Griseofulvin-induced aneuploidy and meiotic delay in female mouse germ cells. II. Cytogenetic analysis of one-cell zygotes.

The effects of griseofulvin (GF) upon the first meiotic division of female mouse germ cells were evaluated by cytogenetic analysis of first-cleavage (1-Cl) zygotes. The present study is an extension of an investigation that began with the cytogenetic analysis of metaphase II (M II) oocytes. Different doses (200, 666, 1332, 2000 mg/kg) were tested by oral administration of GF to superovulated animals either at the time of human chorionic gonadotrophin (HCG) injection or 2 h post HCG. When GF was given at the time of HCG, significant dose-dependent increases of different types of cytogenetically abnormal cells were found. These included zygotes containing ostensibly female-derived M I or M II arrested chromosomes and polyploid zygotes. The total yields of these aberrations were 2.9, 4.3, 26.2, 60.6, and 64.1% for control, 200, 666, 1332, and 2000 mg/kg, respectively. The origin of these zygotes was attributed to the fertilization of oocytes that had been previously arrested at M I. No significant induction of hyperploidy was detected. Developmentally abnormal zygotes were still observed when GF was administered 2 h post HCG, although their frequencies were significantly lower than in the first series of experiments. The yields of developmentally abnormal zygotes were 49, 10.2, and 23.6% at 200, 666, and 2000 mg/kg. Additionally, a dose-dependent increase in the frequency of hyperploid zygotes was detected up to a maximum of 36.5% at 2000 mg/kg. These results confirm the cytogenetic observations from M II oocytes after GF treatment under the same experimental conditions; namely, a dramatic change in the oocyte target susceptibility to GF occurred within a short time period. Also, the present study demonstrated that most of GF-induced aneuploid oocytes were fertilized and reached first-cleavage metaphase.     

Griseofulvin-induced aneuploidy and meiotic delay in female mouse germ cells. I. Cytogenetic analysis of metaphase II oocytes.

Griseofulvin (GF) was tested in female mouse germ cells for the induction of aneuploidy and meiotic arrest. Superovulated mice were orally treated with 200, 666, 1332 or 2000 mg/kg in olive oil at the time of human chorionic gonadotrophin (HCG) injection and were sacrificed 18 h later. A dose-dependent increase in the frequency of metaphase I (M I) arrested oocytes was observed (maximum of 70%). Aneuploidy was not significantly induced. Also, the kinetics of meiotic progression up to the metaphase II (M II) stage was studied in untreated mice in order to correlate the time of treatment with the time of the first meiotic division. The results demonstrate that the majority of cells was treated with GF approximately 8 h before the M I stage. A second series of experiments were performed to test GF effects at a different treatment time. Doses of 200, 666 or 2000 mg/kg were administered 2 h post HCG. As in the first series of experiments, the animals were sacrificed 18 h post HCG. The results, compared with those obtained in the first experimental series, showed an inverse trend for meiotic arrest and aneuploidy induction. The frequency of M I arrested oocytes dropped from a maximum of 70% to a maximum of 20%, while, at the latest treatment time, a dose-dependent increase in the frequency of hyperploid oocytes was observed up to 56% aberrant cells at 2000 mg/kg. Altogether the results suggest that the arrest of meiotic division and the induction of aneuploidy by GF are caused by interaction with different targets or different developmental stages of the same target. In conclusion, GF has been shown to induce aneuploidy during the first meiotic division in a dose-related manner, together with other effects such as polyploidy, developmental delay and meiotic arrest. Also, these findings demonstrate that the sensitivity of the oocyte target(s) may be restricted to a specific time period and that a correct experimental protocol is critical for assessing the aneugenic activity of a chemical.     

Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes

To increase our understanding about the potential risks of chemically-induced aneuploidy, more information about the various mechanisms of aneuploidy induction is needed, particularly in germ cells. Most chemicals that induce aneuploidy inhibit microtubule polymerization. However, taxol alters microtubule dynamics by enhancing polymerization and stabilizing the polymer fraction. We tested the hypothesis that taxol induces meiotic delay, spindle defects, and aneuploidy in mouse oocytes and zygotes. Super-ovulated ICR mice received 0 (control), 2.5, 5.0, and 7.5 mg/kg taxol intraperitoneally immediately after HCG. Females were paired (1:1) with males for 17 h after taxol treatment. Mated females were given colchicine 25 h after taxol and their one-cell zygotes were collected 16 h later. Ovulated oocytes from non-mated females were collected 17 h after taxol. Chromosomes were C-banded for cytogenetic analyses. Oocytes were also collected from another group of similarly treated females for in situ chromatin and microtubule analyses. Taxol significantly (p<0.01) enhanced the proportion of oocytes exhibiting parthenogenetic activation, chromosomes displaced from the meiotic spindle, and sister-chromatid separation. Moreover, 7.5 mg/kg taxol significantly (p<0.01) increased the proportions of metaphase I and diploid oocytes and polyploid zygotes. A significant (p<0.01) dose response for taxol-induced hyperploidy in oocytes and zygotes was found. These results support the hypothesis that taxol-induced meiotic delay and spindle defects contribute to aneuploid mouse oocytes and zygotes.     

Cytogenetic analysis of mouse oocytes and one-cell zygotes as a potential assay for heritable germ cell aneuploidy

Assays are needed for detecting chemically-induced aneuploidy, for investigating the mechanisms of aneuploidy production, and for obtaining heritable germ cell data that can be used to formulate human risk estimates. In this report, we describe the results of experiments designed to study aneuploidy in metaphase II (MII) oocytes induced by intraperitoneal (i.p.) or oral dosages of colchicine, and to investigate the proportion of aneuploid oocytes transmitted to one-cell (1C) zygotes following oral administration of colchicine immediately following HCG. The proportions (and percentages) of hyperploid MII oocytes were: 1/606 (0.2), 37/504 (7.3), 152/731 (20.8) and 75/319 (23.5) for control, 0.2, 0.3 and 0.4 mg/kg, respectively for i.p. administration of colchicine; and 3/216 (1.4), 8/539 (1.5), 81/511 (15.9), 71/398 (17.8) and 98/391 (25.1) for control, 1.0, 2.0, 3.0 and 4.0 mg/kg, respectively for oral administration of colchicine. The proportions of hyperploid 1C zygotes were 2/327 (0.6), 21/389 (5.4), 62/435 (14.3) and 69/438 (15.8) for control, 2.0, 3.0 and 4.0 mg/kg, respectively for oral colchicine. The proportions of hyperploid MII oocytes and 1C zygotes were significantly higher (Chi-square, P less than 0.01) at each i.p. or oral dose (except 1.0 mg/kg oral) than in the controls. The frequencies of hyperploidy induced by oral doses of colchicine were greater in MII oocytes than in 1C zygotes. We also found that the frequency of developmentally delayed and polyploid 1C zygotes increased with the dose of oral colchicine. Developmentally delayed zygotes contained male-derived chromosomes and female-derived fragmented pronuclei and pronuclei with decondensed chromosomes. These results indicate that higher doses of oral colchicine are needed to induce comparable levels of aneuploidy found after i.p. administration, and that aneuploid oocytes are fertilized and reach first cleavage metaphase. In addition, colchicine induces a spectrum of events including aneuploidy, polyploidy and developmentally delayed oocytes and zygotes.     

Age‐dependent aneuploidy in mammalian oocytes instigated at the second meiotic division

Ageing severely affects the chromosome segregation process in human oocytes resulting in aneuploidy, infertility and developmental disorders. A considerable amount of segregation errors in humans are introduced at the second meiotic division. We have here compared the chromosome segregation process in young adult and aged female mice during the second meiotic division. More than half of the oocytes in aged mice displayed chromosome segregation irregularities at anaphase II, resulting in dramatically increased level of aneuploidy in haploid gametes, from 4% in young adult mice to 30% in aged mice. We find that the post‐metaphase II process that efficiently corrects aberrant kinetochore‐microtubule attachments in oocytes in young adult mice is approximately 10‐fold less efficient in aged mice, in particular affecting chromosomes that show small inter‐centromere distances at the metaphase II stage in aged mice. Our results reveal that post‐metaphase II processes have critical impact on age‐dependent aneuploidy in mammalian eggs.     

