Melatonin has an important role as anti-oxidative effect and reducing of endoplasmic reticulum(ER)-stress on oocyte maturation and embryo development. Under ER-stress condition, unfolding protein response (UPR) is a defence mechanism in mammalian cells. Recently, regulation of UPR signaling genes are involved in oocyte maturation, embryo development and female reproduction. However, there is no report on the role of melatonin for UPR signaling and ER-stress mediated apoptosis during pig oocyte maturation progression. Moreover, the changes of UPR genes expression according to the porcine oocyte maturation is not yet fully understood. Here, we investigated the changes of UPR signal (BIP/GRP78, ATF4, p90/p50ATF6, and XBP1) and ER-stress apoptotic factor CHOP genes expressions in porcine oocyte maturation by Western blot and RT-PCR analysis. During oocyte maturation, UPR marker and CHOP genes expressions were significantly increased in matured oocytes or cumulus-oocyte complexes (COCs). UPR markers expressions were significantly increased by ER-stress inducer, tunicamycin (Tm), treated (1, 5, 10 μg/ml) groups in a dose-dependent manner compared with control group. To confirm the reducing of ER-stress by melatonin (0.1 μM), we were compared to the effects of ER-stress inhibitor, TUDCA (200 μM), after pre-treated Tm (5 μg/ml) for 22 h maturation. Expressions of UPR markers and meiotic maturation were recovered by melatonin (0.1 μM) in COCs. And, we observed the role of Grp78/Bip as UPR signaling beginning marker using siRNA. In result, reduction of Grp78/Bip gene expression by siRNA was induced the inhibition of oocyte maturation (32.5±10.1 vs control; 77.8±5.3), and p50ATF6 protein level was significantly decreased (p<0.001) in cultured COCs for 44 h. In addition, these results were recovered through the addition of melatonin (0.1 μM) or TUDCA (200 μM) in maturation medium. These results demonstrated that the regulation of UPR signaling via Grp78/Bip gene induction plays a critical role in porcine oocyte maturation in vitro. Furthermore, this present study first confirmed a functional link between inhibition effect of ER-stress by melatonin and regulating of UPR signaling in porcine oocyte maturation. In conclusion, melatonin improves the oocyte maturation and cumulus cells expansion of COCs through the regulation of UPR signal pathway by BIP/GRP78 against the ER-stress during porcine oocyte maturation periods.

Changes in stress-associated biomolecules can be used as an important criterion for assessing the levels of environmental pollution because living organisms demonstrate contamination-stimulated stress responses. This study was conducted to determine the e

본 연구는 nPr-BTP/nitrobenzene 추출 계에 의한 악티나이드(III)의 선택적 분리로, 우선 자연친화적 CHN 형 의 nPr-BTP (2.6-Bis-(5.6-n-propyl-1.2.4-triazin-3-yl)-pyridine)를 합성하고, 이의 희석제에 대한 용해성 및 질산에 대한 안정성 등을 평가하였다. 악티나이드(III)의 대표원소로는 Am을 선정하였으며, 0.1M nPr-BTP/nitrobenzene-1M , O/A=2의 조건에서 Am은 약 85%, RE 원소는 Eu가 8%, 기타 Nd, Ce, Y 등은 3% 이하가 추출되어 (이때 Am/Eu의 상호분리 계수 약 60정도) 악티나이드(III)의 선택적 추출에는 별 문제가 없을 것으로 판단되었다. 그러나 Am의 역추출의 경우 0.05M 질산용액으로 O/A=1 에서 약43%가 역추출 되었으며, O/A=0.3에서도 65% 정도만이 역추출 되어 질산 이외의 다른 역추출제의 개발이 요구되고 있다.

In the present study, we investigated the role of binding immunoglobulin protein/glucose-regulated protein, 78-kDa (BIP/GRP78)-regulated endoplasmic reticulum (ER)-stress on meiotic maturation and cumulus cells expansion in porcine cumulus-oocyte complexes (COCs). Previously, it has been demonstrated that unfolded protein response (UPR)- related genes, such as molecules involved in ER-stress defense mechanisms, were expressed in matured oocytes and cumulus cells during in vitro maturation (IVM) of porcine oocytes. However, BIP/GRP78-mediated regulation of ER stress in porcine oocytes has not been reported. Firstly, we observed the effects of knockdown of BIP/GRP78 (an UPR initiation marker) using porcine-specific siRNAs (#909, #693, and #1570) on oocyte maturation. Among all siRNAs, siRNA #693 significantly reduced the protein levels of UPR marker proteins (BIP/GRP78, ATF4, and P90ATF6) in porcine COCs observed by Western blotting and immunofluorescence analysis. We also observed that the reduction of BIP/GRP78 levels by siRNA#693 significantly inhibited the meiotic maturation of oocytes (siRNA #693: 32.5±10.1% vs control: 77.8±5.3%). In addition, we also checked the effect of ER-stress inhibitors, tauroursodeoxycholic acid (TUDCA, 200 μM) and melatonin (0.1 μM), in BIP/ GRP78-knockdown oocytes. TUDCA and melatonin treatment could restore the expression levels of ER-stress marker proteins (BIP/GRP78, p-eIF2α, eIF2α, ATF4, and P90ATF6) in siRNA #693-transfected matured COCs. In conclusion, these results demonstrated that BIP/GRP78-mediated regulation of UPR signaling and ER stress plays an important role in in vitro maturation of porcine oocytes.