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。
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.
引文
1. Brues AM, Cohen A. Effects of colchicine and related substances on cell division [J]. Biochemical Journal 1936, 30:1363–8.
2. Jackson H, Dickerson D, Gunther A. The reduction of intracranial pressure in cerebral injury by the intravenous use of hypertonic sucrose solution [J]. Annals of Surgery 1937, 106:161–8.
3. Zieve GW, Turnbull D, Mullins JM, McIntosh JR. Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor nocodazole [J]. nocodazole accumulated mitotic cells. Exp Cell Res 1980, 126:397–405.
4. Hoyt MA, Totis L, Roberts BT. Saccharomyces-Cerevisiae Genes Required for Cell-Cycle Arrest in Response to Loss of Microtubule Function [J]. Cell 1991, 66:507–17.
5. Li R, Murray AW. Feedback-Control of Mitosis in Budding Yeast [J]. Cell 1991, 66:519–31.
6. Musacchio A, Hardwick KG. The spindle checkpoint: structural insights into dynamic signalling [J]. Nat Rev Mol Cell Biol 2002, 3:731–41.
7. Taylor SS, Scott MIF, Holland AJ. The spindle checkpoint: a quality control mechanism which ensures accurate chromosome segregation [J]. Chromosome Research 2004, 12:599–616.
8. Wang WH, Sun QY. Meiotic spindle, spindle checkpoint and embryonic aneuploidy [J]. Front Biosci 2006; 11:620-36.
9. Moroi Y, Peebles C, Fritzler MJ, Steigerwald J, Tan EM. Autoantibody to centromere (kinetochore) in scleroderma sera [J]. Proc Natl Acad Sci USA 1980, 77:1627–31.
10. Moroi Y, Hartman AL, Nakane PK, Tan EM. Distribution of kinetochore (centromere) antigen in mammalian cell nuclei [J]. J Cell Biol 1981, 90:254–9.
11. Warburton PE, Cooke CA, Bourassa S, Vafa O, Sullivan BA, Stetten G, et al. Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres [J]. Curr Biol 1997, 7:901–4.
12. Hudson DF, Fowler KJ, Earle E, Saffery R, Kalitsis P, Trowell H, et al. Centromere protein B null mice are mitotically and meiotically normal but have lower body and testis weights [J]. J Cell Biol 1998, 141:309–19.
13. Craig JM, Earnshaw WC, Vagnarelli P. Mammalian centromeres: DNA sequence, protein composition, and role in cell cycle progression [J]. Exp Cell Res 1999, 246:249–62.
14. Simerly C, Balczon R, Brinkley BR, Schatten G. Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [J]. J Cell Biol 1990, 111:1491–504.
15. Ma W, Hou Y, Sun QY, Sun XF, Wang WH. Localization of centromere proteins and their association with chromosomes and microtubules during meiotic maturation in pig oocytes [J]. Reproduction 2003, 126:731–8.
16. Yen TJ, Compton DA, Wise D, Zinkowski RP, Brinkley BR, Earnshaw WC, et al. Cenp-E, a Novel Human Centromere-Associated Protein Required for Progression from Metaphase to Anaphase [J]. EMBO J 1991, 10:1245–54.
17. Tomascik-Cheeseman L, Marchetti F, Lowe X, Shamanski FL, Nath J, Pedersen RA, et al. CENP-B is not critical for meiotic chromosome segregation in male mice [J]. Mutation Res 2002, 513:197–203.
18. Bernat RL, Borisy GG, Rothfield NF, Earnshaw WC. Injection of Anticentromere Antibodies in Interphase Disrupts Events Required for Chromosome Movement at Mitosis [J]. J Cell Biol 1990, 111:1519–33.
19. Bernat RL, Delannoy MR, Rothfield NF, Earnshaw WC. Disruption of Centromere Assembly during Interphase Inhibits Kinetochore Morphogenesis and Function in Mitosis [J]. Cell 1991, 66:1229–38.
20. Pennisi E. Cell division gatekeepers identified. Science 1998, 279:477–8.
21. Rieder CL, Salmon ED. The vertebrate cell kinetochore and its roles during mitosis [J]. Trends Cell Biol 1998, 8:310–8.