Trichlorfon predisposes to aneuploidy and interferes with spindle formation in in vitro maturing mouse oocytes

The pesticide trichlorfon (TCF) has been implicated in human trisomy 21, and in errors in chromosome segregation at male meiosis II in the mouse. We previously provided evidence that TCF interferes with spindle integrity and cell-cycle control during murine oogenesis. To assess the aneugenic activity of TCF in oogenesis, we presently analysed maturation, spindle assembly, and chromosome constitution in mouse oocytes maturing in vitro in the presence of 50 or 100 microg/ml TCF for 16 h or in pulse-chase experiments. TCF stimulated maturation to meiosis II at 50 microg/ml, but arrested meiosis in some oocytes at 100 microg/ml. TCF at 100 microg/ml was aneugenic causing non-disjunction of homologous chromosomes at meiosis I, a significant increase of the hyperploidy rate at metaphase II, and a significant rise in the numbers of oocytes that contained a 'diploid' set of metaphase II chromosomes (dyads). TCF elevated the rate of precocious chromatid segregation (predivision) at 50 and 100 microg/ml. Pulse-chase experiments with 100 microg/ml TCF present during the first 7 h or the last 9 h of maturation in vitro did not affect meiotic progression and induced intermediate levels of hyperploidy at metaphase II. Exposure to > or =50 microg/ml TCF throughout maturation in vitro induced severe spindle aberrations at metaphase II, and over one-third of the oocytes failed to align all chromosomes at the spindle equator (congression failure). These observations suggest that exposure to high concentrations of TCF induces non-disjunction at meiosis I of oogenesis, while lower doses may preferentially cause errors in chromosome segregation at meiosis II due to disturbances in spindle function, and chromosome congression as well as precocious separation of chromatids prior to anaphase II. The data support evidence from other studies that TCF has to be regarded as a germ cell aneugen.     

Cytostatic Activity Develops during Meiosis I in Oocytes of LT/Sv Mice

Oocytes of wild-type mice are ovulated as the secondary oocytes arrested at metaphase of the second meiotic division. Their fertilization or parthenogenetic activation triggers the completion of the second meiotic division followed by the first embryonic interphase. Oocytes of the LT/Sv strain of mice are ovulated either at the first meiotic metaphase (M I) as primary oocytes or in the second meiotic metaphase (M II) as secondary oocytes. We show here that duringin vitromaturation a high proportion of LT/Sv oocytes progresses normally only until metaphase I. In these oocytes MAP kinase activates shortly after histone H1 kinase (MPF) activation and germinal vesicle breakdown. However, MAP kinase activation is slightly earlier than in oocytes from wild-type F1 (CBA/H × C57Bl/10) mice. The first meiotic spindle of these oocytes forms similarly to wild-type oocytes. During aging, however, it increases in size and finally degenerates. In those oocytes which do not remain in metaphase I the extrusion of first polar bodies is highly delayed and starts about 15 h after germinal vesicle breakdown. Most of the oocytes enter interphase directly after first polar body extrusion. Fusion between metaphase I LT/Sv oocytes and wild-type mitotic one-cell embryos results in prolonged M-phase arrest of hybrids in a proportion similar to control LT/Sv oocytes and control hybrids made by fusion of two M I LT/Sv oocytes. This indicates that LT/Sv oocytes develop cytostatic factor during metaphase I. Eventually, anaphase occurs spontaneously and the hybrids extrude the polar body and form pronuclei in a proportion similar as in controls. In hybrids between LT/Sv metaphase I oocytes and wild-type metaphase II oocytes (which contain cytostatic factor) anaphase I proceeds at the time observed in control LT/Sv oocytes and hybrids between two M I LT/Sv oocytes, and is followed by the parthenogenetic activation and formation of interphase nuclei. Also the great majority of hybrids between M I and M II wild-type oocytes undergoes the anaphase but further arrests in a subsequent M-phase. These observations suggest that an internally triggered anaphase I occurs despite the presence of the cytostatic activity both in LT/Sv and wild-type M I oocytes. Anaphase I triggering mechanism must therefore either inactivate or override the CSF activity. The comparison between spontaneous and induced activation of metaphase I LT/Sv oocytes shows that mechanisms involved in anaphase I triggering are altered in these oocytes. Thus, the prolongation of metaphase I in LT/Sv oocytes seems to be determined by delayed anaphase I triggering and not provoked directly by the cytostatic activity.     

