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