Luo Yi-Bo, Zhang Li, Lin Zi-Li, Ma Jun-Yu, Jia Jialin, Namgoong Suk, Sun Qing-Yuan
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Beijing, China.
Department of Animal Science, Chungbuk National University, Cheongju, Korea.
Histochem Cell Biol. 2016 Jan;145(1):93-104. doi: 10.1007/s00418-015-1369-4. Epub 2015 Oct 14.
LINE-1 is an autonomous non-LTR retrotransposon in mammalian genomes and encodes ORF1P and ORF2P. ORF2P has been clearly identified as the enzyme supplier needed in LINE-1 retrotransposition. However, the role of ORF1P is not well explored. In this study, we employed loss/gain-of-function approach to investigate the role of LINE1-ORF1P in mouse oocyte meiotic maturation. During mouse oocyte development, ORF1P was observed in cytoplasm as well as in nucleus at germinal vesicle (GV) stage while was localized on the spindle after germinal vesicle breakdown (GVBD). Depletion of ORF1P caused oocyte arrest at the GV stage as well as down-regulation of CDC2 and CYCLIN B1, components of the maturation-promoting factor (MPF). Further analysis demonstrated ORF1P depletion triggered DNA damage response and most of the oocytes presented altered chromatin configuration. In addition, SMAD4 showed nuclear foci signal after Orf1p dsRNA injection. ORF1P overexpression held the oocyte development at MI stage and the chromosome alignment and spindle organization were severely affected. We also found that ORF1P could form DCP1A body-like foci structure in both cytoplasm and nucleus after heat shock. Taken together, accurate regulation of ORF1P plays an essential role in mouse oocyte meiotic maturation.
LINE-1是哺乳动物基因组中的一种自主非长末端重复逆转座子，编码ORF1P和ORF2P。ORF2P已被明确鉴定为LINE-1逆转座所需的酶供应者。然而，ORF1P的作用尚未得到充分研究。在本研究中，我们采用功能丧失/获得方法来研究LINE1-ORF1P在小鼠卵母细胞减数分裂成熟中的作用。在小鼠卵母细胞发育过程中，在生发泡（GV）期，ORF1P在细胞质以及细胞核中均有观察到，而生发泡破裂（GVBD）后则定位于纺锤体上。ORF1P的缺失导致卵母细胞停滞在GV期，并下调了成熟促进因子（MPF）的组成成分CDC2和细胞周期蛋白B1。进一步分析表明，ORF1P的缺失引发了DNA损伤反应，大多数卵母细胞呈现出染色质构型改变。此外，注射Orf1p双链RNA后，SMAD4显示出核灶信号。ORF1P的过表达使卵母细胞发育停滞在MI期，染色体排列和纺锤体组织受到严重影响。我们还发现，热休克后ORF1P可在细胞质和细胞核中形成类似DCP1A小体的灶状结构。综上所述，ORF1P的精确调控在小鼠卵母细胞减数分裂成熟中起着至关重要的作用。