Spontaneous and sperm-induced activation of oocytes in LT/Sv strain mice

The oocytes of LT/Sv strain mice are unique in that a high proportion of them (∼40% in this study) are ovulated before reaching metaphase of the second meiotic division (metaphase II). The remaining oocytes of LT/Sv mice are ovulated at metaphase II, as in other strains of mice. When recently ovulated oocytes were cultured in vitro for 11–12 h, those ovulated at metaphase II remained at this stage, whereas those ovulated at metaphase of the first meiotic division (metaphase I) commonly resumed meiosis during in vitro aging. These oocytes extrude the polar body and form a diploid pronucleus. This oocyte activation is not coupled with cortical granule exocytosis. The oocytes ovulated at metaphase II are fully capable of normal fertilization, whereas those ovulated at metaphase I are not. Approximately 50% of metaphase I oocytes penetrated by spermatozoa remain at this stage, and sperm nuclei frequently undergo premature chromosome condensation. Only 13% of spermpenetrated metaphase I oocytes formed a diploid female pronucleus and a haploid male pronucleus by 4 h after insemination. These results demonstrate that the two types of ovulated LT/Sv oocytes have different potentials to undergo either spontaneous or sperm-induced activation.     

Progression of mouse oocytes from metaphase I to metaphase II is inhibited by fusion with G2 cells

We show that in contrast to metaphase II oocytes, metaphase I oocytes cannot be activated by fusion with the zygote. Fusion of metaphase I oocytes with G2 zygotes was followed by premature chromosome condensation, with 60% of the hybrids becoming arrested at metaphase I, the remainder progressing and arresting at metaphase II. Hybrids of metaphase I oocytes and M-phase zygotes underwent accelerated maturation, but all arrested at metaphase II. In both cases the arrest could be overcome by treatment with the parthenogenetic activators ethanol and cycloheximide. We discuss these findings in relation to the possibility that the metaphase I oocyte contains cytostatic factor activity that is activated by its zygotic partner. Alternatively, the G2 zygote may provide an inhibitor of anaphase, normally never present in the metaphase I oocyte and which is absent from the M-phase zygote.     

Tamoxifen-induced alterations in meiotic maturation and cytogenetic abnormalities in mouse oocytes and 1-cell zygotes

Alterations in the rate of oocyte meiotic maturation (OM) and the timing of the metaphase-anaphase transition may predispose oocytes to premature centromere separation (PCS) and aneuploidy. Tamoxifen has the potential for perturbing the rate of OM since it can function as a calcium antagonist by binding to calmodulin and inhibiting the formation of a calcium-calmodulin complex which is needed for activating calmodulin-dependent cAMP phosphodiesterase and initiating OM. The objective of this study was to test the hypothesis that tamoxifen alters the rate of OM and predisposes oocytes to PCS and aneuploidy. Different does of tamoxifen were administered by oral gavage to female mice preovulation. Metaphase II oocyte and 1-cell zygote chromosomes were C-banded and cytogenetically analysed. Tamoxifen treatment resulted in a modest, but significant (p < 0.05), increase in oocytes with PCS. Similar frequencies of hyperploidy and oocytes with unpaired, single chromatids (SC) were found. Metaphase I, diploid and premature anaphase (PA) oocytes were not detected. Hyperploidy, polyploidy, PCS, PA and SC were not detected in zygotes. These data indicate that the levels of tamoxifen-induced PCS found in mouse oocytes did not predispose zygotes to aneuploidy. Tamoxifen did, however, reduce the proportion of females exhibiting oestrus.     

Ultrastructural analysis of abnormalities in the morphology of the second meiotic spindle in ethanol-induced parthenogenones

A high frequency of parthenogenetic activation occurs when ovulated mouse oocytes are briefly exposed to a dilute solution of ethanol in vitro. Cytogenetic analyses of parthenogenones at metaphase of the first cleavage division have confirmed that parthenogenetic activation, per se, does not increase the incidence of chromosome segregation errors during the completion of the second meiotic division. Ethanol-induced activation, however, significantly increases the incidence of aneuploidy. The ultrastructural changes that occur in the morphology and organization of the second meiotic spindle apparatus in ethanol- and hyaluronidase-activated oocytes is reported here. Abnormalities in the arrangement of microtubule arrays and chromosome position were principally observed in ethanol-activated oocytes at anaphase and telophase of the second meiotic division, but were only rarely observed in hyaluronidase-activated oocytes. It is proposed that the abnormalities in spindle morphology and chromosome displacement observed in ethanol-activated oocytes represent the initial events that lead to chromosome segregation errors following exposure to this agent.     

Griseofulvin-induced aneuploidy and meiotic delay in male mouse germ cells: detected by using conventional cytogenetics and three-color FISH.