22. Cimini D, Howell B, Maddox P, Khodjakov A, Degrassi F, Salmon ED. Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells [J]. J Cell Biol 2001, 153:517–27.
23. Brunet S, Maria AS, Guillaud P, Dujardin D, Kubiak JZ, Maro B. Kinetochore fibers are not involved in the formation of the first meiotic spindle in mouse oocytes, but control the exit from the first meiotic M phase [J]. J Cell Biol 1999, 146:1–12.
24. Heald R, Tournebize R, Habermann A, Karsenti E, Hyman A. Spindle assembly in Xenopusegg extracts: respective roles of centrosomes and microtubule self-organization [J]. J Cell Biol 1997, 138:615–28.
25. Polanski Z, Ledan E, Brunet S, Louvet S, Verlhac MH, Kubiak JZ, et al. Cyclin synthesis controls the progression of meiotic maturation in mouse oocytes [J]. Development 1998, 125:4989–97.
26. Taylor SS, Hussein D, Wang Y, Elderkin S, Morrow CJ. Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells [J]. J Cell Sci 2001, 114:4385–95.
27. Vigneron S, Prieto S, Bernis C, Labbe JC, Castro A, Lorca T. Kinetochore localization of spindle checkpoint proteins: who controls whom [J]? Mol Biol Cell 2004, 15:4584–96.
28. Jablonski SA, Chan GK, Cooke CA, Earnshaw WC, Yen TJ. The hBUB1 and hBUBR1 kinases sequentially assemble onto kinetochores during prophase with hBUBR1 concentrating at the kinetochore plates in mitosis [J]. Chromosoma 1998, 107:386–96.
29. Sharp-Baker H, Chen RH. Spindle checkpoint protein Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E, independently of its kinase activity [J]. J Cell Biol 2001, 153:1239–50.
30. Abrieu A, Magnaghi-Jaulin L, Kahana JA, Peter M, Castro A, Vigneron S, et al. Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint [J]. Cell 2001, 106:83–93.
31. Chen RH, Shevchenko A, Mann M, Murray AW. Spindle checkpoint protein Xmad1 recruits Xmad2 to unattached kinetochores [J]. J Cell Biol 1998, 143:283–95.
32. Chen RH. BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1 [J]. J Cell Biol 2002, 158:487–96.
33. Adams RR, Maiato H, Earnshaw WC, Carmena M. Essential roles of Drosophila inner centromere protein (INCENP) and aurora B in histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation [J]. J Cell Biol 2001, 153:865–79.
34. Kallio MJ, McCleland ML, Stukenberg PT, Gorbsky GJ. Inhibition of aurora B kinase blocks chromosome segregation, overrides the spindle checkpoint, and perturbs microtubule dynamics in mitosis [J]. Curr Biol 2002, 12:900–5.
35. Murata-Hori M, Wang YL. The kinase activity of aurora B is required for kinetochore-microtubule interactions during mitosis [J]. Curr Biol 2002, 12:894–9.
36. Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T, et al. Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores [J]. J Cell Biol 2003, 161:267–80.
37. Johnson VL, Scott MIF, Holt SV, Hussein D, Taylor SS. Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome congression [J]. J Cell Sci 2004, 117:1577–89.
38. Liu ST, Chan GKT, Hittle JC, Fujii G, Lees E, Yen TJ. Human MPS1 kinase is required for mitotic arrest induced by the loss of CENP-E from kinetochores [J]. Mol Biol Cell 2003, 14:1638–51.
39. Yen TJ, Li G, Schaar BT, Szilak I, Cleveland DW. Cenp-E Is a Putative Kinetochore Motor That Accumulates Just before Mitosis [J]. Nature 1992, 359:536–9.
40. Cooke CA, Schaar B, Yen TJ, Earnshaw WC. Localization of CENP-E in the fibrous corona and outer plate of mammalian kinetochores from prometaphase through anaphase [J]. Chromosoma 1997, 106:446–55.
