微囊藻毒素microcystin-LR通过调节MAPK信号通路影响斑马鱼卵母细胞减数分裂的机制研究

Cyanobacterial blooms and the associated microcystin (MC) have become a global ecological disaster. MC can strongly inhibit the protein phosphatase PP2A activity with liver and gonad as the first and second important target organs, respectively. Our previous studies show that MC-LR exposure to zebrafish significantly influenced the quantity and quality of eggs，inhibited the PP2A activity, activated the mitogen-activated protein kinase (MAPK) and accelerated the germinal vesicle breakdown (GVBD), an indicator of resumption of first meiotic division. We therefore propose a scientific hypothesis of “MCLR-PP2A-MAPK-Meiosis” and believe that MC-LR leads to oocytes developmental disorders by inhibiting PP2A activity, activating MAPK signaling pathway and influencing the meiosis progress. Zebrafish will be used as experimental subject for evaluating the reproductivity, PP2A activity, phosphorylation levels of MAPK and their cellular localization. An oocyte in vitro culture system will be established to verify the effects of MC-LR on MAPK signaling pathway, GVBD, spindle assembly and chromosomal arrangement, which are very important meiotic events. The present study will further reveal the mechanism of reproductive toxicological effects of microcystin on fish, and provide some academic bases for the protection of biodiversity of polluted water and the evaluation of ecological safety.

蓝藻水华及微囊藻毒素（Microcystin，MC）污染已成为全球性的生态灾难。MC是蛋白磷酸酶PP2A的强效抑制剂，且性腺是MC除肝脏之外的第二靶器官。我们的研究表明MC-LR引起斑马鱼产卵的“量”和“质”下降、PP2A活性抑制、丝裂原活化蛋白激酶（MAPK）激活、生发泡破裂GVBD（第一次减数分裂恢复标志）提前。我们提出“MCLR-PP2A-MAPK-Meiosis”科学假设，认为MC-LR通过抑制PP2A活性、激活MAPK通路并影响减数分裂而造成卵母细胞发育障碍的。项目以斑马鱼为研究对象，研究MC-LR对生殖力、PP2A活性、MAPK磷酸化及细胞定位的影响。项目还拟建立卵母细胞体外培养体系，研究MC-LR对其MAPK信号通路及GVBD、纺锤体组装、染色体排列等减数分裂事件的影响。完成本项目将进一步揭示MC对鱼类生殖毒性的作用机制，为污染水体的生物多样性保护和生态安全评价提供理论参考。

蓝藻水华及微囊藻毒素（Microcystin，MC）污染已成为全球性的生态灾难。MC是蛋白磷酸酶PP2A的强效抑制剂，且性腺是MC除肝脏之外的第二靶器官。我们的研究表明MC-LR引起斑马鱼产卵的“量”和“质”下降、PP2A活性抑制、丝裂原活化蛋白激酶（MAPK）激活、生发泡破裂GVBD（第一次减数分裂恢复标志）提前。我们提出“MCLR-PP2A-MAPK-Meiosis”科学假设，认为MC-LR通过抑制PP2A活性、激活MAPK通路并影响减数分裂而造成卵母细胞发育障碍的。本项目以性成熟雌性斑马鱼和斑马鱼卵母细胞为研究对象，开展了环境相关浓度MC-LR对在体斑马鱼及卵母细胞离体暴露实验。结果表明：MC-LR对雌性斑马鱼暴露导致卵巢PP2A活性受到显著抑制，激活了MAPK信号通路的转录及表达水平，MC-LR通过PP2A-MAPK-MPF信号通路促进卵母细胞发生GVBD，引发卵母细胞成熟进程紊乱；此外，MC-LR通过内质网应激通路引发卵母细胞凋亡，还比较了MC-LR暴露造成斑马鱼肝脏、卵母细胞以及F1代幼鱼凋亡的作用机制的差异；并且，本项目首次全面建立了斑马鱼卵母细胞分离和体外培养体系，并利用该体系，进一步验证了MC-LR在促进斑马鱼卵母细胞成熟过程中，MAPK发挥着重要的作用。完成项目研究任务的同时，发表高水平学术论文7篇，指导研究生（博士生和硕士生）7名。总之，本研究进一步揭示MC对鱼类生殖毒性的作用机制，较好地完成了基金申请书中既定的研究任务，为污染水体的生物多样性保护和生态安全评价提供理论参考。