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

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 microM 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 SMC1beta 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) but neither reduced non-disjunction nor induced aneuploidy in aged oocytes. Polyploidy was high in FF-MAS-stimulated maturation, in particular in the aged oocytes (P > 0.001). Relative levels of Smc1beta 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 Smc1beta 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.

Effects of low O2 and ageing on spindles and chromosomes in mouse oocytes from pre-antral follicle culture.

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% O2. Prolonged culture and the presence of only 5% O2 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.

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

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 microg/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 microg/ml DZ and became significantly elevated in oocytes matured with 25 microg/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 microg/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 spatio-temporal 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.

Consumption of trichlorfon-poisoned fish by women in a small Hungarian village has been associated with trisomy resulting from an error of meiosis II in oogenesis. We therefore examined mouse oocytes exposed for 3 h during fertilization to 50 microg/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 II. 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.

Mechanisms of spontaneous and chemically-induced aneuploidy in mammalian oogenesis: basis of sex-specific differences in response to aneugens and the necessity for further tests.

The basis for sex-specific differences in chemically-induced and age-related aneuploidy of mammalian germ cells is still unknown. We have analysed the maturation of isolated mouse oocytes to characterize the mechanisms underlying drug-induced aneuploidy produced by two compounds, chloral hydrate (CH) and diazepam (DZ). When administered in vivo both drugs increase hyperploidy in male but not in female germ cells. In the assay presented here we show that both CH and DZ caused meiotic delay in in vitro maturing mouse oocytes. CH blocked meiotic progression irreversibly, affecting even dictyate stage oocytes. DZ-exposure slowed down maturation but many oocytes may eventually develop to metaphase II. Under the influence of CH asymmetric spindles were formed. Often chromosomes failed to align properly. This appeared to be responsible for triggering a meiotic checkpoint which arrests oocytes in meiosis I. Many oocytes escaping the block became 'diploid'. Lagging of chromosomes at anaphase I may contribute to significant rises in hypoploidy, while the scattering of chromosomes at metaphase II and the premature decondensation of chromatin may also predispose oocytes to the formation of structural and numerical chromosomal aberrations during meiosis II. In contrast, diazepam appeared to enhance the resumption of meiosis in immature oocytes at pharmacologically relevant doses, and only at high concentrations lead to a prominent meiotic arrest/delay. Importantly, several oocytes matured for 16 h in 25 micrograms/ml DZ displayed scattered chromosomes on the spindle and were hyperploid. Concomitantly, precocious separation of homologues occurred after DZ, and oocytes contained uneven numbers of chromatids, suggesting equational division at anaphase I. Cytoplasmic maturation. e.g., association of mitochondria with the spindle was disturbed by DZ. We compared the potential of the in vitro test to evaluate the aneugenic potential, the targets, threshold concentrations and long-lasting effects of relevant environmental pollutants on mammalian oogenesis with the in vivo findings, and evaluated the basis for sex-specific responses to aneugens.

Chloral hydrate induced spindle aberrations, metaphase I arrest and aneuploidy in mouse oocytes.

In view of tissue- and sex-specific differences in chemically-induced aneuploidy, we analysed the effects of chloral hydrate (CH) on cell-cycle progression, spindle formation and aneuploidy in in vitro maturing mouse oocytes with cytogenetic and immunofluorescent methods. CH blocks oocyte maturation irreversibly and concentration dependently. During culture in 125 micrograms/ml CH, germinal vesicle breakdown is delayed, and most oocytes become arrested in meiosis I with bivalent chromosomes. Their spindles are asymmetric or attain fusiform poles. Oocytes which progress to metaphase II also possess astral instead of barrel-shaped, anastral spindles as characteristic for the controls. Resolution of chiasmata without polar body extrusion during exposure to 50 and 125 micrograms/ml CH results in significant rises in 'diploid' metaphase II oocytes. Hyperploidy rates do not increase significantly at any concentration of CH, but hypoploidy levels are elevated at 125 micrograms/ml CH. CH induces lagging of chromosomes during telophase I, inhibits spindle elongation in anaphase B and causes chromosome displacement from the spindle equator in metaphase I and II. Oocytes also become irreversibly arrested in maturation when exposed to CH prior to resumption of maturation, or when CH is present during the first or second 8 h of maturation. Therefore, these data show unequivocally that CH is a potent aneugen in female germ cells, affecting spindle shape, cytokinesis and cell-cycle progression. However, a metaphase I checkpoint in mammalian oogenesis sensing disturbances in the spindle appears to prevent nondisjunction efficiently in most oocytes. In this in vitro model the aneugenic activity of drugs, critical periods in oocyte maturation, threshold concentrations, and targets of drug action can be directly assessed.