41. Wood KW, Sakowicz R, Goldstein LSB, Cleveland DW. CENP-E is a plus end-directed kinetochore motor required for metaphase chromosome alignment [J]. Cell 1997, 91:357–66.
42. Lombillo VA, Nislow C, Yen TJ, Gelfand VI, Mcintosh JR. Antibodies to the Kinesin Motor Domain and Cenp-E Inhibit Microtubule Depolymerization-Dependent Motion of Chromosomes in-Vitro [J]. J Cell Biol 1995, 128:107–15.
43. Chan GK, Schaar BT, Yen TJ. Characterization of the kinetochore binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F and hBUBR1 [J]. J Cell Biol 1998, 143:49–63.
44. Chan GKT, Jablonski SA, Sudakin V, Hittle JC, Yen TJ. Human BUBR1 is a mitotic checkpoint kinase that monitors CENP-E functions at kinetochores and binds the cyclosome/APC [J]. J Cell Biol 1999, 146:941–54.
45. Yao XB, Anderson KL, Cleveland DW. The microtubule-dependent motor centromere-associated protein E (CENP-E) is an integral component of kinetochore coronafibers that link centromeres to spindle microtubules [J]. J Cell Biol 1997, 139:435–47.
46. Yao XB, Abrieu A, Zheng Y, Sullivan KF, Cleveland DW. CENP-E forms a link between attachment of spindle microtubules to kinetochores and the mitotic checkpoint [J]. Nature Cell Biology 2000, 2:484–91.
47. Mao YH, Abrieu A, Cleveland DW. Activating and silencing the mitotic checkpoint through CENP-E-dependent activation/inactivation of BubR1 [J]. Cell 2003, 114:87–98.
48. Weaver BAA, Bonday ZQ, Putkey FR, Kops GJPL, Silk AD, Cleveland DW. Centromere-associated protein-E is essential for the mammalian mitotic checkpoint to prevent aneuploidy due to single chromosome loss [J]. J Cell Biol 2003, 162:551–63.
49. Taylor SS, Ha E, Mckeon F. The human homologue of Bub3 is required for kinetochore localization of Bub1 and a Mad3/Bub1-related protein kinase [J]. J Cell Biol 1998, 142:1–11.
50. Martinez-Exposito MJ, Kaplan KB, Copeland J, Sorger PK. Retention of the Bub3 checkpoint protein on lagging chromosomes [J]. Proc Natl Acad Sci USA 1999, 96:8493–8.
51. Taylor SS, McKeon F. Kinetochore localization of murine Bub1 is required for normal mitotic timing and checkpoint response to spindle damage [J]. Cell 1997, 89:727–35.
52. Sudakin V, Chan GKT, Yen TJ. Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2 [J]. J Cell Biol 2001, 154:925–36.
53. Fraschini R, Beretta A, Sironi L, Musacchio A, Lucchini G, Piatti S. Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores [J]. EMBO J 2001, 20:6648–59.
54. Fang GW. Checkpoint protein BubR1 acts synergistically with Mad2 to inhibit anaphase-promoting complex [J]. Mol Biol Cell 2002, 13:755–66.
55. Wu HY, Lan ZD, Li WQ, Wu SC, Weinstein J, Sakamoto KM, et al. p55CDC/hCDC20 is associated with BUBR1 and may be a downstream target of the spindle checkpoint kinase [J]. Oncogene 2000, 19:4557–62.
56. Chen RH, Brady DM, Smith D, Murray AW, Hardwick KG. The spindle checkpoint of budding yeast depends on a tight complex between the Mad1 and Mad2 proteins [J]. MolBiol Cell 1999, 10:2607–18.
57. Sironi L, Mapelli M, Knapp S, De Antoni A, Jeang KT, Musacchio A. Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of 'safety belt' binding mechanism for the spindle checkpoint [J]. EMBO J 2002, 21:2496–506.