Griseofulvin (GF) was tested in male mouse germ cells for the induction of meiotic delay and aneuploidy. Starved mice were orally treated with 500, 1000 and 2000 mg/kg of GF in corn oil and testes were sampled 22 h later for meiotic delay analysis and chromosome counting in spermatocytes at the second meiotic metaphase (MMII). A dose-related increase in meiotic delay by dose-dependently arresting spermatocytes in first meiotic metaphase (MMI) or/and prolonging interkinesis was observed. Hyperhaploid MMII cells were not significantly increased. Sperm were sampled from the Caudae epididymes 22 days after GF-treatment of the males for three-color fluorescence in situ hybridization (FISH). The frequencies of diploidies were 0.01-0.02% in sperm of the solvent control animals and increased dose-dependently to 0.03%, 0.068% and 0.091%, respectively, for 500, 1000 and 2000 mg/kg of GF. The frequencies of disomic sperm were increased significantly above the controls in all GF-treated groups but showed no dose response. The data for individual classes of disomic sperm indicated that MII was more sensitive than MI to GF-induced non-disjunction in male mice. A comparison of the present data from male mice and literature data from female mice suggests that mouse oocytes are more sensitive than mouse spermatocytes to GF-induced meiotic delay and aneuploidy.     

Analysis of mouse metaphase II oocytes as an assay for chemically induced aneuploidy

Our initial objective was to develop an in vivo mammalian, female aneuploid assay that is consistent, time efficient, and that yields a large number of oocytes amenable to objective analyses. Subsequently, we desired to use such an assay for identifying chemicals and dosages that could increase the incidence of aneuploidy in mouse metaphase II oocytes. The experimental protocol involved superovulating CD-1 mice with PMS; HCG was given 48 h later. At the time of HCG injection, different dosages od diethylstilbestrol diphosphate, cadmium chloride, chloral hydrate, or colchicine were injected intraperitoneally. 17 h later, oocytes were collected and fixed prior to C-banding the chromosomes. The procedure required about 3 h to process oocytes from 25 mice and yielded over 100 analyzable metaphase II oocytes. Colchicine was the only compound tested that resulted in a statistically significant (P less than 0.01) increase in hyperploid (N greater than 20) oocytes over controls. The incidence of hyperploid oocytes in the colchicine group was 2/167, 1/182, 21/220, and 38/202, for control, 0.1 mg/kg, 0.2 mg/kg, and 0.3 mg/kg, respectively. This assay appears sensitive for aneuploidy detection but requires further validation.     

Participation of the ubiquitin-proteasome pathway in rat oocyte activation

The role of the ubiquitin-proteasome pathway (UPP) in mitosis is well known. However, its role in meiotic division is still poorly documented, especially in the activation of mammalian oocytes. In this study, the role of proteasome in the spontaneous and parthenogenetic activation of rat oocytes was investigated. We found that ALLN, an inhibitor of proteasome, when applied to metaphase II oocytes, inhibited spontaneous activation, blocked extrusion of the second polar body (PB) and caused the withdrawal of the partially extruded second PB. ALLN also inhibited the parthenogenetic activation induced by cycloheximide, but had no effect on the formation of pronuclei in activated eggs. In metaphase and anaphase, ubiquitin and proteasome localized to the meiotic spindle, concentrating on both sides of the oocyte-second PB boundary during PB extrusion. This pattern of cellular distribution suggests that UPP may have a role in regulating nuclear division and cytokinesis. Ubiquitin was seen to form a ring around the pronucleus, whereas proteasome was evenly distributed in the pronuclear region. Taken together, our results indicate that (1) UPP is required for the transitions of oocytes from metaphase II to anaphase II and from anaphase II to the end of meiosis; and (2) the UPP plays a role in cytokinesis of the second meiotic division.     

The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF.

