Elsevier

Reproductive Toxicology

Volume 19, Issue 4, March–April 2005, Pages 459-471
Reproductive Toxicology

Non-invasive method to assess genotoxicity of nocodazole interfering with spindle formation in mammalian oocytes

https://doi.org/10.1016/j.reprotox.2004.09.007Get rights and content

Abstract

Trisomies due to nondisjunction in oogenesis are still a major cause of genetic diseases in humans. In this study, we analysed spindle morphology of in vitro matured nocodazole-exposed mouse oocytes by novel non-invasive Polscope-microscopy, and compared images to those obtained by anti-tubulin immunofluorescence of fixed oocytes. Polscope revealed a reduction in the numbers of oocytes expressing a birefringent spindle, and alterations in spindle morphology at concentrations of nocodazole below those inducing detectable aberrations in immunofluorescence. Hyperploidy increased significantly at a concentration of 40 nM nocodazole in mouse metaphase II oocytes, similar to thresholds inducing nondisjunction in cultured human lymphocytes. In conclusion, Polscope represents a novel highly sensitive, non-invasive method to identify chemicals inducing severe spindle aberrations that predispose mammalian oocytes to nondisjunction. Polscope may provide information on the functionality of the spindle in experimental studies but is also compatible with clinical trials in human assisted reproduction due to its non-invasive nature.

Introduction

Microtubule inhibitors are frequently used as cytostatic drugs in cancer treatment since they cause spindle depolymerisation, cell cycle arrest and/or apoptosis by interfering with microtubule polymerisation kinetics [1]. However, most microtubule inhibitors also comprise indirectly acting mutagens, since they can induce spindle defects, which may lead to nondisjunction, chromosome loss, and aneuploidy in somatic and germ cells [2], [3], [4], [5], [6]. A transient cell cycle arrest by cellular feedback controls may be activated, frequently termed the “spindle checkpoint” to prevent errors in chromosome segregation at mitosis and meiosis, to ensure the order of events in cell division, and coordinate bipolar attachment (recently reviewed by [7], [8], [9], [10]). The presence of unattached kinetochores, and/or absence of tension on chromosomes trigger the spindle checkpoint in most mitotically-dividing cells and in meiosis [11], [12]. This induces a transient delay in M-phase to anaphase progression so that the cell has a chance to assemble all chromosomes at the spindle equator (termed: chromosome congression) and centromeres of sister chromatids of each replicated chromosome, or homologous chromosomes in a bivalent in meiosis face and attach to opposite spindle poles prior to their segregation at anaphase. It has been suggested that mammalian oocytes may have a rather leaky checkpoint control under certain conditions, and that this could contribute to the special susceptibility for meiotic errors [13], [14], [15], [16].

Generally, indirectly acting mutagens are expected to exhibit a biological threshold as shown for microtubule-depolymerising chemicals like colchicine, vinblastine, benomyl or nocodazole [4], [6], [17], [18], [19], [20]. Nocodazole is a classical aneugen, which binds to beta-tubulin with high affinity and affects polymerisation kinetics even at very low concentrations [21], [22]. In addition, chemicals like colchicine or nocodazole may alter the morphology of centromeres and kinetochores, the sites of attachment for spindle microtubules on the chromosome, and induce malorientation and lagging of chromosomes in mitotic and meiotic cells [23], [24]. It is conceivable from the different types of disturbances of spindles and chromosome behaviour caused by aneugens, that the thresholds for aneugenic processes differ depending on the endpoint in cytogenetic analysis (e.g. nondisjunction versus chromosome loss) [4]. Differences in thresholds may also be expected between species or at the level of the tissues depending on expression of targets and cellular defences [20], [25]. There are only few studies comparing thresholds for induction of aneuploidy in meiotic versus mitotic cells. For instance, studies comparing somatic with meiotic errors in experimental animals were performed in the male (e.g. by analysis of nondisjunction in bone marrow as compared to aneuploidy in sperm of chemically-exposed mice) [26], [27]. Sperm can be obtained in large numbers, and can be effectively analysed by fluorescent in situ hybridisation (FISH) with chromosome-specific probes for interphase analysis of nondisjunction, even in humans [27], [28], [29]. In contrast, only a limited number of oocytes arrested meiotically at metaphase II is ovulated in each cycle in mammals like humans and rodents, and oogenesis is a long and complex process, which is not easily amenable to analysis of chemically-induced aneuploidy [30], [31], [32]. In an attempt to study thresholds for chemically induced aneuploidy and mechanisms of action of aneugens in the mammalian oocyte in comparison to somatic cells we decided to study the response to nocodazole-exposure in in vitro maturing mouse oocytes [20].