58. Chung E, Chen RH. Spindle checkpoint requires Mad1-bound and Mad1-free Mad2 [J]. Mol Biol Cell 2002, 13:1501–11.
59. Nasmyth K. Segregating sister genomes: The molecular biology of chromosome separation [J]. Science 2002, 297:559–65.
60. Uhlmann F. Chromosome cohesion and separation: From men and molecules [J]. Curr Biol 2003, 13:R104–R14.
61. Uhlmann F. Separase regulation during mitosis [J]. Biochem Soc Symp 70 2003, 70:243–51.
62. Page SL, Hawley RS. Chromosome choreography: The meiotic ballet [J]. Science 2003, 301:785–9.
63. Tang Z, Bharadwaj R, Li B, Yu H. Mad2-Independent inhibition of APCCdc20 by the mitotic checkpoint protein BubR1 [J]. Dev Cell 2001, 1:227–37.
64. Fang GW, Yu HT, Kirschner MW. The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation [J]. Genes Dev 1998, 12:1871–83.
65. Howell BJ, McEwen BE, Canman JC, Hoffman DB, Farrar EM, Rieder CL, et al. Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation [J]. J Cell Biol 2001, 155:1159–72.
66. Pfarr CM, Coue M, Grissom PM, Hays TS, Porter ME, Mcintosh JR. Cytoplasmic Dynein Is Localized to Kinetochores during Mitosis [J]. Nature 1990, 345:263–5.
67. Echeverri CJ, Paschal BM, Vaughan KT, Vallee RB. Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis [J]. J Cell Biol 1996, 132:617–33.
68. King JM, Hays TS, Nicklas RB. Dynein is a transient kinetochore component whose binding is regulated by microtubule attachment, not tension [J]. J Cell Biol 2000, 151:739–48.
69. Basto R, Gomes R, Karess RE. Rough deal and Zw10 are required for the metaphase checkpoint in Drosophila [J]. Nat Cell Biol 2000, 2:939–43.
70. Chan GK, Jablonski SA, Starr DA, Goldberg ML, Yen TJ. Human Zw10 and ROD are mitotic checkpoint proteins that bind to kinetochores [J]. Nat Cell Biol 2000, 2:944–7.
71. Scaerou F, Aguilera I, Saunders R, Kane N, Blottiere L, Karess R. The rough deal protein is a new kinetochore component required for accurate chromosome segregation in Drosophila [J]. J Cell Sci 1999, 112:3757–68.
72. Karess R. Rod-Zw10-Zwilch: a key player in the spindle checkpoint [J]. Trends Cell Biol 2005, 15:386–92.
73. Griffis ER, Stuurman N, Vale RD. Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore [J]. J Cell Biol 2007, 177:1005–15.
74. Gassmann R, Essex A, Hu JS, Maddox PS, Motegi F, Sugimoto A, et al. A new mechanism controlling kinetochore-microtubule interactions revealed by comparison of two dynein-targeting components: SPDL-1 and the Rod/Zwilch/Zw10 complex [J]. Genes Dev 2008, 22:2385–99.
75. Yamamoto TG, Watanabe S, Essex A, Kitagawa R. SPDL-1 functions as a kinetochore receptor for MDF-1 in Caenorhabditis elegans [J]. Journal of Cell Biology 2008, 183:187–94.
76. Hassold T, Hunt P. To err (meiotically) is human: the genesis of human aneuploidy [J]. Nat Rev Genet 2001, 2:280–91.
77. Jallepalli PV, Lengauer C. Chromosome segregation and cancer: cutting through the mystery (Reprinted from Nature Reviews Cancer, vol 1, pg 109-117, 2001) [J]. Nat Rev Cancer 1 2001:S36–S44.
78. Matsuura S, Ito E, Tauchi H, Komatsu K, Ikeuchi T, Kajii T. Chromosomal instability syndrome of total premature chromatid separation with mosaic variegated aneuploidy is defective in mitotic-spindle checkpoint [J]. Am J Hum Genet 2000, 67:483–6.
79. Bharadwaj R, Yu H. The spindle checkpoint, aneuploidy, and cancer [J]. Oncogene 2004, 23:2016–27.
80. Zenzes MT, Casper RF. Cytogenetics of human oocytes, zygotes, and embryos after in vitrofertilization [J]. Hum Genet 1992, 88:367–75.
