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Event: 721

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Disorganization, Meiotic Spindle

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Disorganization, Meiotic Spindle

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Cellular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
eukaryotic cell

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
spindle organization spindle decreased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Tubulin binding and aneuploidy KeyEvent Francesco Marchetti (send email) Open for citation & comment EAGMST Under Review

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
mouse Mus musculus High NCBI
human Homo sapiens High NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
All life stages High

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Term Evidence
Mixed High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

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].

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

Spindle abnormalities in its structure and shape 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].

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

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

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.

Evidence for Perturbation by Stressor

Colchicine

In vitro treatment with 0.4 micrograms/mL (1 microM) induces reduction of spindle size and lowe microtubule density; cytoskeleton remodeling  is also observed (Ibanez et al 2003). In addtion, colchicine treament results in abnormal spindle localization of several proteins that are essential for chormosome segregation, such as: Aurora A (Yao et al 2004); Polo-like-kinase I (Yao et al 2003; Tong et al 2002); GTPase Ran (Cao et al, 2005)

Nocodazole

In vitro expsoure of oocytes to 20 microgram/mL (67 microM) Nocodazole  causes a gradual disassembly of the spindle , which is completed within 15 minutes (Xu et al 2002)

Colcemid

After in vitro treatment wiht 0.4 microgram/mL (1 microgramM) reduction of spindle size and lower microtubule density is detected  in activated oocytes with respect to controls; cytoskeleton remodelling is also observed (Ibanez et al 2003).

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

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. 2011. 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.