclear transferred embryos derived from fetal kidney cells. The developmental rate of porcine cloned embryos was investigated by using cloned fetal kidney cells and neonatal ear fibroblasts (control) as donor nuclei. Results showed a significantly higher percentage of reconstructed oocytes (80.6% vs 69.8%; p<0.05) produced in fetal kidney cells group than control. Both donor cells types had no significant difference in the pregnancy rates and farrowing rates but kidney cells group showed a trend towards improved farrowing rate in gilts (11.7%) and non-ovulated group of recipients (16.6%) although non-significant, perhaps due to the less number of recipients. Whereas, in control group no farrowing was observed in previously mentioned recipients. The cleavage rate of in-vitro developed reconstructed embryos did not differed significantly, whereas the rate of blastocyst development was significantly more in cloned fetal kidney cell group (21.3 vs. 15.3%; p<0.05). The gene expression of different reprogramming and apoptosis related genes did not showed any significant change in cloned fetal kidney cell group compared to control. It was concluded from results that cloned fetal kidney cells have significantly higher in-vitro cloned blastocyst production but no significant improvement was observed for in-vivo production of cloned piglets. However, cloned fetal kidney cells have shown a tendency for improving the cloned piglet production. Increasing the number of surrogate recipients may positively affect the development rate of kidney cell group.

In this study, we tried to generate porcine induced pancreatic β-cells (piPan β-cells) from alpha 1,3 galactosyltransferase knockout (GalTKO) and membrane cofactor protein (MCP) knock-in porcine derived bone marrow mesenchymal stem cell (pBM-MSCs). GalT is involved in synthesis of Alpha-1,3-Galactose, which is main culprit of hyperacute rejection in xenotransplantation, while MCP is main part of complement system and protect host cells from damage. pBM-MSCs were induced with pancreatic differentiation media (bFGF, Activin A, retinoic acid), until maturation. pBM-MSCs attained definitive endoderm morphology after Activin A treatment and formed exact pancreatic clusters after 8 weeks in maintenance media. Dithizone staining showed moderate expression in piPan β-cells clusters, which confirmed the presence of zinc ion in the islet’s beta cells. After 8 weeks, pancreatic specific protein expressions were analyzed in piPan β-cells by immunofluorescence. Furthermore, to reveal the functional characteristics of piPan β-cells, glucose stimulating insulin secretion (GSIS) analysis were performed using low (2 mM) and high glucose (20 mM) concentration by real time PCR. The newly produced piPan β-cells exhibited higher expression of pancreatic markers (insulin, c-peptide, PDX1) as well as showed an elevation in insulin expression at both concentrations upon GSIS analysis. Morphological changes and expression of pancreatic markers revealed successfully differentiation of pBM-MSCs into piPan β-cells; however, more in depth studies for its functional characteristic and in vivo maintenance are still in progress. In conclusion; in near future, these induced piPan β-cells can be used to replace the beta cells in human diabetic patients with minimum risk of immune rejection.This work was supported by “Cooperative Research Program for Agriculture Science & Technology Development (PJ 01100203)” and 2018 RDA fellowship program of NIAS, RDA, Republic of Korea.

The organs of alpha 1, 3-galactosyltransferase homozygous knock out (GT) pigs are assumed to be the potential donor to solve the problem of big awaiting list of patients for xenotransplantation. In this study, we analyzed the morphometric parameters of heart and kidney of three, two and four GT pigs corresponding to the ages of 1-, 3- and 7-months. We underwent laparotomy under general anesthesia to collect kidney and heart of the GT pigs. Subsequently, we measured the weight, lengths of coronal, sagittal and axial planes of a heart and both kidneys. The result showed significantly increased body weight according to the age at 7-month (38.2 ± 1.8 kg), from 1-month (5.2 ± 0.3 kg) and 3-month (12.2 ± 0.3 kg). We observed, on weight of heart, significant differences depending on growing ages at 1-month 33.3 ± 3.3, 3-month 67.9 ± 2.6 and 7-months197.4 ± 1.8. The weights of kidney were 33.3 ± 4.4, 33.8 ± 4.9 and 80.3 ± 11.3 g at 1-, 3- and 7-months, respectively, showing significant increase of the weight at 7-month. The statistically significant differences were not observed between the left and right kidney weights. The size of kidney also identified as significant increase at 7-month, which showing similar pattern of weight growing. Interestingly, lengths of axial and coronal planes of 7-month kidney were shown significantly increase, but we observed sagittal plane was not increase, likely that the porcine kidney might grow in size prominently to axial and coronal directions. The index of kidney weight relative to body weight at 1-, 3- and 7-months showed decrease at 3- and 7-months compared with 1-month, whereas there were significances among the three ages. Finally, we found strong correlations between age and measured values, between body weight and, weight and size of the organs. Taken together, we provide standard data to allow prediction of the matched-size of heart and kidney depending on ages of GT pigs for pig to non-human primate transplantation.