81. Cowchock FS, Gibas Z, Jackson LG. Chromosome Errors as a Cause of Spontaneous-Abortion - the Relative Importance of Maternal Age and Obstetric History [J]. Fertil Steril 1993, 59:1011–4.
82. Hassold T, Abruzzo M, Adkins K, Griffin D, Merrill M, Millie E, et al. Human aneuploidy: incidence, origin, and etiology [J]. Environ Mol Mutagen 1996, 28:167–75.
83. Bolton VN, Hawes SM, Taylor CT, Parsons JH. Development of Spare Human Preimplantation Embryos Invitro - an Analysis of the Correlations among Gross Morphology, Cleavage Rates, and Development to the Blastocyst [J]. J In Vitro Fertil Embryo Transfer 1989, 6:30–5.
84. Rubio C, Simon C, Vidal F, Rodrigo L, Pehlivan T, Remohi J, et al. Chromosomal abnormalities and embryo development in recurrent miscarriage couples [J]. Hum Reprod 2003, 18:182–8.
85. Robinson WP, McFadden DE, Stephenson MD. The origin of abnormalities in recurrent aneuploidy/polyploidy [J]. Am J Hum Genet 2001, 69:1245–54.
86. Bruyere H, Rupps R, Kuchinka BD, Friedman JM, Robinson WP. Recurrent trisomy 21 in a couple with a child presenting trisomy 21 mosaicism and maternal uniparental disomy for chromosome 21 in the euploid cell line [J]. Am J Med Genet 2000, 94:35–41.
87. Tseng LH, Chuang SM, Lee TY, Ko TM. Recurrent Downs-Syndrome Due to Maternal Ovarian Trisomy-21 Mosaicism [J]. Arch Gyn Ob 1994, 255:213–6.
88. Amon A. The spindle checkpoint [J]. Curr Opin Genet Dev 1999, 9:69–75.
89. Nicklas RB. How cells get the right chromosomes [J]. Science 1997, 275:632–7.
90. Hoyt MA. A new view of the spindle checkpoint [J]. J Cell Biol 2001, 154:909–11.
91. Karsenti E, Vernos I. The mitotic spindle: a self-made machine [J]. Science 2001, 294:543–7.
92. Vernos I, Karsenti E. Chromosomes take the lead in spindle assembly [J]. Trends Cell Biol 1995, 5:297–301.
93. Li GP, Liu Y, Bunch TD, White KL, Aston KI. Asymmetric division of spindle microtubules and microfilaments during bovine meiosis from metaphase I to metaphase II [J]. Mol Reprod Dev 2005, 71:220–6.
94. Sun XF, Zhang WH, Chen XJ, Xiao GH, Mai WY, Wang WH. Spindle dynamics in living mouse oocytes during meiotic maturation, ageing, cooling and overheating: a study by polarized light microscopy [J]. Zygote 2004, 12:241–9.
95. Fan HY, Sun QY. Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals [J]. Biol Reprod 2004, 70:535–47.
96. Dujardin D, Wacker UI, Moreau A, Schroer TA, Rickard JE, De Mey JR. Evidence for a role of CLIP-170 in the establishment of metaphase chromosome alignment [J]. J Cell Biol 1998, 141:849–62.
97. Stricker SA. Comparative biology of calcium signaling during fertilization and egg activation in animals [J]. Dev Biol 1999, 211:157–76.
98. Zhang D, Li M, Ma W, Hou Y, Li YH, Li SW, et al. Localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during the first meiosis and its functions as a spindle checkpoint protein [J]. Biol Reprod 2005, 72:58–68.
99. Hardwick KG, Murray AW. Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast [J]. J Cell Biol 1995, 131:709–20.
100. Campbell MS, Chan GK, Yen TJ. Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase [J]. J Cell Sci 2001, 114:953–63.
101. Zhang D, Ma W, Li YH, Hou Y, Li SW, Meng XQ, et al. Intra-oocyte localization of MAD2 and its relationship with kinetochores, microtubules, and chromosomes in rat oocytes during meiosis [J]. Biol Reprod 2004, 71:740–8.