In unfertilized eggs from vertebrates, the cell cycle is arrested in metaphase of the second meiotic division (metaphase II) until fertilization or activation. Maintenance of the long-term meiotic metaphase arrest requires mechanisms preventing the destruction of the maturation promoting factor (MPF) and the migration of the chromosomes. In frog oocytes, arrest in metaphase II (M II) is achieved by cytostatic factor (CSF) that stabilizes MPF, a heterodimer formed of cdc2 kinase and cyclin. At the metaphase/anaphase transition, a rapid proteolysis of cyclin is associated with MPF inactivation. In Drosophila, oocytes are arrested in metaphase I (M I); however, only mechanical forces generated by the chiasmata seem to prevent chromosome separation. Thus, entirely different mechanisms may be involved in the meiotic arrests in various species. We report here that in mouse oocytes a CSF-like activity is involved in the M II arrest (as observed in hybrids composed of fragments of metaphase II-arrested oocytes and activated mitotic mouse oocytes) and that the high activity of MPF is maintained through a continuous equilibrium between cyclin B synthesis and degradation. In addition, the presence of an intact metaphase spindle is required for cyclin B degradation. Finally, MPF activity is preferentially associated with the spindle after bisection of the oocyte. Taken together, these observations suggest that the mechanism maintaining the metaphase arrest in mouse oocytes involves an equilibrium between cyclin synthesis and degradation, probably controlled by CSF, and which is also dependent upon the three-dimensional organization of the spindle.     

Improvement of male pronuclear formation during cross‐fertilization between Chinese hamster spermatozoa and Syrian hamster oocytes by nocodazole,and chromosome analysis of hybrid zygotes

During cross‐fertilization between Chinese hamster spermatozoa and Syrian hamster oocytes, incorporated sperm heads frequently fail to develop into male pronuclei, whereas the group of oocyte chromosomes develop into female pronuclei. The present study applies this cross‐fertilization system to the cytogenetic investigation of mammalian hybrid embryos. Immediately after insemination, oocytes were exposed to 0.1 μg/ml nocodazole for 1 hr (1 hr group) or 2 hr (2 hr group), then further cultured. Although the rates of sperm penetration in the 1 hr (48.0%) and 2 hr (75.8%) groups were significantly lower than that in the control group (89.8%), the ratios of male pronuclear formation were higher in both exposed groups (79.4% and 74.2%, respectively) than in the control group (10.6%). These results were apparently due to sperm head decondensation induced during the meiotic arrest of oocytes at metaphase II by nocodazole. Chromosomes of hybrid zygotes obtained after nocodazole exposure were analyzed at the first cleavage metaphase. The incidence of structural chromosome aberrations in the Chinese hamster genome of hybrid zygotes was high in the control (42.1%) and 1 hr (48.8%) groups. This incidence was reduced to 14.4% in the 2 hr group. Because the lag of sperm head decondensation behind the second meiotic division of oocytes was greater in the control and 1 hr groups than in the 2 hr group, untimely sperm head decondensation may be implicated in occurrence of structural chromosome aberrations in the male genomes of hybrid zygotes. Mol. Reprod. Dev. 52:117–124, 1999. © 1999 Wiley‐Liss, Inc.     

Evaluation of aneugenic effects of bisphenol A in somatic and germ cells of the mouse

Bisphenol A (BPA) is a synthetic monomer widely used to polymerize polycarbonate plastics and resins. It is shown in vitro to interfere with microtubules, producing aberations in mitotic and meiotic spindles. An increase of meiotic abnormalities in untreated female mice from an experimental colony was temporally correlated with the accidental release of BPA from polycarbonate cages and bottles damaged by inadvertent treatment with harsh alkaline detergents [P.A. Hunt, K.E. Koehler, M. Susiarjo, C.A. Hodges, A. Ilagan, R.C. Voigt, S. Thomas, B.F. Thomas, T.J. Hassold, Bisphenol A exposure causes meiotic aneuploidy in the female mouse, Curr. Biol. 13 (2003) 546-553]. In the present study, potential aneugenic effects of BPA on mouse male and female germ cells and bone marrow cells have been evaluated after acute, sub-chronic or chronic in vivo exposure. Female mice were orally treated with a single BPA dose, with 7 daily administrations or exposed for 7 weeks to BPA in drinking water. No significant induction of hyperploidy or polyploidy was observed in oocytes and zygotes at any treatment condition. The only detectable effect was a significant increase of metaphase II oocytes with prematurely separated chromatids after chronic exposure; this effect, however, had no irreversible consequence upon the fidelity of chromosome segregation during the second meiotic division, as demonstrated by the normal chromosome constitution of zygotes under the same exposure condition. With male mice, no delay of meiotic divisions was found after six daily oral doses of BPA with the BrdU assay. Similarly, no induction of hyperploidy and polyploidy was shown in epydidimal sperm hybrized with probes for chromosomes 8, X and Y, 22 days after six daily oral BPA doses. Finally, two daily oral BPA doses did not induce any increase of micronucleus frequencies in polychromatic erythrocytes of mouse bone marrow. In conclusion, our results do not add evidence to the suspected aneugenic activity of BPA and suggest that other factors or co-factors should be considered to explain the unexpected burst of meiotic abnormalities previously attributed to accidental BPA exposure.     

