This study aimed to investigate changes in the activity and mRNA expression of plasminogen activators (PAs) induced by 17β-estradiol (E₂), human chorionic gonadotropin (hCG), and interleukin-1β (IL-1β) in porcine endometrial cells. Endometrial cells were isolated from the epithelium and cultured to 80% confluence. They were then treated for 24 h with E₂ (0.2, 2, 20, and 200 ng/mL), IL-1β (0.1, 1, 10, and 100 ng/mL), and hCG (0.5, 1, 1.5 and 2 IU/mL). mRNA expressions of urokinase-type (uPA) and tissue-type (tPA) PAs were analyzed using reverse transcription PCR, and activities were measured using a PA activity assay. mRNA expressions of uPA and tPA increased with E₂ treatment; however, this was not significant. Similarly, treatment with hCG did not influence the mRNA expressions of PAs. Interestingly, treatment with 0.1 ng/mL IL-1β significantly reduced the mRNA expression of uPA, but did not affect that of tPA. Treatment with 2, 20, and 200 ng/mL E₂ increased PA activity compared with the control group; treatment with 0.1 and 1 ng/mL IL-1β significantly increased PA activity compared with the other IL-1β treatment groups, whereas treatment with 10 and 100 ng/mL IL-1β decreased. Treatment with 2 IU/mL hCG increased PA activity compared with the other treatment groups, although there were no significant differences between the hCG and control groups. In conclusion, the activity and mRNA expression of PAs were differently regulat-ed by the hormone/cytokine and its concentration in porcine endometrial cells. Therefore, understanding PA regulatory mechanisms may help to improve the reproductive potential of domestic animals.

Unlike mouse results, cloning efficiency of nuclear transfer from porcine induced pluripotent stem cells (piPSCs) is very low. The present study was performed to investigate the effect of cell cycle inhibitors on the cell cycle synchronization of piPSCs. piPSCs were generated using combination of six human transcriptional factors under stem cell culture condition. To examine the efficiency of cell cycle synchronization, piPSCs were cultured on a matrigel coated plate with stem cell media and they were treated with staurosporine (STA, 20 nM), daidzein (DAI, 100 μM), roscovitine (ROSC, 10 μM), or olomoucine (OLO, 200 μM) for 12 h. Flow Cytometry (FACs) data showed that piPSCs in control were in G1 (37.5±0.2%), S (34.0±0.6%) and G2/M (28.5±0.4%). The proportion of cells at G1 in DAI group was significantly higher than that in control, while STA, ROSC and OLO treatments could not block the cell cycle of piPSCs. Both of viability and apoptosis were affected by STA and ROSC treatment, but there were no significantly differences between control and DAI groups. Real-Time qPCR and FACs results revealed that DAI treatment did not affect the expression of pluripotent gene, Oct4. In case of OLO, it did not affect both of viability and apoptosis, but Oct4 expression was significantly decreased. Our results suggest that DAI could be used for synchronizing piPSCs at G1 stage and has any deleterious effect on survival and pluripotency sustaining of piPSCs.

Pluripotent cells are categorized as either "naive" or "primed" based upon their pluripotent status. According to previous studies, embryonic stem cells and embryonic germ cells are identified as naive pluripotent stem cells and epiblast stem cells are identified as primed pluripotent stem cells. In a permissive species such as the mouse, naive and primed pluripotent stem cells can be derived from embryos without genetic manipulations. In non-permissive species such as humans and pigs, primed pluripotent cells are only established from embryos. However, previous studies have shown that the embryonic germ cells of non-permissive species share similar morphology and features with naive pluripotent cells. For these reasons porcine embryonic germ cells (pEGCs) may provide a useful cell source for comparative studies on naive pluripotent cells in non-permissive species. In this study, we attempted to establish and characterize porcine embryonic germ cells. Consequently, an embryonic germ cell line was derived from the genital ridges of a porcine dpc 30 fetus in media containing LIF and bFGF. After establishment, this cells were cultured and stabilized in LIF or bFGF contained media. This cell lines displayed a dome-shaped colony morphology in both culture condition. The cell lines were maintained in both condition over an extended time period and were able to differentiate into the three germ layers in vitro. Interestingly, cell lines cultured in LIF or bFGF expressed different pluripotency markers. LIF-dependent pEGCs expressed naive-pluripotency markers such as OCT4, SOX2, NANOG and SSEA1, while bFGF-dependent pEGCs expressed primed-pluripotency markers such as OCT4, SOX2, NANOG and SSEA4. However, as a result of analysis of XCI, two cell lines showed hemi-methylated pattern similarly in XIST promoter regions. In conclusion, we were able to successfully derive embryonic germ cells from genital ridges of a porcine fetus. Pluripotent state of pEGCs were regulated by modulation of culture condition. In LIF supplement, pEGCs showed naive-pluripotency expressing SSEA1, while pEGCs show primed-pluripotency expressing SSEA4 in bFGF condition. This cell line could potentially be used as naive pluripotent cell source for comparative study with porcine embryonic stem cells and other pluripotent cell lines. As porcine pluripotent cells, pEGCs could be useful candidates for preliminary studies of human disease as well as a source for generating transgenic animals.