102. Wassmann K, Niault T, Maro B. Metaphase I arrest upon activation of the Mad2-dependent spindle checkpoint in mouse oocytes [J]. Curr Biol 2003, 13:1596–608.
103. Ma W, Zhang D, Hou Y, Li YH, Sun QY, Sun XF, Wang WH. Reduced expression of MAD2, BCL2, and MAP kinase activity in pig oocytes after in vitro aging are associated with defects in sister chromatid segregation during meiosis II and embryo fragmentation after activation [J] . Biol Reprod 2005, 72(2):373–83.
104. Brunet S, Pahlavan G, Taylor S, Maro B. Functionality of the spindle checkpoint during the first meiotic division of mammalian oocytes [J]. Reproduction 2003, 126:443–50.
105. Millband DN, Hardwick KG. Fission yeast Mad3p is required for Mad2p to inhibit the anaphase-promoting complex and localizes to kinetochores in a Bub1p-, Bub3p-, andMph1p-dependent manner [J]. Mol Cell Biol 2002, 22:2728–42.
106. Wang X, Babu JR, Harden JM, Jablonski SA, Gazi MH, Lingle WL, et al. The mitotic checkpoint protein hBUB3 and the mRNA export factor hRAE1 interact with GLE2p-binding sequence (GLEBS)-containing proteins [J]. J Biol Chem 2001, 276:26559–67.
107. Li M, Li S, Yuan J, Wang ZB, Sun SC, Schatten H, et al. Bub3 is a spindle assembly checkpoint protein regulating chromosome segregation during mouse oocyte meiosis [J]. PLoS One 2009, 4:e7701.
108. Campbell L, Hardwick KG. Analysis of Bub3 spindle checkpoint function in Xenopus egg extracts [J]. J Cell Sci 2003, 116:617–28.
109. Logarinho E, Resende T, Torres C, Bousbaa H. The human spindle assembly checkpoint protein Bub3 is required for the establishment of efficient kinetochore-microtubule attachments [J]. Mol Biol Cell 2008, 19:1798–813.
110.Hardwick KG, Johnston RC, Smith DL, Murray AW. MAD3 encodes a novel component of the spindle checkpoint which interacts with Bub3p, Cdc20p, and Mad2p [J]. J Cell Biol 2000, 148:871–82.
111. Hwang LH, Lau LF, Smith DL, Mistrot CA, Hardwick KG, Hwang ES, et al. Budding yeast Cdc20: a target of the spindle checkpoint [J]. Science 1998, 279:1041–4.
112. Howell BJ, Moree B, Farrar EM, Stewart S, Fang G, Salmon ED. Spindle checkpoint protein dynamics at kinetochores in living cells [J]. Curr Biol 2004, 14:953–64.
113. Kallio M, Mustalahti T, Yen TJ, Lahdetie J. Immunolocalization of alpha-tubulin, gamma-tubulin, and CENP-E in male rat and male mouse meiotic divisions: pathway of meiosis I spindle formation in mammalian spermatocytes [J]. Dev Biol 1998, 195:29–37.
114. Lee J, Miyano T, Dai Y, Wooding P, Yen TJ, Moor RM. Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes [J]. Mol Reprod Dev 2000, 56:51–62.
115. Duesbery NS, Choi T, Brown KD, Wood KW, Resau J, Fukasawa K, et al. CENP-E is an essential kinetochore motor in maturing oocytes and is masked during mos-dependent, cell cycle arrest at metaphase II [J]. Proc Natl Acad Sci U S A 1997, 94:9165–70.
116. Minshull J, Sun H, Tonks NK, Murray AW. A MAP kinase-dependent spindle assemblycheckpoint in Xenopus egg extracts [J]. Cell 1994, 79:475–86.
117.Takenaka K, Gotoh Y, Nishida E. MAP kinase is required for the spindle assembly checkpoint but is dispensable for the normal M phase entry and exit in Xenopus egg cell cycle extracts [J]. J Cell Biol 1997, 136:1091–7.
118. Wang XM, Zhai Y, Ferrell JE, Jr. A role for mitogen-activated protein kinase in the spindle assembly checkpoint in XTC cells [J]. J Cell Biol 1997, 137:433–43.
