API

Event: 721

Key Event Title

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Disorganization, Spindle

Short name

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Disorganization, Spindle

Biological Context

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Level of Biological Organization
Cellular

Cell term

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Cell term
female germ cell


Organ term

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Key Event Components

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Process Object Action
spindle organization spindle decreased

Key Event Overview


AOPs Including This Key Event

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AOP Name Role of event in AOP
Tubulin binding and aneuploidy KeyEvent

Stressors

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Taxonomic Applicability

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Life Stages

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Sex Applicability

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Key Event Description

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The spindle is a cytoskeletal structure present in every eukaryotic cell that must form before cell division in order to properly separate chromosomes between daughter cells [Prosser and Pelletier, 2017]. The spindle organizes itself in a bipolar configuration within the cell prior to cell division. Several hundred proteins are required to assemble a functioning spindle, and microtubules are the most abundant components of the machinery. Although the function of the spindle is similar between mitotic and meiotic cells, spindle formation occurs via distinct mechanisms in female germ cells with respect to other cell types (including male germ cells) [Dumont and Desai, 2012]. This is because spindle formation is generally driven by centrioles, which are lacking in eggs [Szollosi et al., 1972; Manandhar et al., 2005]. The processes in somatic cells and male germ cells versus those operating in oocytes are briefly described below. In this key event, a bipolar spindle configuration is not achieved. Alternatively, there may be some spindle fibers that are not of the appropriate length, shape or structure to ensure that chromosomes can be properly aligned at metaphase and equally distributed between daughter cells.

 

Somatic cells and male germ cells:

The spindle of mitotic cells and that of male germ cells is organized by the centrosome which is composed by a pair of centrioles surrounded by an amorphous pericentriolar material containing more than 100 proteins [Andersen et al., 2003]. Many proteins that are involved in regulating microtubule dynamics and spindle assembly checkpoint (SAC) are contained in the centrosome. The centrosome is the principal microtubule-organizing center (MTOC) in mammalian cells and plays a major role in controlling microtubule dynamics, nucleation, and kinetochore–microtubule attachments [Conduit et al., 2015]. Errors in these processes lead to structural and functional abnormalities in the mitotic spindle [Rivera-Rivera and Saavedra, 2016].

 

Centriole and centrosome duplication are tightly coordinated with DNA replication, mitosis, and cytokinesis and play key roles in regulating transitions through the cell cycle [Chan, 2011]. The centrioles, cylindrical particles composed by nine triplet microtubules [Gogendeau et al., 2015], duplicate by forming daughter centrioles oriented at right angles with respect to the parent centrioles and then become surrounded by separate pericentriolar material during S-phase [Bettencourt-Dias and Glover, 2007]. Before mitosis, the newly formed centrosomes move to the opposite site of the nucleus and originate the two poles of the mitotic spindle [Kellog, 1989; Paintrand et a.l, 1992; Chavali et al., 2012]. Microtubules begin to radiate away from the centrosome and move toward the metaphase plate forming the mitotic spindle. During the assembly of the mitotic spindle, some microtubule fibers attach to the kinetochores on chromosomes, some radiate from the spindle poles toward the cell cortex and others extend past the metaphase plate forming a region of overlap with spindle fibers originating from the opposite centrosome [Cassimeris and Skibbens, 2003; Prosser and Pelletier, 2017]. Although a bipolar spindle can be formed in the absence of centrosomes, having too many centrosomes can result in a morphologically abnormal spindle and increase the chance of chromosome missegration [Hinchcliffe, 2014; Nigg and Holland, 2018].

Oocytes:
In mammalian oocytes, centrioles and centrosomes are absent [Manandhar et al.. 2005] and the meiotic spindle starts its growth from several MTOCs that substitute for the conventional centrosome pair. A mouse oocyte can have up to 80 of these MTOCs [Dumont and Desai, 2012]. These MTOCs gradually coalesce and surround the chromosomes [Schuh and Ellenberg, 2007]. Then, microtubules elongate forming a barrel-shape bipolar spindle. Recent data suggest that MTOCs undergo a three-step decondensation and fragmentation process that facilitate their equal distribution to the spindle poles [Clift and Schuh, 2015]. In addition, recent evidence has shown the presence of actin fibers in the mammalian oocyte spindle that are important for ensuring proper chromosome segregation [Mogessie and Schuh, 2017]. Evidence is also emerging about differences in spindle assembly between rodent and human oocytes. Specifically, human oocytes may lack MTOCs and spindle assembly is mediated by chromosomes and the small guanosine triphosphate Ran [Holubcová et al., 2015].

