摘要保罗样激酶1(polo-like kinase 1, Plk1)在有丝分裂过程中扮演多种角色，对于中心体复制和分离、纺锤体组装及染色体分离等事件具有重要的调节作用。但目前对其在卵细胞减数分裂过程中的表达与功能仍知之甚少。本实验旨在探究猪(Sus scrofa)卵母细胞第一次减数分裂过程中 Plk1的动态表达及其对第一次减数分裂中期(metaphase Ⅰ, MⅠ)纺锤体组装的影响。首先，通过间接免疫荧光方法结合激光共聚焦显微成像技术检测了Plk1的动态表达及亚细胞定位，在此基础之上，采用Plk1特异性抑制剂GSK461364抑制实验，研究了Plk1抑制对猪卵母细胞成熟及MⅠ期染色体排列与纺锤体结构的影响。结果表明，在生发泡破裂期(germinal vesicle breakdown, GVBD)、MⅠ期和第一次减数分裂后末期(anaphase I and telophase I, ATI)Plk1均有表达，并且与α微管蛋白(α-tubulin)的亚细胞定位非常相似；采用Plk1特异性抑制剂GSK461364处理后，卵母细胞的第一极体排出率呈抑制剂浓度依赖性下降，当GSK461364浓度达0.3 μmol/L时，第一极体排出率极显著下降(39.37±5.46)% vs (81.10±7.69)% (P＜0.01)，并伴随着染色体排列的紊乱；进一步通过激光共聚焦显微成像技术检测发现，Plk1特异性抑制后，卵母细胞纺锤体结构异常的比例极其显著升高(P＜0.001)。本研究结果表明，在猪卵母细胞第一次减数分裂过程中，Plk1的亚细胞定位与α-tubulin密切相关，对于MⅠ纺锤体组装具有重要的调节作用。本研究为深入了解卵母细胞减数分裂的调节机制、进一步提高猪卵母细胞体外成熟效率提供了实验参考。
Abstract：Polo-like kinase 1 (Plk1) has been determined to have a variety of roles in the precise regulation of centrosome duplication and separation, spindle assembly, chromosome detachment, however, much less is known about its expression and function during the meiotic progression in oocytes. The present study was conducted to investigate the dynamic expression and the possible roles of Plk1 in the metaphase Ⅰ (MⅠ) stage spindle formation in porcine oocytes. Immunofluorescent staining coupled with confocal microscopy imaging techniques was used to examined the dynamic changes of Plk1. Then, the specific inhibitor GSK461364 was used to explore the potential functions of Plk1 during the meiotic maturation of porcine oocytes. Results showed that Plk1 was observed at germinal vesicle breakdown (GVBD), MⅠ and anaphase I and telophase I (ATI) stage, with a similar distribution of α-tubulin location. GSK461364 treatment resulted in the failure of polar body extrusion in pig oocytes in a concentration-dependent manner, and the first polar body emission rate was significantly decreased (39.37+5.46)% VS (81.10+7.69)% (P＜0.01) when the concentration of GSK461364 reached 0.3 mol/L, which accompanied by misaligned chromosomes. Additional confocal microscopy results showed that GSK461364 treatment resulted in a significantly higher percentage of the treated oocytes with aberrant spindles during the first meiotic division. This study show that distribution of Plk1 is closely associated with the α-tubulin expression, and the regulation is related to its effects on spindle formation during the first meiotic division in porcine oocytes. The results of this study will provide an experimental basis for further understanding the regulation mechanism of oocyte meiosis and further improve the in vitro maturation efficiency of porcine oocytes.