For in vitro maturation, oocytes are isolated from large antral follicles at the dictyate stage, placed into appropriate culture media with or without drugs, and then spontaneously progress from the G2-phase to metaphase II (e.g. [5], [33], [34], [35], [36]). The mechanism of action and the most sensitive stage of exposures to aneugens have been studied by this in vitro model [14], [37]. However, in the past it was necessary to fix oocytes to obtain information on mechanisms behind chemically-induced aneuploidy, in particular, the induction of spindle aberrations. Such methods provide only a static image of the highly dynamic nature of the cellular cytoskeleton and cannot be directly combined with other types of analysis like assessment of numerical or structural chromosomal aberrations [5], [38], [39], [40], [41], [42], [43]. Due to ethical considerations only limited numbers of human oocytes have been studied for spindle aberrations by immunofluorescence, and these were mainly donated, spare or unfertilised, aged human metaphase II oocytes from assisted reproduction [43], [44], [45], [46].

Thus, it is essential to establish new, efficient methods for studying the causal factors and mechanisms of meiotic errors in the female gamete since the mammalian oocyte is especially prone to errors in chromosome segregation at meiosis, particularly during anaphase I [32], [47], [48], [49]. Aneuploidy in the oocyte gives rise to monosomy or trisomy after fertilization and can cause implantation failure [50], congenital abnormalities [49], [51] or spontaneous abortion [52], [53]. The major unambiguously identified ethological factor in oocyte aneuploidy in humans is maternal age but it is still unknown whether and to what extent factors like environmental exposures, pharmaca, or life style also contribute. Although human oocytes exhibit rates of nondisjunction by far exceeding those in other species [54], [55], [56], for obvious reasons it is difficult to obtain sufficient material for prospective or retrospective analysis of causal factors for the genesis of spindle aberrations in human oogenesis. To establish non-invasive methods for spindle analysis might therefore be very helpful.

In the present study, we therefore used a novel non-invasive method to quantify spindle disturbances in mouse oocytes induced by exposure to a classical aneugen, nocodazole. Enhanced polarizing microscopy (Polscope/SpindleView™) provides images of the highly ordered microtubular arrays of the spindle in a living oocyte [43], [57], [58], [59], [60], [61]. We wanted to know whether Polscope microscopy is sensitive enough and useful to detect spindle abnormalities in oocytes in response to exposures to aneugenic chemicals, as this question has not been systematically addressed so far. Therefore, we compared disturbances by exposures to nocodazole in spindles of in vitro maturing mouse oocytes detected by Polscope non-invasively with those observed after fixation and staining of oocytes by conventional anti-tubulin immunofluorescence. Concentrations of nocodazole inducting cell cycle delay, cell cycle arrest, spindle aberrations and disturbances in chromosome congression were compared to those increasing hyperploidy. This includes segregation of chromosomes in the absence of polar body formation inducing “diploidy” at metaphase II. Diploidy was defined as “meiotic slippage” in oocytes, when they progress through first anaphase to metaphase II in absence of cytokinesis (thus they contain 40 instead of 20 dyads). This may contribute to the formation of digynic or chaotic preimplantation embryos, which cannot develop to term and may give rise to implantation failure after fertilization [62], [63]. Our observations support earlier qualitative assessments on threshold for nocodazole-induced nondisjunction in oocytes [20]. Moreover, they suggest that even minor changes in spindle integrity causing risks of meiotic disturbances and aneuploidy can be detected by Polscope, which may not be obvious and are difficult to quantify by conventional immunofluorescence.

Section snippets

Animals and oocytes

MF1 mice originally obtained from Harlan Winkelmann (Borchen, Germany) were housed in the animal facilities of the faculty under constant temperature and humidity under a regular 12 h photo-period (7 a.m.–7 p.m.), and were fed ad libitum. Ovaries were isolated from sexually mature MF1 mice (7–20 weeks of age) on the day of diestrous of the natural cycle, and placed into M2 medium [64] containing 14 mg/ml bovine serum albumin (BSA, Sigma, Deisenhofen, Germany). In vitro maturation of denuded mouse

Nocodazole-induced meiotic delay and arrest

Kinetics of meiotic progression of mouse oocytes was analysed by scoring oocytes emitting a first polar body (PB) at hourly intervals starting from 8 h of maturation in vitro (Fig. 1). In the control and solvent control, around 80% of oocytes emitted a PB by 16 h of culture. Meiotic progression was delayed in all nocodazole-exposed groups. For instance, at 11 h of culture only 38.7, 38.1, and 21.9% of the oocytes exposed to 20, 30, and 40 nM nocodazole, respectively, exhibited PB formation, in

Polscope in analysis of spindle morphology and function

Spindle analysis in fixed oocytes processed for anti-tubulin immunofluorescence fully confirmed the observations by non-invasive Polscope microscopy in living mouse oocytes and demonstrated that even 20 nM nocodazole had the potential to influence spindle morphology in oocytes. Maturation in presence of 20–40 nM nocodazole significantly and dose-dependently reduced spindle length at meiosis I and II. Average spindle length for the control and the solvent control at meiosis I was 27.7 ± 2.7 and 28.8 ±

Acknowledgements

The work has been supported by EU (QCRT-2000-00058).

References (84)

  • S.N. London et al.

    Clomiphene citrate-induced perturbations during meiotic maturation and cytogenetic abnormalities in mouse oocytes in vivo and in vitro

    Fertil Steril

    (2000)
  • C.M. Combelles et al.

