Park Hyo-Jin, Park Jae-Young, Kim Jin-Woo, Yang Seul-Gi, Jung Jae-Min, Kim Min-Ji, Park Joung Jun, Koo Deog-Bon
Dept. of Biotechnology, College of Engineering, Daegu University, Gyeongsan 38453, Republic of Korea.
Saewha Hospital, Dongnae, Busan 47822, Republic of Korea.
Dev Reprod. 2017 Dec;21(4):407-415. doi: 10.12717/DR.2017.21.4.407. Epub 2017 Dec 31.
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 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 maturation of porcine oocytes.
在本研究中，我们探究了结合免疫球蛋白蛋白/葡萄糖调节蛋白78千道尔顿（BIP/GRP78）调控的内质网（ER）应激对猪卵丘-卵母细胞复合体（COCs）减数分裂成熟和卵丘细胞扩展的作用。此前已证明，未折叠蛋白反应（UPR）相关基因，如参与内质网应激防御机制的分子，在猪卵母细胞体外成熟（IVM）过程中的成熟卵母细胞和卵丘细胞中表达。然而，BIP/GRP78介导的猪卵母细胞内质网应激调控尚未见报道。首先，我们观察了使用猪特异性小干扰RNA（siRNAs）（#909、#693和#1570）敲低BIP/GRP78（一种UPR起始标记物）对卵母细胞成熟的影响。在所有siRNAs中，通过蛋白质免疫印迹和免疫荧光分析观察到，siRNA #693显著降低了猪COCs中UPR标记蛋白（BIP/GRP78、ATF4和P90ATF6）的蛋白水平。我们还观察到，siRNA#693降低BIP/GRP78水平显著抑制了卵母细胞的减数分裂成熟（siRNA #693：32.5±10.1% 对比对照组：77.8±5.3%）。此外，我们还检测了内质网应激抑制剂牛磺熊去氧胆酸（TUDCA，200 μM）和褪黑素（0.1 μM）对BIP/GRP78敲低卵母细胞的影响。TUDCA和褪黑素处理可恢复siRNA #693转染的成熟COCs中内质网应激标记蛋白（BIP/GRP78、p-eIF2α、eIF2α、ATF4和P90ATF6）的表达水平。总之，这些结果表明，BIP/GRP78介导的UPR信号调控和内质网应激在猪卵母细胞成熟中起重要作用。