119. Zecevic M, Catling AD, Eblen ST, Renzi L, Hittle JC, Yen TJ, et al. Active MAP kinase in mitosis: localization at kinetochores and association with the motor protein CENP-E [J]. J Cell Biol 1998, 142:1547–58.
120.Gordo AC, He CL, Smith S, Fissore RA. Mitogen activated protein kinase plays a significant role in metaphase II arrest, spindle morphology, and maintenance of maturation promoting factor activity in bovine oocytes [J]. Mol Reprod Dev 2001, 59:106–14.
121. Inoue M, Naito K, Nakayama T, Sato E. Mitogen-activated protein kinase translocates into the germinal vesicle and induces germinal vesicle breakdown in porcine oocytes [J]. Biol Reprod 1998, 58:130–6.
122. Verlhac MH, de Pennart H, Maro B, Cobb MH, Clarke HJ. MAP kinase becomes stably activated at metaphase and is associated with microtubule-organizing centers during meiotic maturation of mouse oocytes [J]. Dev Biol 1993, 158:330–40.
123. Lee J, Miyano T, Moor RM. Localisation of phosphorylated MAP kinase during the transition from meiosis I to meiosis II in pig oocytes [J]. Zygote 2000, 8:119–25.
124. Vaisberg EA, Koonce MP, McIntosh JR. Cytoplasmic dynein plays a role in mammalian mitotic spindle formation [J]. J Cell Biol 1993, 123:849–58.
125. Van Kooij RJ, Looman CW, Habbema JD, Dorland M, te Velde ER. Age-dependent decrease in embryo implantation rate after in vitro fertilization [J]. Fertil Steril 1996, 66:769–75.
126. Schvarzstein M, Wignall SM, Villeneuve AM. Coordinating cohesion, co-orientation, and congression during meiosis: lessons from holocentric chromosomes [J]. Genes Dev 2010, 24:219–28.
127. Jones GH. The control of chiasma distribution [J]. Symp Soc Exp Biol 1984, 38:293–320.
128. Ghabrial A, Schupbach T. Activation of a meiotic checkpoint regulates translation ofGurken during Drosophila oogenesis [J]. Nat Cell Biol 1999, 1:354–7.
129. Bhalla N, Dernburg AF. A conserved checkpoint monitors meiotic chromosome synapsis in Caenorhabditis elegans [J]. Science 2005, 310:1683–6.
130. Handel MA, Schimenti JC. Genetics of mammalian meiosis: regulation, dynamics and impact on fertility [J]. Nat Rev Genet 2010, 11:124–36.
131. Roeder GS, Bailis JM. The pachytene checkpoint [J]. Trends Genet 2000, 16:395–403.
132. Wu HY, Burgess SM. Two distinct surveillance mechanisms monitor meiotic chromosome metabolism in budding yeast [J]. Curr Biol 2006, 16:2473–9.
133. Li X, Schimenti JC. Mouse pachytene checkpoint 2 (Trip13) is required for completing meiotic recombination but not Synapsis [J]. Plos Genetics 2007, 3:1365–76.
134. Barchi M, Mahadevaiah S, Di Giacomo M, Baudat F, de Rooij DG, Burgoyne PS, et al. Surveillance of different recombination defects in mouse spermatocytes yields distinct responses despite elimination at an identical developmental stage [J]. Mol Cell Biol 2005, 25:7203–15.
135. Turner JM, Mahadevaiah SK, Ellis PJ, Mitchell MJ, Burgoyne PS. Pachytene asynapsis drives meiotic sex chromosome inactivation and leads to substantial postmeiotic repression in spermatids [J]. Dev Cell 2006, 10:521–9.
136. Yin S, Sun XF, Schatten H, Sun QY. Molecular insights into mechanisms regulating faithful chromosome separation in female meiosis [J]. Cell Cycle 2008, 7:2997–3005.
137. Wei L, Liang XW, Zhang QH, Li M, Yuan J, Li S, et al. BubR1 is a spindle assembly checkpoint protein regulating meiotic cell cycle progression of mouse oocyte [J]. Cell Cycle 2010, 9:1112–21.