    Centrosome-specific perturbations during in vitro maturation of mouse oocytes exposed to cocaine

    Exp Cell Res

    (2000)
  • P.A. Hunt et al.

    Bisphenol: a exposure causes meiotic aneuploidy in the female mouse

    Curr Biol

    (2003)
  • U. Eichenlaub-Ritter et al.

    Manipulation of the oocyte: possible damage to the spindle apparatus

    Reprod Biomed Online

    (2002)
  • M. Plachot

    Chromosomal abnormalities in oocytes

    Mol Cell Endocrinol

    (2001)
  • M.V. Zaragoza et al.

    Parental origin and phenotype of triploidy in spontaneous abortions: predominance of diandry and association with the partial hydatidiform mole

    Am J Hum Genet

    (2000)
  • A. Kuliev et al.

    Chromosomal abnormalities in a series of 6,733 human oocytes in preimplantation diagnosis for age-related aneuploidies

    Reprod Biomed Online

    (2003)
  • W.H. Wang et al.

    The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes

    Fertil Steril

    (2001)
  • D. Keefe et al.

    Imaging meiotic spindles by polarization light microscopy: principles and applications to IVF

    Reprod Biomed Online

    (2003)
  • S. Munne et al.

    Chromosome mosaicism in cleavage-stage human embryos: evidence of a maternal age effect

    Reprod Biomed Online

    (2002)
  • C.P. Silva et al.

    The first polar body does not predict accurately the location of the metaphase II meiotic spindle in mammalian oocytes

    Fertil Steril

    (1999)
  • K. Wassmann et al.

    I arrest upon activation of the Mad2-dependent spindle checkpoint in mouse oocytes

    Curr Biol

    (2003)
  • W.H. Wang et al.

    Prediction of chromosome misalignment among in vitro matured human oocytes by spindle imaging with the PolScope

    Fertil Steril

    (2002)
  • W.M. Generoso et al.

    Chromosome malsegregation and embryonic lethality induced by treatment of normally ovulated mouse oocytes with nocodazole

    Mutat Res

    (1989)
  • A. Baumgartner et al.

    Detection of aneuploidy in rodent and human sperm by multicolor FISH after chronic exposure to diazepam

    Mutat Res

    (2001)
  • M.A. Jordan

    Mechanism of action of antitumor drugs that interact with microtubules and tubulin

    Curr Med Chem Anti-Canc Agents

    (2002)
  • M.A. Jordan et al.

    Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindlesImplications for the role of microtubule dynamics in mitosis

    J Cell Sci

    (1992)
  • A. Elhajouji et al.

    Indications for a threshold of chemically-induced aneuploidy in vitro in human lymphocytes

    Environ Mol Mutagen

    (1995)
  • A. Elhajouji et al.

    Indication for thresholds of chromosome non-disjunction versus chromosome lagging induced by spindle inhibitors in vitro in human lymphocytes

    Mutagenesis

    (1997)
  • U. Eichenlaub-Ritter et al.

    Nocodazole sensitivity, age-related aneuploidy, and alternations in the cell cycle during maturation of oocytes

    Cytogenet Cell Genet

    (1989)
  • A. Musacchio et al.

    The spindle checkpoint: structural insights into dynamic signalling

    Nat Rev Mol Cell Biol

    (2002)
  • C.L. Rieder et al.

    The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores

    J Cell Biol

    (1995)
  • R.B. Nicklas et al.

    Checkpoint signals in grasshopper meiosis are sensitive to microtubule attachment, but tension is still essential

    J Cell Sci

    (2001)
  • R. LeMaire-Adkins et al.

    Lack of checkpoint control at the metaphase/anaphase transition: a mechanism of meiotic nondisjunction in mammalian females

    J Cell Biol

    (1997)
  • H. Yin et al.

    Trichlorfon exposeure, spindle aberrations and nondisjunction in mammalian oocytes

    Chromosoma

    (1998)
  • C.A. Hodges et al.

    Experimental evidence that changes in oocyte growth influence meiotic chromosome segregation

    Hum Reprod

    (2002)
  • N. Steuerwald et al.

    Association between spindle assembly checkpoint expression and maternal age in human oocytes

    Mol Hum Reprod

    (2001)
  • K.S. Bentley et al.

    Evaluation of thresholds for benomyl- and carbendazim-induced aneuploidy in cultured human lymphocytes using fluorescence in situ hybridization

    Mutat Res

    (2001)
  • D.L. Sackett et al.

    Molecular mechanism of colchicine action: induced local unfolding of beta-tubulin

    Biochemistry

    (1993)
  • T. Neumann et al.

    Determination of the net exchange rate of tubulin dimer in steady-state microtubules by fluorescence correlation spectroscopy

    Biol Chem

    (2001)
  • K.S. Ladrach et al.

    Malorientation and abnormal segregation of chromosomes during recovery from colcemid and nocodazole

    Cell Motil Cytoskeleton

    (1986)
  • D. Cimini et al.

    Merotelic kinetochore orientation versus chromosome mono-orientation in the origin of lagging chromosomes in human primary cells

    J Cell Sci

    (2002)
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