体细胞核替代生发泡重构卵母细胞减数分裂异常的机理研究

Studies have demonstrated that abnormal oocyte meiosis is a major problem that causes apogeny, spontaneous abortion and birth defect. Nuclear transfer into prophase oocytes can be used as an approach not only to studying the cell cycle control and nuclear-cytoplasmic interactions during oocyte meiosis, but also to achieving somatic cell haploidization and treating sterility caused by complete absence of oocytes or spermatozoa. Up to date, however, normal maturation and activation have not been achieved in oocytes reconstructed by replacing germinal vesicles (GV) of oocytes with somatic cell nuclei. Therefore, mechanism studies on meiotic anomalies in oocytes manipulated to replace GV with somatic cell nuclei will contribute not only to our understanding of the mechanism of germ cell meiosis, but also to the establishment of techniques for somatic cell haploidization. Based on our findings that oocytes manipulated to replace GV with primary spermatocyte (PS) nuclei did not undergo cortical polarization and showed an unequal MAPK allocation with their polar bodies, this project will (i) study the effects of spindle migration, cortical polarization, chromosome condensation and MAPK allocation between oocyte and polar bodies on the generation of giant polar bodies, polar body degeneration and pronuclear formation after maturation and/or activation of reconstructed oocytes with GV replaced with PS nuclei; (ii) quantify expression of the molecules involved in regulation of spindle migration, chromosome condensation/separation and cleavage furrow positioning in the reconstructed oocytes; (iii) determine the effects of RNAi or over expression of the related molecules on the related meiotic events, to try to improve the developmental potential of the oocytes with GV replaced with somatic cell nuclei.

研究表明，卵母细胞减数分裂异常是导致不孕、自发流产和先天缺陷的主要原因之一。用体细胞核替代生发泡（GV）重构卵母细胞是研究减数分裂细胞周期调控和核质互作机理不可替代的模型，也是实现体细胞单倍体化，治疗由于卵母细胞或精子完全缺乏而导致不育的重要方法。然而，目前用体细胞核替代GV重构的卵母细胞还不能正常成熟和激活。因此，深入研究这种重构卵母细胞减数分裂异常的机理既有助于了解减数分裂，也有助于建立体细胞单倍体化技术。我们将在发现初级精母细胞核替代GV重构卵母细胞皮质不极化和MAPK在极体和卵之间分配不均的基础上，深入研究1）重构卵纺锤体迁移、皮质极化、染色体凝集及MAPK分配对大极体产生、极体退化和原核形成的影响；2）参与调控纺锤体迁移、染色体凝集/分离和分裂沟定位等相关分子在重构卵中的表达；3）通过RNAi和基因过表达研究相关分子在调控重构卵减数分裂中的作用，力争通过某些分子过表达使重构卵发育

将体细胞核注射到GV卵母细胞质中重构卵母细胞出现减数分裂异常，说明GV物质在减数分裂中有不可替代作用，但具体机制不清楚。本项目研究发现，将初级精母细胞核（PSN）注射到前中期I卵胞质中的重构卵母细胞不发生减数分裂异常，但将PSN注射到去GV卵胞质中的重构（PSG）卵母细胞发生减数分裂异常。在所观察到的异常中，纺锤体组装异常伴有染色体凝集不充分和出现染色质桥。纺锤体迁移异常、皮质不极化及异常的纺锤体导致分裂沟随机定位、大第一极体（PB1）形成和MAPK在卵母细胞和PB1间分配不均。纺锤体组装检验点（SAC）激活但不阻止异常分裂。MAPK不均等分离造成原核形成以及PB1退化的差异：得到较多MAPK的卵形成原核能力更强，而得到较多MAPK的PB1则更容易退化。抑制Top2通过激活Aurora B和SAC阻碍小鼠卵母细胞核成熟，说明Top2和Aurora B的激活可能与GV物质有关。总之，使用PSG卵母细胞作为实验模型，本项目揭示出缺少GV物质造成减数分裂异常的分子机理，对于进一步研究GV物质在卵母细胞减数分裂调控中的作用具有重要意义。