Lee SeungHoon, Hiradate Yuuki, Hoshino Yumi, Tanemura Kentaro, Sato Eimei
Laboratory of Animal Reproduction,Graduate School of Agricultural Science,Tohoku University,Aobaku,Sendai 981-8555,Japan.
Laboratory of Animal Reproduction,Graduate School of Agricultural Science,Tohoku University,1-1 Tsutsumidori-Amamiyamachi,Aobaku,Sendai 981-8555,Japan.
Zygote. 2014 Aug;22(3):404-10. doi: 10.1017/S0967199413000269. Epub 2013 Jun 12.
Microtubule-associated protein light chain 3 (LC3)-II is a marker of autophagosome. In this study, LC3-II expression was used to identify autophagy, during the in vitro maturation of porcine oocytes. In a time-course experiment, cumulus-oocyte complexes (COCs) were cultured in NCSU23 medium for 0 h, 14 h, 28 h or 42 h. The cumulus cells were removed and denuded oocytes were processed for western blotting or immunostaining. Western blotting showed that the LC3-II levels changed over time, with maximum levels observed at 14 h and minimum levels at 42 h. Immunostaining of LC3 showed the signals with dot shapes and ring shapes in oocytes at every group that probably represent autophagosomes. To ascertain whether autophagic induction and degradation were occurring, we treated the cultures with autophagic inhibitors. Lysosomal protease inhibitor E64d and pepstatin A increased the LC3-II levels and wortmannin, inhibitor of autophagic induction, decreased the LC3-II levels. Western blotting and immunostaining demonstrated that LC3-II is present in porcine oocytes cultured in vitro. The decreased LC3-II levels after wortmannin treatment suggest that it is newly generated in porcine oocytes, a phenomenon that represents autophagic induction. Furthermore, increased LC3-II levels after E64d and pepstatin A addition imply that LC3-II is degraded by lysosomal proteases, an indication of autophagic degradation. Our results suggest that autophagy, which is a dynamic process whereby autophagosomes are newly generated and subsequently degraded, is probably occurring in porcine oocytes during in vitro maturation.
微管相关蛋白轻链3（LC3）-II是自噬体的标志物。在本研究中，LC3-II的表达被用于鉴定猪卵母细胞体外成熟过程中的自噬。在一个时间进程实验中，将卵丘-卵母细胞复合体（COCs）在NCSU23培养基中培养0小时、14小时、28小时或42小时。去除卵丘细胞，对裸卵进行蛋白质免疫印迹或免疫染色处理。蛋白质免疫印迹显示LC3-II水平随时间变化，在14小时时达到最高水平，在42小时时达到最低水平。LC3的免疫染色显示每组卵母细胞中都有斑点状和环状信号，可能代表自噬体。为了确定是否发生了自噬诱导和降解，我们用自噬抑制剂处理培养物。溶酶体蛋白酶抑制剂E64d和胃蛋白酶抑制剂A增加了LC3-II水平，而自噬诱导抑制剂渥曼青霉素降低了LC3-II水平。蛋白质免疫印迹和免疫染色表明LC3-II存在于体外培养的猪卵母细胞中。渥曼青霉素处理后LC3-II水平降低表明它是在猪卵母细胞中新生成的，这一现象代表自噬诱导。此外，添加E64d和胃蛋白酶抑制剂A后LC3-II水平升高意味着LC3-II被溶酶体蛋白酶降解，这是自噬降解的一个指标。我们的结果表明，自噬是一个自噬体新生成并随后被降解的动态过程，可能发生在猪卵母细胞体外成熟过程中。
