Diazepam induces meiotic delay, aneuploidy and predivision of homologues and chromatids in mammalian oocytes

Yin H, Baart E, Betzendahl I, Eichenlaub-Ritter U. Diazepam induces meiotic delay, aneuploidy and predivision of homologues and chromatids in mammalian oocytes. MUTAGENESIS. 1998;13(6):567-580.The tranquilizer and anti-convulsant diazepam (DZ) is a suspected aneugen. In order to assess its aneugenic potential in mammalian oogenesis we exposed in vitro maturing mouse oocytes to the drug. Spindle formation and cell cycle progression, the behaviour of chromosomes and the distribution of mitochondria were characterized with respect to induction of numerical chromosomal aberrations. A concentration of 25 mu g/ml DZ induced a pronounced delay in maturation and blocked a high percentage of oocytes in meiosis I. This arrest was partly reversible. Hyperploidy was slightly increased in oocytes matured in the presence of 5 mu g/ml DZ and became significantly elevated in oocytes matured with 25 mu g/ml DZ, relative to controls. Concomitantly, DZ induced spindle aberrations and displacement of chromosomes from the equator, but unlike in mitosis and in male meiosis most oocytes still possessed bipolar spindles. A significant fraction of meiotically delayed, metaphase I-blocked oocytes exposed to 25 mu g/ml DZ contained univalents. Some DZ-treated oocytes progressing to meiosis II exhibited one or multiple single chromatids, Precocious chiasma resolution and equational segregation of chromatids from functional univalents in first anaphase (predivision) may be responsible for this condition, a mechanism also discussed in the aetiology of maternal age-related aneuploidy, DZ disturbed the spatiotemporal distribution of mitochondria during oocyte maturation, possibly by binding to peripheral-type benzodiazepine receptors on mitochondria, thus affecting the availability of ATP and calcium homeostasis. Blocks in maturation may also relate to binding of DZ to calmodulin. Data suggest that DZ exposes mammalian oocytes to predivision and aneuploidy, Thresholds, long lasting effects of DZ in vivo and sex-specific sensitivities in chemically induced aneuploidy of mammalian germ cells are critically evaluated

Trichlorfon exposure, spindle aberrations and nondisjunction in mammalian oocytes

Yin H, Cukurcam S, Betzendahl I, Adler ID, Eichenlaub-Ritter U. Trichlorfon exposure, spindle aberrations and nondisjunction in mammalian oocytes. CHROMOSOMA. 1998;107(6-7):514-522.Consumption of trichlorfon-poisoned fish by women in a small Hungarian village has been associated with trisomy resulting from an error of meiosis B in oogenesis. We therefore examined mouse oocytes exposed for 3 h during fertilization to 50 mu g/ml trichlorfon. Spindle morphology was not visibly altered by the pesticide. Chromosomes segregated normally at anaphase II with no induction of aneuploidy. However, formation of a spindle was disturbed in many oocytes resuming meiosis I in the presence of trichlorfon. In spite of the spindle aberrations and the failure of bivalents to align properly at the equator, oocytes did not become meiotically arrested but progressed to metaphase B. At this stage, spindles were highly abnormal, and chromosomes were often totally unaligned, unattached or dispersed on the elongated and disorganized spindle. By causing spindle aberrations and influencing chromosome congression, trichlorfon appears, therefore, to predispose mammalian oocytes to random chromosome segregation, especially when they undergo a first division and develop to metaphase II during exposure. This is the first case in which environmentally induced human trisomy can be correlated with spindle aberrations induced by chemical exposure. Our observations suggest that oocytes may not possess a checkpoint sensing displacement of chromosomes from the equator at meiosis I and may therefore be prone to nondisjunction

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

Shen Y, Betzendahl I, Sun FY, Tinneberg HR, Eichenlaub-Ritter U. Non-invasive method to assess genotoxicity of nocodazole interfering with spindle formation in mammalian oocytes. REPRODUCTIVE TOXICOLOGY. 2005;19(4):459-471.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 11 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. (c) 2004 Elsevier Inc. All rights reserved

Effects of low O-2 and ageing on spindles and chromosomes in mouse oocytes from pre-antral follicle culture