The assembly of the mitotic or meiotic spindle requires unaltered microtubule dynamics. Chemicals that bind to tubulin induce microtubule depolymerization and affect the proper assembly of the spindle resulting in various abnormalities in its structure and shape. The major abnormalities that can be recorded are: reduction of microtubule density, loss of barrel shape, monopolar or multipolar spindle, reduced distance between the poles [Ibanez et al., 2003; Shen et al., 2005; Eichenlaub-Ritter et al., 2007; Xu et al., 2012]. In addition, the use of enhanced polarizing microscope (Polscope/SpindleViewTM) allows the detection of reduction in the birefringency and reduced light retardance of the spindle, which are indicators of loss of organization, at doses below which spindle abnormalities are detected with more conventional immunofluorescence methods [Shen et al., 2005].


In mammalian oocytes, centrioles are absent [Manandhar et al. 2005] and the meiotic spindle starts its growth from several Microtubule-Organizing Centers (MTOCs) that substitute for the conventional centrosome pair. These MTOCs gradually coalesce and surround the chromosomes [Schuh and Ellenberg 2007]. Then, microtubules elongate forming a barrel-shape bipolar spindle. Recent data suggest that MTOCs undergo a three-step decondensation and fragmentation process that facilitate their equal distribution to the spindle poles [Clift and Schuh, 2015] and that human oocytes may lack MTOCs and that spindle assembly is mediated by chromosomes and the small guanosine triphosphate Ran [Holubcová et al., 2015]. The major abnormalities which can be recorded are: reduction of microtubule density, loss of barrel shape, monopolar or multipolar spindle, reduced distance between the poles [Ibanez et al. 2003; Shen et al. 2005; Eichenlaub-Ritter et al. 2007; Xu et al. 2012].


How It Is Measured or Detected

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Spindle organization is generally assessed by fluorescent immunodetection of its components and confocal microscopy [Ibanez et al., 2003; Shen et al., 2005; Eichenlaub-Ritter et al., 2007; Xu et al., 2012]. Localization of proteins with a known role in spindle function is also assessed [Tong et al., 2002; Yao et al., 2004; Cao et al., 2005]. 3D live imaging of cells expressing fluorescent-tagged proteins provides the possibility to follow spindle function at high resolution, and to describe and measure abnormal parameters (e.g., spindle morphology, altered distance between the two poles, mono- or multipolarity) [Schuh and Ellenberg, 2007]. Enhanced polarizing microscope has also been used to assess spindle integrity in human oocytes during in vitro fertilization techniques [Wang et al., 2001a,b; Keefe et al., 2003; Staessen et al., 1997].


Domain of Applicability

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All eukaryotic cells possess a spindle that must be properly organized for normal cellular division. Thus, this key event, although typically measured in mouse and human cells, is theoretically relevant to any eukaryotic cell type.


References

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Andersen JS, Wilkinson CJ, Mayor T, Mortensen P, Nigg EA, Mann M. 2003. Proteomic characterization of the human centrosome by protein correlation profiling. Nature 426:570-574.

Bettencourt-Dias M, Glover DM. 2007. Centrosome biogenesis and function: centrosomics brings new understanding. Nat Rev Mol Cell Biol 8:451-463.

Cao Y-K, Zhong Z-S, Chen D-Y, Zhang G-X, Schatten H, Sun Q-Y. 2005. Cell cycle-dependent localization and possible roles of the small GTPase Ran in mouse oocyte maturation, fertilization and early cleavage. Reproduction 130:431-440.

Cassimeris L, Skibbens RV. 2003. Regulated assembly of the mitotic spindle: a perspective from two ends. Curr Issues Mol Biol 5:99-112.

Chan JY. A clinical overview of centrosome amplification in human cancers. Int J Biol Sci 7:1122-1144.

Chavali PL, Peset I, Gergely F. 2012. Centrosomes and mitotic poles: a recent liason?  Biochem Soc Trans 43:13-18.

Conduit PT, Wainman A, Raff JW. 2015. Centrosome function and assembly in animal cells. Nat Rev Mol Cell Biol 16:611-624.

Clift D, Schuh M. 2015. A three-step MTOC fragmentation mechanism facilitate bipolar spindle assembly in mouse oocytes. Nat Commun 6:7217, 10.1038/ncomm8217.

Dumont J, Desai A. 2012. Acentrosomal spindle assembly and chromosome segregation during oocyte meiosis. Trends Cell Biol 22: 241-249.

