【作者】 张清华；

【导师】 安利国； 孙青原；

【作者基本信息】 山东师范大学 ， 动物学， 2010， 博士

【摘要】 与在有丝分裂中一样,减数分裂中的纺锤体检验点也是通过抑制后期的起始来保证染色体的精确分离,即使还剩一条染色体没有排列在赤道板上也会产生足够的后期延迟信号来保持纺锤体检验点的活性。纺锤体检验点信号通路包括Mad1, 2, 3(Mad:mitotic arrest deficient)和Bub1, 2, 3(Bub:budding unperturbed by benzimidazole)蛋白等主要成分。它们除了构成检验点的主要结构成份之外,还有负责放大检验点信号并调控其他检验点蛋白的活性,如Mps1和Aurora B等。在多细胞生物中,有另外3种蛋白:ZW10,Rough Deal(Rod)和Zwilch(三者合称为RZZ复合体),也在有丝分裂纺锤体检验点中具有至关重要的作用。最近,人们认为多细胞生物中的这3种蛋白会形成一种复合体,在募集动粒元件(dynein/dynactin复合体, Mad1和Mad2)到动粒的过程中发挥作用。Spindly是一种新近才被发现的纺锤体检验点蛋白,它首先是在果蝇的S2细胞中被发现的,它在线虫和人类中的同源蛋白分别被命名为SPDL-1和Hs Spindly/hSpindly。在不同的物种中,Spindly及其同源蛋白都具有在体细胞的有丝分裂中募集dynein或dynein/dynactin复合体到动粒的功能,进而在动粒-微管连接的建立中发挥作用,而且作用的发挥被认为是同RZZ复合体协同合作的过程。Spindly的缺失会导致果蝇、线虫和人体细胞中广泛的中期停滞。在果蝇中,Spindly在募集dynein到未与微管连接的动粒及转运Rod和Mad2离开中期动粒的过程中发挥作用。在线虫中,SPDL-1负责募集dynei-dynactin复合体和MDF-1(线虫中Mad1的同源蛋白)到动粒。而在人类的HeLa细胞中,Hs Spindly募集dynein/dynactin复合体到动粒,却并不参与检验点蛋白往动粒的募集过程。但是,这种蛋白在减数分裂中的作用尚未见有人报道,也未见有人报道过这种蛋白的同源物在小鼠细胞分裂过程的表达、分布和功能等情况。本实验首先使用Western Blot和免疫荧光的方法,鉴定了Spindly的同源蛋白(mSpindly)在小鼠卵母细胞减数分裂过程中的表达情况及不同时期的亚细胞定位情况。然后,又用过表达和RNAi等方法研究了mSpindly在小鼠卵母细胞减数分裂中的功能。本实验使用的卵母细胞取自4~6周龄的雌性昆明白小鼠,并在取卵48h前注射7.5～10个单位孕马血清促性腺激素(pregnant mare serum gonadotropin,PMSG)进行超数排卵。我们收取6个典型时期(GV期、GVBD期、pro-MⅠ期、MⅠ期、AⅠ-TⅠ期以及MⅡ期)的小鼠卵母细胞作为研究对象,通过Western Blot,发现mSpindly在这6个典型时期均有表达,且表达水平没有明显变化,说明在小鼠卵母细胞的减数分裂过程中有Spindly的同源蛋白mSpindly的表达,而且其的表达量在GV期后的整个减数分裂过程中没有明显变化。接下来,我们提取小鼠卵母细胞的总RNA后进行反转录,得到小鼠卵母细胞的cDNA。再以此cDNA为模板,用巢式RCR的方法扩增出mSpindly的CDS区全长片段,将此基因片段连接到过表达载体上,体外转录得到带有Myc标签的mSpindly的mRNA。通过显微注射的方法将此mRNA注射入GV期小鼠卵母细胞质中,在外源的mRNA翻译成蛋白后,用FITC标记的抗Myc抗体孵育,并用激光共聚焦显微镜观察mSpindly在小鼠卵母细胞减数分裂GV期及以后各时期中的亚细胞定位变化情况。我们发现mSpindly在GVBD(germinal vesicle breakdown)期染色质开始浓缩时定位在动粒上,而在MⅠ期和MⅡ期,mSpindly在动粒和纺锤体的两极均有定位。在得到mSpindly在小鼠卵母细胞减数分裂的各时期表达和亚细胞定位结果之后,我们通过显微注射的方法将高浓度的mSpindly mRNA注射入GV期小鼠卵母细胞,使mSpindly蛋白在小鼠卵母细胞内过量表达。通过激光共聚焦显微镜观察mSpindly在小鼠卵母细胞中的定位,同时统计过表达mSpindly的小鼠卵母细胞排出第一极体的时间与对照组的差异。并对排出第一极体的小鼠卵母细胞进行染色体铺片观察,看过表达对染色体的正确分离是否有影响。我们发现人为地使mSpindly过表达不会改变mSpindly在小鼠卵母细胞减数分裂过程中的亚细胞定位,并且也不会缩短第一极体的排出时间;染色体铺片结果显示过表达mSpindly对染色体的正确分离也没有产生显著影响。接下来我们又通过显微注射的方法将mSpindly Morpholino注射入GV期小鼠卵母细胞,干涉mSpindly蛋白的表达,并用激光共聚焦显微镜观察mSpindly缺失的小鼠卵母细胞的纺锤体形态,染色体在中期赤道板上的排列,以及第一极体的排出率。我们发现mSpindly的缺失会造成pro-MⅠ/MⅠ期的停滞,卵母细胞不能进入后期,同时也影响第一极体的排放,并且伴随着异常形态的纺锤体及不能正常排列的染色体。我们的结果表明,在小鼠卵母细胞的减数分裂过程中,有Spindly同源蛋白mSpindly的表达和特殊定位,并且通过功能研究,我们发现mSpindly参与了减数分裂纺锤体检验点的沉默,在减数分裂过程从pro-MⅠ期到MⅠ期,再到AⅠ期的发展过渡中起着必不可少的重要作用。根据对现有资料的分析,我们认为它可能在纺锤体的形成过程中扮演着募集或/和转运检验点蛋白的角色,而且在这个过程中mSpindly需要和其他因子一起发挥作用。本实验首次在减数分裂过程中发现Spindly同源蛋白的定位和功能,证明了Spindly在减数分裂纺锤体检验点中发挥必不可少的功能,它的缺失会导致减数分裂纺锤体检验点的持续激活,第一次减数分裂的中期延长,异常的纺锤体形态和染色体排列以及第一极体不能正常排出。以上现象均说明在mSpindly缺失的情况下,小鼠卵母细胞不能正常成熟,进而影响到之后的受精及胚胎发育过程。该研究也为哺乳动物不孕不育症的研究及治疗提供了一定的理论基础。更多还原