Pluripotent cells are categorized as either "naive" or "primed" based upon their pluripotent status. According to previous studies, embryonic stem cells and embryonic germ cells are identified as naive pluripotent stem cells and epiblast stem cells are identified as primed pluripotent stem cells. In a permissive species such as the mouse, naive and primed pluripotent stem cells can be derived from embryos without genetic manipulations. In non-permissive species such as humans and pigs, primed pluripotent cells are only established from embryos. However, previous studies have shown that the embryonic germ cells of non-permissive species share similar morphology and features with naive pluripotent cells. For these reasons porcine embryonic germ cells (pEGCs) may provide a useful cell source for comparative studies on naive pluripotent cells in non-permissive species. In this study, we attempted to establish and characterize porcine embryonic germ cells. Consequently, an embryonic germ cell line was derived from the genital ridges of a porcine dpc 30 fetus in media containing bFGF. This cell line displayed a dome-shaped colony morphology. The cell line was maintained in a stable condition over an extended time period and was able to differentiate into the three germ layers in vitro. Pluripotency markers such as OCT4, SOX2, NANOG and SSEA4 were expressed in these pEGCs. Similar with pESCs, Mek/Erk signaling pathway were activated by bFGF in the cultured pEGCs. In conclusion, we were able to successfully derive embryonic germ cells from genital ridges of a porcine fetus. Unlikely naive pluripotent cells such as mESCs, pluripotency of pEGCs were regulated by Mek/Erk signaling pathway activated by bFGF. This cell line could potentially be used as naive pluripotent cell source for comparative study with porcine embryonic stem cells and other pluripotent cell lines. As porcine pluripotent cells, pEGCs could be useful candidates for preliminary studies of human disease as well as a source for generating transgenic animals.

돼지 중간엽 줄기세포를 Dimethyl sulfoxide(DMSO), Ethylene glycol(EG), 그리고 DMSO/EG을 이용하여 세포동결을 유도한 후 적절한 동결보호제를 알아보았다. 2개월 이내 돼지 골수에서 중간엽 줄기세포를 분리하여 colony 형성 및 alkaline phosphatase(AP) 활성을 확인하고, 지방 세포로의 분화 유도에 의한 줄기세포의 능력을 확인하였다. 이들 중간엽 줄기세포의 완만 동결을 위해, DMEM에 각각 10

The objective of this study was to investigate the effects of oxygen tension during in vitro maturation of porcine oocytes on the nuclear maturation and differences in gene expression. Cumulus-oocyte complexes (COCs) were collected from ovaries obtained at a local slaughterhouse, matured for 44 hours in TCM199 supplemented with porcine follicular fluid (pFF) under 5% or 20% oxygen concentration. In results, oxygen tension had no significant effects on nuclear maturation. Relative poly(A) mRNA abundance of MnSOD, CCNB1, LDHA, G6PD, BCL, GPX1, IGFR2, GLUT1, BAX, GREM, PTGS2 was analysed in cumulus cells. GLUT1, G6PD, LDHA were up-regulated in the cumulus cells matured in low oxygen, suggesting a higher glucose uptake and an increase in anaerobic glycolysis, whereas CCNB, MnSOD were up-regulated in the cumulus cells matured in high oxygen, which suggest a higher activity of mitosis-promoting factor and antioxidant response. In conclusion, cumulus cells increase in glucose metabolism via anaerobic glycolysis under low oxygen concentration and show significant change in antioxidant against oxidant damage or apoptotic response under high oxygen concentration. For such an effect of cumulus cells, oocytes could be matured normally regardless of various oxygen concentration.

One of the problems associated with in vitro culture of primordial gern cells (PGCs) is the large loss of cells during the initial period of culture. This study characterized the initial loss and determined the effectiveness of two classes of apoptosis inhibitors, protease inhibitors and antioxidants, on the ability of the porcine PGCs to survive in culture. Results from electron microscopic analysis and in situ DNA fragmentation assay indicated that porcine PGCs rapidly undergo apoptosis when placed in culture. Additionally, \ulcorner2-macroglobulin, a protease inhibitor and cytokine carrier, and N-acetylcysteine, an antioxidant, increased the survival of PGCs in vitro. While other protease inhibitors tested did not affect survival of PGCs, all antioxidants tested improved survival of PGCs (p<0.05). Further results indicated that the beneficial effect of the antioxidants was critical only during the initial period of culture. Finally, it was determined that in short-term culture, in the absence of feeder layer, antioxidants could partially replace the effect(s) of growth factors and reduce apoptosis. Collectively, these results indicate that the addition of \ulcorner2-macroglobulin and antioxidatns can increase the number of PGCs in vitro by suppressing apoptosis.