Cyclin B2/CDK1通过APC/CCDH1调控卵母细胞减数分裂恢复的机制研究

Maturation-promoting factor (MPF), which is classically recognized to consist of Cyclin B1 and Cyclin-dependent kinase 1(CDK1), promotes the resumption of meiosis directly in mammalian oocytes. We previously reported that conditional knockout of Cyclin B1 had no effect on meiosis I progression in mouse oocytes because Cyclin B2 compensated for Cyclin B1’s function, and knockout of Cyclin B2 prevented the meiotic resumption instead. So, it is of great significance to study the function of Cyclin B2 in regulation of meiotic resumption. ..Our preliminary results show that Cyclin B1 is declined dramatically in Cyclin B2-null oocytes, and supplement of stable Cyclin B1 can rescue and promote meiotic resumption. It is indicated that Cyclin B2/CDK1 may affect the stability of Cyclin B1 through regulating the activity of anaphase-promoting complex/cyclosome (APC/C), the upstream of Cyclin B1. ..In this project, using Cyclin B2 knockout and Cyclin B1 conditional knockout mice, we expect to study the mechanism of MPF regulation by Cyclin B2/CDK1-APC/C pathway, and to investigate the interaction between Cyclin B2 and CDH1, the co-activator of APC/C. The project will not only reveal the role of Cyclin B2/CDK1 in regulation of oocyte meiotic resumption, but also provide new theoretical reference for clinical therapy of female reproductive disease.

成熟促进因子MPF一直被认为是由CyclinB1和CDK1所构成。我们已报道：CyclinB1的敲除并不影响小鼠卵母细胞减数分裂的恢复，而CyclinB2的敲除反而阻滞了减数分裂的恢复。因此，研究CyclinB2在减数分裂恢复中的功能具重要意义。我们的预实验表明：CyclinB1在敲除CyclinB2的卵母细胞中发生了显著下调，补充CyclinB1可以促进减数分裂的恢复。这提示我们：CyclinB2/CDK1可能通过调控后期促进复合物APC/C的活性影响了CyclinB1的稳定性。本项目拟利用CyclinB2敲除和CyclinB1条件性敲除小鼠模型，研究CyclinB2/CDK1通过APC/C来调控MPF活性的分子机制，探讨CyclinB2与APC/C激活因子CDH1的作用关系。本项目不仅可揭示CyclinB2调控卵母细胞减数分裂恢复的分子机制，也可为临床女性生殖疾病的诊疗提供新的参考。

本项目基于CyclinB2敲除卵母细胞减数分裂恢复严重受阻及其卵母细胞中的后期促进复合物APC/C活性升高的表型提出了CyclinB2/CDK1可能通过影响APC/CCDH1活性调控了卵母细胞减数分裂的恢复这一科学假说。为了解答该科学问题，本项目对CyclinB2敲除卵母细胞的减数分裂进程、CDK1磷酸化状态、CyclinB1蛋白水平变化、后期促进复合物APC/C的活性、CDH1蛋白的磷酸化水平变化以及CyclinB2与CDH1的蛋白间互作进行了全面分析，并通过激活卵母细胞中的CDK1、上调CyclinB1的蛋白水平、改变CDH1的磷酸化状态来补救CyclinB2敲除卵母细胞减数分裂的恢复，进而通过质谱分析和磷酸化位点突变手段研究分析了CyclinB2/CDK1在CDH1蛋白上的具体磷酸化作用位点。通过本项目的研究，我们发现CyclinB2敲除卵母细胞生发泡中CyclinB1的蛋白过低是造成减数分裂恢复受阻的主要原因，进一步研究发现CyclinB2与CDH1之间存在特异性的蛋白互作并且CyclinB2表达变化会影响CDH1蛋白的磷酸化水平。本项目的研究结果揭示了在卵母细胞减数分裂恢复过程中，CyclinB2/CDK1复合体通过影响CDH1的磷酸化状态调控了卵母细胞中APC/CCDH1活性的分子机制。本项目的研究成果丰富了卵母细胞减数分裂成熟调控的分子机制，也为临床女性卵母细胞成熟障碍的病因寻找提供了新的参考。