Eichenlaub-Ritter U, Winterscheidt U, Vogt E, Shen Y, Tinneberg HR, Sorensen R. 2007. 2-methoxyestradiol induces spindle aberrations, chromosome congression failure, and nondisjunction in mouse oocytes. Biol Reprod 76:784-793.

Gogendeau D, Guichard P, Tassin AM. 2015. Purification of centrosomes from mammalian cell lines. Methods Cell Biol. 129:171-189.

Hinchcliffe EH. 2014. Centrosomes and the art of mitotic spindle maintenance. Int Rev Cell Mol Biol 313:179-217.

Holubcová Z, Blayney M, Elder K, Schuh M. 2015. Error-prone chromosome-mediated spindle assembly favors chromosome segregation defects in human oocytes. Science 348:1143-1147.

Ibanez E, Albertini DF, Overstrom EW. 2003. Demecolcine-induced oocyte enucleation for somatic cell cloning: coordination between cell-cycle egress, kinetics of cortical cytoskeletal interactions, and second polar body extrusion. Biol Reprod 68:1249-1258.

Keefe D, Liu L, Wang W, Silva C. 2003. Imaging meiotic spindles by polarization light microscopy: principles and applications to IVF. Reprod Biomed Online 7:24–29.

Kellog DR. 1989. Centrosomes. Organizing cytoplasmic events. Nature 340:99-100.

Manandhar G, Schatten H, Sutovsky P. 2005. Centrosome reduction during gametogenesis and its significance. Biol Reprod 72:2-13.

Marchetti F, Massarotti A, Yauk CL, Pacchierotti F, Russo A. 2016. The adverse outcome pathway (AOP) for chemical binding to tubulin in oocytes leading to aneuploid offspring. Environ Mol Mutagen 57:87-113.

Mogessie B, Schuh M. 2017. Actin protects mammalian eggs against chromosome segregation errors. Science Aug 25;357(6353). pii: eaal1647.

Nigg EA, Holland AI. 2018. Once and only once: mechanisms of centriole duplication and their deregulation in disease. Nat Rev Mol Cell BIol 19:297-312.

Paintrand M, Moudjou M, Delacroix H, Bornens M. 1992. Centrosome organization and centriole architecture: their sensitivity to divalent cations. J Struct Biol 108:107–128.

Prosser SL, Pelletier L. 2017. Mitotic spindle assembly in animal cells: a fine balancing act. Nat Rev Mol Cell Biol 18:187-201.

Rivera-Rivera Y, Saavedra HI. Centrosome - a promising anti-cancer target. 2016. Biologics 10:167-176.

Schuh M, Ellenberg J. 2007. Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes. Cell 130:484-498.

Shen Y, Betzendahl I, Sun F, Tinneberg HR, Eichenlaub-Ritter U. 2005. Non-invasive method to assess genotoxicity of nocodazole interfering with spindle formation in mammalian oocytes. Reprod Toxicol 19:459-471.

Staessen C, Van Steirteghem AC. 1997. The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intracytoplasmic sperm injection and and conventional in-vitro fertilization. Hum Reprod 12:321–327.

Szollosi D, Calarco P, Donahue RP. 1972. Absence of centrioles in the first and second meiotic spindles of mouse oocytes. J Cell Sci 11:521-541.

Tong C, Fan H-Y, Lian L, Li S-W, Chen D-Y, Schatten H, Sun Q-Y. 2002. Polo-like kinase-1 is a pivotal regulator of microtubule assembly during mouse oocyte meiotic maturation, fertilization, and early embryonic mitosis. Biol Reprod 67:546-554.

Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. 2001a. The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertil Steril 75:348–353.

 

Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. 2001b. Limited recovery of meiotic spindles in living human oocytes after cooling–rewarming observed using polarized light microscopy. Hum Reprod 16:2374–2378.

Xu XL, Ma W, Zhu YB, Wang C, Wang BY, An N, An L, Liu Y, Wu ZH, Tian JH. 2012. The microtubule-associated protein ASPM regulates spindle assembly and meiotic progression in mouse oocytes. PLoS One 7:e49303.

Yao LJ, Fan HY, Tong C, Chen DY, Schatten H, Sun QY. 2003. Polo-like kinase-1 in porcine oocyte meiotic maturation, fertilization and early embryonic mitosis. Cell Mol Biol 49:399-405.

Yao L-J, Zhong Z-S, Zhang L-S, Chen D-Y, Schatten H, Sun Q-Y. 2004. Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos. Biol Reprod 70:1392-1399.