【Abstract】 As in mitosis, the spindle assembly checkpoint is ensuring correct segregation of chromosomes or sister chromatids by preventing the onset of anaphase. Even a single misaligned chromosome is sufficient to generate an anaphase waiting signal to maintain SAC activation and delay the anaphase onset. SAC signal pathway includes Mad1, 2, 3 (Mad for mitotic arrest deficient) and Bub1, 2, 3 (Bub for budding unperturbed by benzimidazole) as major components. Besides these proteins, Mps1 and Aurora B amplify the SAC signal and regulate mitotic checkpoint complex formation. In metazoan cells there are three additional proteins, Zw10, Rough Deal (Rod) and Zwilch, that play a crucial role in mitotic spindle checkpoint functions. Recently, it was determined that the proteins form a complex (the RZZ complex), which is required for recruitment of kinetochore components (dynein/dynactin complex, Mad1, and Mad2). Spindly is a newly found spindle assembly checkpoint protein which was first identified in S2 cells of Drosophila; its homologues are termed SPDL-1 in Caenorhabditis elegans and Hs Spindly/hSpindly in humans. In all these species, Spindly and its homologues execute the function of recruiting dynein or dynein/dynactin complex to kinetochores and as a kinetochore-microtubules association regulator by cooperating with RZZ complex in mitosis of somatic cells. Depletion of Spindly causes an extensive metaphase arrest during somatic mitoses in Drosophila, C. elegans and humans. In Drosophila, Spindly is required for recruiting dynein to unattached kinetochore and shedding of Rod and Mad2 from the kinetochores in metaphase; in C. elegans, SPDL-1 presides over the recruitment of dynein/dynactin complex and MDF-1 to the kinetochores; in humans, Hs Spindly is required for recruiting both dynein and dynactin to kinetochores but it is dispensable for removal of checkpoint proteins from kinetochores. To data, we have not seen any report on study of Spindly homologues in meiosis of any animal, and no study on mouse at all.The present study was designed to investigate the localization and function of the Spindly homologue (mSpindly) during mouse oocyte meiotic maturation by immunofluorescent analysis, and by overexpression and knockout of mSpindly. Firstly, by using Western Bolt and immunofluorescence we identified the expression and subcellular localization of mSpindly in the meiosis of mouse oocytes. And then, we used overexpression and RNAi techniques to study the function of mSpindly in the meiotic process of mouse oocytes.Female mice Kunming white,4- to 6-week-old, were used in our study. Before oocytes collection the mice were injected with 7.5～10 U PMSG for the superovulation.In the experiments, we collected 6 typical stage samples of mouse oocytes: GV, GVBD, pro-MⅠ, MⅠ, AⅠ-TⅠand MⅠstages. With Western blot, we found that mSpindly protein was expressed from GV to MⅠstages, without detectable change in expression level in the whole process. This implies that the Spindly homologue, mSpindly, was exist in mouse oocyte meiosis, and the level of its expression has been kept in a balanced level during the meiotic process. Next, we extracted