College of Animal Science and Technology, Nanjing Agricultural University, 210095, Nanjing, China.
Reprod Biol Endocrinol. 2021 Apr 19;19(1):57. doi: 10.1186/s12958-021-00740-1.
In mitotic cells, WAPL acts as a cohesin release factor to remove cohesin complexes from chromosome arms during prophase to allow the accurate chromosome segregation in anaphase. However, we have recently documented that Wapl exerts a unique meiotic function in the spindle assembly checkpoint (SAC) control through maintaining Bub3 stability during mouse oocyte meiosis I. Whether this noncanonical function is conserved among species is still unknown.
We applied RNAi-based gene silencing approach to deplete WAPL in porcine oocytes, validating the conserved roles of WAPL in the regulation of SAC activity during mammalian oocyte maturation. We also employed immunostaining, immunoblotting and image quantification analyses to test the WAPL depletion on the meiotic progression, spindle assembly, chromosome alignment and dynamics of SAC protein in porcine oocytes.
We showed that depletion of WAPL resulted in the accelerated meiotic progression by displaying the precocious polar body extrusion and compromised spindle assembly and chromosome alignment. Notably, we observed that the protein level of BUB3 was substantially reduced in WAPL-depleted oocytes, especially at kinetochores.
Collectively, our data demonstrate that WAPL participates in the porcine oocyte meiotic progression through maintenance of BUB3 protein levels and SAC activity. This meiotic function of WAPL in oocytes is highly conserved between pigs and mice.
在有丝分裂细胞中，WAPL 作为黏连蛋白释放因子，在前期从染色体臂上移除黏连蛋白复合物，以确保后期准确分离染色体。然而，我们最近的研究表明，Wapl 在小鼠卵母细胞减数分裂 I 过程中通过维持 Bub3 的稳定性，发挥独特的有丝分裂功能来控制纺锤体组装检查点（SAC）。这种非典型功能在物种间是否保守尚不清楚。
我们应用 RNAi 基因沉默方法在猪卵母细胞中耗尽 WAPL，验证了 WAPL 在哺乳动物卵母细胞成熟过程中调节 SAC 活性的保守作用。我们还采用免疫染色、免疫印迹和图像定量分析方法，检测 WAPL 耗竭对猪卵母细胞减数分裂进程、纺锤体组装、染色体排列和 SAC 蛋白动力学的影响。
我们发现 WAPL 的耗竭导致减数分裂进程加速，表现为提前极体挤出和纺锤体组装及染色体排列受损。值得注意的是，我们观察到 WAPL 耗竭的卵母细胞中 BUB3 蛋白水平显著降低，尤其是在着丝粒处。
总之，我们的数据表明，WAPL 通过维持 BUB3 蛋白水平和 SAC 活性参与猪卵母细胞的减数分裂进程。WAPL 在卵母细胞中的这种减数分裂功能在猪和小鼠之间高度保守。
