Key Event Overview
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AOPs Including This Key Event
|AOP Name||Event Type||Essentiality|
|Chemical binding to tubulin in oocytes leading to aneuploid offspring||KE|
Level of Biological Organization
How this Key Event works
Although this key event occurs in all eukaryotic cell types, spindle formation occurs via distinct mechanisms in female germ cells with respect to other cell types (including male germ cells). This is because spindle formation is generally driven by centrioles, which are lacking in eggs. The two processes are briefly described below.
Somatic cells and male germ cells: INSERT TEXT.
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
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].
Consider the following criteria when describing each method: 1. Is the assay fit for purpose? Yes 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? Directly. 3. Is the assay repeatable? Yes 4. Is the assay reproducible? Yes
Evidence Supporting Taxonomic Applicability
All eukaryotic cells possess spindle that must be properly organized for cellular division. Thus, this key event, although typically measured in mouse and human cells, is theoretically relevant to any eukaryotic cell type.
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.
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.
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.
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.
Manandhar G, Schatten H, Sutovsky P. 2005. Centrosome reduction during gametogenesis and its significance. Biol Reprod 72:2-13.
Marchetti A, Massarotti A, Yauk CL, Pacchierotti F, Russo A. Submitted. The adverse outcome pathway (AOP) for chemical binding to tubulin in oocytes leading to aneuploid offspring. Environ Mol Mutagen.
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.
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.
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.