Hu Y, Betzendahl I, Cortvrindt R, Smitz J, Eichenlaub-Ritter U. Effects of low O-2 and ageing on spindles and chromosomes in mouse oocytes from pre-antral follicle culture. HUMAN REPRODUCTION. 2001;16(4):737-748.To assess their quality, spindles were analysed in mouse oocytes from pre-antral follicle culture. High or low oxygen tension was present during the last 16 or 20 h post human chorionic gonadotrophin (HCG)/epidermal growth factor (EGF) addition. Most oocytes from pre-antral follicle culture possessed typical anastral spindles with flat poles resembling those of ovulated, in-vivo-matured oocytes of sexually mature mice, while denuded oocytes in-vitro matured to metaphase II (MII) formed significantly longer, slender spindles with pointed, narrow poles. Spindles in oocytes from follicle culture were only slightly shorter and less compact at the equator as compared with those of oocytes matured in vivo. Chromosomes were well aligned at the equator in MII oocytes obtained from follicle culture with high oxygen. Maturation rate was significantly reduced by lowering oxygen tension to 5% O-2. Prolonged culture and the presence of only 5% O-2 dramatically increased the percentage of MII oocytes with unaligned chromosomes. These observations indicate that sufficient oxygen supply and time of retrieval after initiation of resumption of maturation by HCG as well as the microenvironment and cell-cell interactions between oocytes and their somatic compartment are critical in affecting the oocyte's capacity to mature to MII, to form a functional spindle, and to align chromosomes correctly

The effect of alternative propellants on the electron drift instability in Hall-effect thrusters: Insight from 2D particle-in-cell simulations

International audienceHall-effect thrusters (HETs) operated with xenon are one of the most commonly used electric propulsion technologies for a wide range of space missions, including drag compensation in low Earth orbit, station-keeping, and orbital insertion, as access to space becomes more affordable. Although anomalous electron transport, the electron drift instability (EDI), and secondary electron emission (SEE) have been studied experimentally and numerically in xenon-based HETs, the impact of alternative propellants is still poorly characterized. In this work, a two-dimensional particle-in-cell/Monte Carlo collision code is used to model the (r – θ) plane of a HET operated separately with four different noble gases: xenon, krypton, argon, and helium. Models for electron induced secondary electron emission (SEE) and dielectric walls are implemented in order to investigate the coupling between the propellant choice and the radial thruster walls. For all conditions and propellants studied, an EDI and enhanced electron cross-field transport are observed. The frequency of the instability, as well as the electron mobility, is compared with analytical expressions from a recently developed kinetic theory. Confirming this theory, it is shown that while the frequency of the EDI depends on the propellant mass, the electron mobility appears to be almost independent of the propellant choice

Influence of follicular fluid meiosis-activating sterol on aneuploidy rate and precocious chromatid segregation in aged mouse oocytes

Cukurcam S, Betzendahl I, Michel G, et al. Influence of follicular fluid meiosis-activating sterol on aneuploidy rate and precocious chromatid segregation in aged mouse oocytes. HUMAN REPRODUCTION. 2007;22(3):815-828.BACKGROUND: Follicular fluid meiosis-activating sterol (FF-MAS) protects young oocytes from precocious chromatid separation (predivision). Reduced expression of cohesion and checkpoint proteins and predivision has been hypothesized to occur in age-related aneuploidy in oocytes. METHODS: To know whether FF-MAS also protects aged oocytes from predivision and from age-related non-disjunction, we analysed chromosome constitution in mouse oocytes matured spontaneously with or without 10 mu M FF-MAS and in hypoxanthine (HX)-arrested young and aged oocytes induced to resume maturation by FF-MAS. Messenger RNA for checkpoint protein MAD2 and cohesion protein SMC1 beta was compared between oocytes matured with or without FF-MAS. RESULTS: Aged oocytes possessed many bivalents with single distal chiasma at meiosis I. Predivision was especially high in aged oocytes cultured sub-optimally to metaphase II in alpha-minimum essential medium (alpha-MEM). FF-MAS reduced predivision significantly (P 0.001). Relative levels of Smc1 beta mRNA appeared increased by maturation in FF-MAS, and mitochondrial clustering was restored. CONCLUSIONS: Sister chromatids of aged oocytes appear to be highly susceptible to precocious chromatid separation, especially when maturation is under sub-optimal conditions, e.g. in the absence of cumulus and FF-MAS. This may relate to some loss of chromatid cohesion during ageing. FF-MAS protects aged oocytes from predivision during maturation, possibly by supporting Smc1 beta expression, thus reducing risks of meiotic errors, but it cannot prevent age-related non-disjunction. Aged oocytes appear prone to loss of co-ordination between nuclear maturation and cytokinesis suggesting age-related relaxed cell cycle control