the total RNA from mouse oocytes, and got the cDNA after RT-PCR. We used the cDNA as template to clone the full length mSpindly gene sequence and cloned this sequence to pCS2+MT vector which already has a Myc-tag in it. Then we got the Myc-mSpindly mRNA after transcription invitro. We microinjected the Myc-mSpindly mRNA into cytoplasm of GV stage mouse oocytes. After the exogenous mRNA was translated into Myc-mSpindly protein, we used FITC-anti-Myc antibody to incubate the oocytes, and then observed the subcellular localization of mSpindly in the mouse oocytes with confocal. We found that when the oocytes developed to GVBD stage, mSpindly was specifically localized to kinetochores. In MⅠand MⅠstage oocytes, mSpindly was typically localized to both the kinetochores and the spindle poles.As our result showed that mSpindly do express and take specific subcellular localization during meiosis of mouse oocytes; we then microinjected high concentration of Myc-mSpindly mRNA into mouse oocytes at GV stage to overexpress mSpindly, and observed the localization of the overexpressed Myc-mSpindly with confocal; we also calculated the time of the first polar body emission (PBE) and spread the chromosomes of the PBE oocytes to see that whether overexpression of mSpindly influenced exact segregation of the chromosomes. We found that overexpression of mSpindly factitiously did not change the subcellular localization of mSpindly in mouse oocytes meiosis, and did not promote the first polar extrude, too. In the chromosome spreading samples we observed no influence of overexpression of mSpindly on exact segregation of chromosomes. Next, we microinjected the mSpindly Morpholino into the GV stage mouse oocytes to depleted mSpindly, and then made observations on the spindle shape, chromosome alignment and the percentage of PBE. We found that depletion of mSpindly caused an arrest of MⅠstage. Most of the mSpindly-depleted oocytes couldn’t enter anaphase and failed to extrude the first polar body, accompanied with abnormal spindle and misalignment of chromosomes.Our data suggested that mSpindly participates in SAC silencing and in spindle formation, the mechanism of its function might be as a recruiter and/or a transporter of kinetochore proteins in mouse oocytes, but it would need to cooperate with other factors to fulfill its function.The present study found the Spindly homologues in meiosis for the first time, and proved that this Spindly homologue participates in meiotic SAC, and depletion of it caused constant SAC activation, pro-MⅠ/ MⅠstage arrest, abnormal spindle shape, unaligned chromosomes and failed extrude the first polar body. All the above phenomena suggested that the mSpindly-depleted mouse oocytes couldn’t maturate normally, and it would affect the following fertilization and embryonic development. Our study also provides some basic materials for the therapy of mammal infertility.更多还原