大鼠卵母细胞体外发育过程中的微管组装研究(英)

大鼠卵母细胞的发育具有许多与其他哺乳动物不同的特点,用激光共聚焦显微术研究了大鼠卵母细胞发育过程中微管的组装过程,以及一些因素对微管组装过程的影响.结果表明,在大鼠卵母细胞减数分裂的细胞周期进程中,细胞微管系统发生广泛而剧烈的重组,紫杉醇、星形孢菌素和冈田酸等药物能显著改变卵母细胞内的微管组织状态.大鼠卵母细胞可以在体外发生自发的孤雌活化,也可以被细胞松弛素诱导发生胞质假分裂.因此,研究了在这些过程中微管结构的特殊构象,以期更加深入认识这些特殊细胞事件的生化机理.     

大鼠卵母细胞体外发育过程中的微管组装研究

大鼠卵母细胞的发育具有许多与其他哺乳动物不同的特点,用激光共聚焦显微术研究了大鼠卵母细胞发育过程中微管的组装过程,以及一些因素对微管组装过程的影响.结果表明,在大鼠卵母细胞减数分裂的细胞周期进程中,细胞微管系统发生广泛而剧烈的重组,紫杉醇、星形孢菌素和冈田酸等药物能显著改变卵母细胞内的微管组织状态.大鼠卵母细胞可以在体外发生自发的孤雌活化,也可以被细胞松弛素诱导发生胞质假分裂.因此,研究了在这些过程中微管结构的特殊构象,以期更加深入认识这些特殊细胞事件的生化机理.     

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.     

孤雌活化诱导恢复的小鼠减数分裂期间细胞骨架的动态与作用

减数分裂的顺利完成是胞质分裂和核分裂在时间和空间上的协调结果,细胞骨架系统在减数分裂的一系列事件中具有重要的调节作用.实验通过孤雌活化诱导小鼠MⅡ期卵减数分裂恢复,采用激光共聚焦显微术检测了减数分裂期间的微管、微丝和核的动态变化,并通过细胞骨架药物处理,以分析微管和微丝在减数分裂事件中的不同作用.结果显示：纺锤体微管为核的定位、分离和运动所必需;纺锤体从与质膜平行旋转至与质膜垂直是极体排放的前提;微丝是控制纺锤体旋转的关键因素;纺锤体旋转完成后微丝随即解聚,不参与极体的最后排出,形成原核后再重新组装.     

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.     

Kinesin-14 Pkl1 targets γ-tubulin for release from the γ-tubulin ring complex (γ-TuRC) ‬‬‬‬‬‬‬

The γ-tubulin ring complex (γ-TuRC) is a key part of microtubule-organizing centers (MTOCs) that control microtubule polarity, organization and dynamics in eukaryotes. Understanding regulatory mechanisms of γ-TuRC function is of fundamental importance, as this complex is central to many cellular processes, including chromosome segregation, fertility, neural development, T-cell cytotoxicity and respiration. The fission yeast microtubule motor kinesin-14 Pkl1 regulates mitosis by binding to the γ-tubulin small complex (γ-TuSC), a subunit of γ-TuRC. Here we investigate the binding mechanism of Pkl1 to γ-TuSC and its functional consequences using genetics, biochemistry, peptide assays and cell biology approaches in vivo and in vitro. We identify two critical elements in the Tail domain of Pkl1 that mediate γ-TuSC binding and trigger release of γ-tubulin from γ-TuRC. Such action disrupts the MTOC and results in failed mitotic spindle assembly. This study is the first demonstration that a motor protein directly affects the structural composition of the γ-TuRC, and we provide details of this mechanism that may be of broad biological importance.     

Changes at the hamster oocyte surface from the germinal vesicle stage to ovulation

Immature and ovulated hamster oocytes were studied with the scanning electron microscope. Immature oocytes at the germinal vesicle stage have their surface uniformly covered by microvilli. When meiosis has progressed to the first meiotic metaphase the overlying surface shows the differentiation of a circular area 19 μm in diameter with a low density of microvilli. Later, from this region the first polar body emerges, and the oocyte surface at the point from which it was extruded shows a cluster of cytoplasmic, conical projections. When the zona-free oocytes are cultured at 37°C for 5 minutes these projections disappear and the oocyte surface at that point becomes smooth. However, when the oocytes remain in the oviduct for several hours after ovulation these projections remain unchanged. The in vitro interactions of capacitated hamster sperm with the immature oocyte was always seen at microvillus surfaces and never associated with the differentiated regions.     

烟草小孢子母细胞减数分裂过程中微管分布变化

应用间接免疫荧光标记技术和激光共聚焦扫描显微镜成像技术观察了烟草小孢子母细胞减数分裂过程中微管的分布变化。在减数分裂前期,小孢子母细胞中的微管较短,随机分散在细胞质中。在减数分裂中期,细胞质中微管形成纺锤体,控制染色体的分布。进入减数分裂I后期,部分纺锤体微管将两组染色体拉向两级。在减数分裂Ⅱ中期,细胞中的微管又形成两个纺锤体。在减数分裂Ⅱ后期,纺锤体微管解聚为微管蛋白分散在细胞质中。胞质分裂发生在四个细胞核形成之后,通过细胞核之间的质膜向内缢缩分隔四个细胞核,产生四个小孢子。