p38MAPK和BMPs在鱼类卵母细胞成熟过程中的作用及机制研究

Oocyte maturation is complex events, requiring the coordinated action of hormone, growth factors, and enviroment factors. p38MAPK is a member of mitogen-activated protein kinase(MAPK)superfamily playing crucial roles in oocyte development and maturation in mammals. However, to date, no published information is available in regarding to the mechanisms of action for p38MAPK in teloests.A number of techniques,such as confocal scanning, Western blot, quantitative real time PCR, microinjection, and oocyte maturation in vitro, will be used in this study. In present study,we will firstly determine that expression, localization,and activation of p38MAPK in oocyte maturation of zebrafish.Secondly, point mutations of p38MAPK and MKK3 will be generated by a two-step PCR protocol. With or without mutations of p38MAPK and MKK3 will be subcloned to the pCS2+ expression vector, and mRNA will be synthesized in vitro using the linearzed plasmid DNA of the constructs as a templete. In order to determine the effects of p38MAPK on BMPs induced-oocyte maturation, transcripts synthesized mRNA in vitro will be used to microinjection in follicle-enclosed oocytes.The expression of some factors of BMPs singal pathway will be analyzed. To identify whether BMPs can activate p38MAPK, Western blot analyses will be done to determine protein and phosphorylated of p38MAPK by BMPs-induced or inhibitor of BMPs type I receptor. Lastly, the mechanism of p38MAPK and BMPs regulated oocyte maturation under different photoperiod will be preliminarily clarified. The results of this study will not only reveal the biological effects of p38MAPK and BMPs on oocyte mautration of zebrafish, will be also important to improve fish artificial breeding technology and further clarify the vertebrate oocyte maturation mechanism.

硬骨鱼类卵母细胞成熟受激素、生长因子和环境因子等多因素调控。p38MAPK属于丝裂原活化蛋白激酶超家族，其在硬骨鱼类卵母细胞成熟中的作用及机制还未完全清楚。本课题拟用激光共聚焦、Western blot、定量PCR、显微注射、卵母细胞体外成熟等方法，以斑马鱼为对象研究卵母细胞成熟过程中p38MAPK的表达、定位及活性变化；通过构建p38MAPK突变体、添加BMPs或其受体阻断剂，探讨p38MAPK失活或持续激活对卵母细胞生发泡迁移和破裂及BMPs通路中信号分子的影响，并分析BMPs与p38MAPK在卵母细胞成熟过程中的相互作用；最后进一步探讨p38MAPK和BMPs响应光周期变化调控卵母细胞成熟的机制。上述研究，不仅可揭示p38MAPK和BMPs调节鱼类卵母细胞成熟的作用机理，对进一步改善鱼类人工繁殖技术也有重要意义，同时可为研究脊椎动物卵母细胞成熟提供重要理论资料。

硬骨鱼类卵母细胞成熟受激素、生长因子和环境因子等多因素调控。p38MAPK属于丝裂原活化蛋白激酶超家族，其在硬骨鱼类卵母细胞成熟中的作用及机制还未完全清楚。在本课题中，我们研究了p38MAPK在斑马鱼卵母细胞体内及体外成熟过程中的表达、定位及活性变化。发现p38MAPKmRNA和p38MAPK蛋白在斑马鱼不同发育时期的卵母细胞中均有表达，而p-p38MAPK水平在早期卵母细胞中较低，随卵母细胞发育增加但在卵母细胞成熟后降低，在滤泡细胞和卵母细胞细胞质和细胞核中均可检测到p-p38MAPK。DHP诱导斑马鱼卵母细胞成熟过程中，p38MAPK磷酸化水平先升高随后显著降低，证明p38MAPK参与调控卵母细胞减数分裂恢复。进一步研究发现SB203580抑制p38MAPK激活或p38突变失活p38MAPK，可促进卵母细胞GVBD发生，p38MAPK通过调控Mos、CyclinB、BMPs信号通路影响卵母细胞成熟。蛋白质组分析发现，斑马鱼卵母细胞自发成熟或DHP诱发成熟过程中，p38MAPK通过调控Cdc42、PGMCR1、MAP2K2A等蛋白的表达影响卵母细胞成熟。同时，本研究发现不同光周期影响斑马鱼卵母细胞发育，在响应光周期变化过程中p38MAPKmRNA及磷酸化水平发生变化，BMPs信号分子的表达变化存在一定差异。本项目的研究成果对进一步理解硬骨鱼类卵母细胞最终成熟机制，改善鱼类人工繁殖技术具有重要的参考价值。