Chen Li, Ouyang Ying-Chun, Li Jian, Qiao Jing-Yi, Gu Lin-Jian, Wang Zhen-Bo, Hou Yi, Schatten Heide, Sun Qing-Yuan
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China.
J Cell Physiol. 2022 Jan;237(1):730-742. doi: 10.1002/jcp.30498. Epub 2021 Jun 30.
In mammals, oocytes are arrested at G2/prophase for a long time, which is called germinal vesicle (GV) arrest. After puberty, fully-grown oocytes are stimulated by a gonadotropin surge to resume meiosis as indicated by GV breakdown (GVBD). CCNB1 is accumulated to a threshold level to trigger the activation of maturation promoting factor (MPF), inducing the G2/M transition. It is generally recognized that the anaphase-promoting complex/cyclosome (APC/C) and its cofactor CDH1 (also known as FZR1) regulates the accumulation/degradation of CCNB1. Here, by using small interfering RNA (siRNA) and messenger RNA (mRNA) microinjection, immunofluorescence and confocal microscopy, immunoprecipitation, time-lapse live imaging, and immunoblotting analysis, we showed that Septin 4 regulates the G2/M transition by regulating the accumulation of CCNB1 via APC/C . Depletion of Septin 4 caused GV arrest by reducing CCNB1 accumulation. Unexpectedly, the expression level of CDC20 was higher in Septin 4 siRNA-injected oocytes than in control oocytes, but there was no significant change in the expression level of CDH1. Importantly, the reduced GVBD after Septin 4 depletion could be rescued not only by over-expressing CCNB1 but also could be partially rescued by depleting CDC20. Taken together, our results demonstrate that Septin 4 may play a critical role in meiotic G2/M transition by indirect regulation of CCNB1 stabilization in mouse oocytes.
在哺乳动物中，卵母细胞会在G2期/前期停滞很长时间，这被称为生发泡（GV）停滞。青春期后，完全成熟的卵母细胞会受到促性腺激素激增的刺激，从而恢复减数分裂，这表现为GV破裂（GVBD）。CCNB1积累到阈值水平以触发成熟促进因子（MPF）的激活，诱导G2/M期转换。人们普遍认为后期促进复合物/细胞周期体（APC/C）及其辅因子CDH1（也称为FZR1）调节CCNB1的积累/降解。在这里，我们通过使用小干扰RNA（siRNA）和信使RNA（mRNA）显微注射、免疫荧光和共聚焦显微镜、免疫沉淀、延时实时成像以及免疫印迹分析表明，Septin 4通过APC/C调节CCNB1的积累来调控G2/M期转换。Septin 4的缺失通过减少CCNB1的积累导致GV停滞。出乎意料的是，注射Septin 4 siRNA的卵母细胞中CDC20的表达水平高于对照卵母细胞，但CDH1的表达水平没有显著变化。重要的是，Septin 4缺失后GVBD的降低不仅可以通过过表达CCNB1来挽救，而且通过敲低CDC20也可以部分挽救。综上所述，我们的结果表明，Septin 4可能通过间接调节小鼠卵母细胞中CCNB1的稳定性在减数分裂G2/M期转换中发挥关键作用。