The APC is dispensable for first meiotic anaphase in Xenopus oocytes

Here we show that segregation of homologous chromosomes and that of sister chromatids are differentially regulated in Xenopus and possibly in other higher eukaryotes. Upon hormonal stimulation, Xenopus oocytes microinjected with antibodies against the anaphase-promoting complex (APC) activator Fizzy or the APC core subunit Cdc27, or with the checkpoint protein Mad2, a destruction-box peptide or methylated ubiquitin, readily progress through the first meiotic cell cycle and arrest at second meiotic metaphase. However, they fail to segregate sister chromatids and remain arrested at second meiotic metaphase when electrically stimulated or when treated with ionophore A34187, two treatments that mimic fertilization and readily induce chromatid segregation in control oocytes. Thus, APC is required for second meiotic anaphase but not for first meiotic anaphase.     

Trichlorfon-induced polyploidy and nondisjunction in mouse oocytes from preantral follicle culture

Trichlorfon (TCF), an organophosphate insecticide and potent inhibitor of choline esterases, was previously shown to induce first meiotic nondisjunction and spindle aberrations in isolated, follicle cell-denuded mouse oocytes maturing in vitro. To explore dose-response and direct and indirect, potentially synergistic effects of TCF on the somatic cells and the oocyte within a follicle, we presently employed preantral follicle culture. 100 microg/ml TCF added at the time of hormonally stimulated resumption of meiosis of follicle cell-enclosed mouse oocytes, 16 h before in vitro ovulation, induced significant rises in first meiotic nondisjunction in oocytes from preantral follicle culture. Lower concentrations (6 microg/ml TCF) disturbed polar body formation. Nuclear maturation to meiosis II in absence of cytokinesis resulted in significant increases in polyploidy. Oocytes maturing in follicles in the presence of TCF had aberrant second meiotic spindles. Influences of TCF on somatic cell function were evident by reduced follicular mucification in vitro and deceased progesterone production. In contrast to TCF, acetylcholine (0.1-100 microM) increased progesterone production. The observations therefore suggest that TCF influences oocyte maturation and folliculogenesis directly and indirectly. High TCF is aneugenic at first meiotic division in oocytes, irrespective of the presence or absence of follicle cells. At lower concentrations TCF interferes with spindle formation, chromosome congression at meiosis II, and coordination of nuclear and cytoplasmic maturation, posing risks for second meiotic errors. The observations suggest that chronic TCF exposure during maturation in the follicle may predispose oocytes to the formation of chromosomally unbalanced preimplantation embryos after fertilization.     

Defective calcium release during in vitro fertilization of maturing oocytes of LT/Sv mice

Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in contrast to the oocytes of wild-type mice, a significant fraction of LT/Sv oocytes remains arrested at the metaphase of the first meiotic division and is unable to undergo sperm-induced activation when fertilized 15 hours after the resumption of meiosis. We also show that LT/Sv oocytes experimentally induced to resume meiosis and to reach metaphase II are unable to undergo activation in response to sperm penetration. However, the ability for sperm-induced activation developed during prolonged in vitro culture. Both types of LT/Sv oocytes, i.e. metaphase I and those that were experimentally induced to reach metaphase II, underwent activation when they were fertilized 21 hours after germinal vesicle breakdown (GVBD). Thus, the ability of LT/Sv oocytes to become activated by sperm depends on cytoplasmic maturation rather than on nuclear maturation i.e. on the progression of meiotic division. We also show that sperm penetration induces fewer Ca(2+) transients in LT/Sv oocytes than in control wild-type oocytes. In addition, we found that the levels of mRNA encoding different isoforms of protein kinase C (alpha, delta and zeta), that are involved in meiotic maturation and signal transduction during fertilization, differed between metaphase I LT/Sv oocytes which cannot be activated by sperm, and those which are able to undergo activation after fertilization. However, no significant differences between these oocytes were found at the level of mRNA encoding IP(3) receptors which participate in calcium release during oocyte fertilization.