138. Tripathi A, Kumar KV, Chaube SK. Meiotic cell cycle arrest in mammalian oocytes [J]. J Cell Physiol 2010, 223:592–600.
139. Hupalowska A, Kalaszczynska I, Hoffmann S, Tsurumi C, Kubiak JZ, Polanski Z, et al. Metaphase I arrest in LT/Sv mouse oocytes involves the spindle assembly checkpoint [J]. Biol Reprod 2008, 79:1102–10.
140. Brunet S, Maro B. Cytoskeleton and cell cycle control during meiotic maturation of the mouse oocyte: integrating time and space [J]. Reproduction 2005, 130:801–11.
141. Homer H, Gui L, Carroll J. A spindle assembly checkpoint protein functions in prophase Iarrest and prometaphase progression. Science 2009, 326:991–4.
142. Musacchio A, Salmon ED. The spindle-assembly checkpoint in space and time [J]. Nat Rev Mol Cell Biol 2007, 8:379–93.
143. McGuinness BE, Anger M, Kouznetsova A, Gil-Bernabe AM, Helmhart W, Kudo NR, et al. Regulation of APC/C activity in oocytes by a Bub1-dependent spindle assembly checkpoint [J]. Curr Biol 2009, 19:369–80.
144. Holt JE, Jones KT. Control of homologous chromosome division in the mammalian oocyte. Mol Hum Reprod 2009, 15:139–47.
145. Yamamuro T, Kano K, Naito K. Functions of FZR1 and CDC20, activators of the anaphase-promoting complex, during meiotic maturation of swine oocytes [J]. Biol Reprod 2008, 79:1202–9.
146. Marangos P, Carroll J. Securin regulates entry into M-phase by modulating the stability of cyclin B [J]. Nat Cell Biol 2008, 10:445–51.
147. Starr DA, Williams BC, Hays TS, Goldberg ML. ZW10 helps recruit dynactin and dynein to the kinetochore. J Cell Biol 1998, 142:763–74.
148. Buffin E, Emre D, Karess RE. Flies without a spindle checkpoint [J]. Nature Cell Biology 2007, 9:565–U150.
149. Cheeseman IM, Desai A. Molecular architecture of the kinetochore-microtubule interface [J]. Nature Reviews Molecular Cell Biology 2008, 9:33–46.
150. Yang ZY, Tulu US, Wadsworth P, Rieder CL. Kinetochore dynein is required for chromosome motion and congression independent of the spindle checkpoint [J]. Current Biology 2007, 17:973–80.
151. Przewloka MR, Glover DM. The kinetochore and the centromere: a working long distance relationship [J]. Annu Rev Genet 2009, 43:439–65.
152. Vorozhko VV, Emanuele MJ, Kallio MJ, Stukenberg PT, Gorbsky GJ. Multiple mechanisms of chromosome movement in vertebrate cells mediated through the Ndc80 complex and dynein/dynactin [J]. Chromosoma 2008, 117:169–79.
153. Civril F, Musacchio A. Spindly attachments [J]. Genes Dev 2008, 22:2302–7.
154. Chan YW, Fava LL, Uldschmid A, Schmitz MH, Gerlich DW, Nigg EA, et al. Mitotic control of kinetochore-associated dynein and spindle orientation by human Spindly [J]. JCell Biol 2009, 185:859–74.
155. Stein KK, Davis ES, Hays T, Golden A. Components of the spindle assembly checkpoint regulate the anaphase-promoting complex during meiosis in Caenorhabditis elegans [J]. Genetics 2007, 175:107–23.
156. Kitagawa R, Rose AM. Components of the spindle-assembly checkpoint are essential in Caenorhabditis elegans [J]. Nat Cell Biol 1999, 1:514–21.
157. Zhang QH, Wei L, Tong JS, Qi ST, Li S, Ou XH, et al. Localization and function of mSpindly during mouse oocyte meiotic maturation [J]. Cell Cycle 2010, 9: 2230–2236.
