Although numerous hypotheses have been proposed to describe the etiology of aneuploidy in human gametes, the only steadfast association remains maternal age 12. Besides this association, very little information is available about the numerous potential mechanisms that may disrupt normal chromosome segregation in oocytes. Based on studies involving compounds that alter microtubular kinetics and induce both cell-cycle delay and aneuploidy, several groups have proposed that temporal perturbations during mammalian oocyte maturation (OM) predispose oocytes to aneuploidy 3456. In addition to faulty microtubule kinetics, perturbations during OM may also stem from abnormal function of centrosomes, kinetochores, spindle checkpoints proteins, the anaphase-promoting complex (APC), the securin-separin-cohesion complex proteins, and the proteasome 7.

OM encompasses the nuclear and cytoplasmic changes that occur during the transition from the dictyotene stage of meiosis I to metaphase II (MII). During this transition, tightly-regulated post-translational phosphorylation-dephosphorylation events and proteasome-mediated proteolytic reactions regulate the activation and inactivation of signal transduction pathways that control chromatin condensation, nuclear membrane dissolution, microtubule nucleation, and formation of a haploid oocyte 8910. Several kinases that exert major roles during OM include maturation promoting factor (MPF) 911, mitogen-activated protein kinases (MAPKs) 1213, and the product of the c-mos protooncogene Mos kinase 1415. Also, during OM oocytes undergo two highly-regulated metaphase-anaphase transitions (MAT) in which homologous chromosomes are equally and randomly segregated to an oocyte and first polar body and a subsequent division in which equational division of sister chromatids results in a haploid oocyte and a second polar body. The MATs are predicated upon the coordinated activities of the spindle checkpoint 1617, the anaphase-promoting complex (APC) or cyclosome 1819, the proteasome 2021, and the cohesion-complex proteins involved with chromosome cohesion and separation 222324. Alterations in the temporal sequence of these coordinated activities may potentially predispose cells to faulty chromosome segregation.

Abnormal chromosome segregation has been observed in mice lacking the Mad2 checkpoint protein 25. The spindle checkpoint utilizes sensory proteins that detect kinetochore-microtubule tension and occupancy and transiently block anaphase until all of the chromosomes are properly attached to microtubules 162627. Although the mammalian spindle checkpoint appears to differ between mitosis and meiosis, the two meiotic divisions, and male and female germ cells 28, anaphase subsequently follows stable kinetochore-microtubule attachments 1729 in both vertebrate mitotic 30 and meiotic 31 cells.

The APC is a large protein complex that ubiquinates mitotic cyclins and other regulatory proteins that are destined for timely proteolysis by proteasomes 1020. Proteasomes are multicatalytic 26S proteases consisting of a 20S central core catalytic subunit bordered by two 19S components 3233 which hydrolyze C-terminal peptide bonds to acidic, basic, and hydrophobic amino-acid residues 2034. Proteasomes proteolyze securins which inhibit separase activity. Separase is needed for inactivating cohesions and enabling sister chromatid separation 353637 in both fission yeast 38 and mammalian cells 39. Although differences have been reported among species 40 and cell types 41, it appears that the majority of cohesion is removed from mammalian chromosomes during prophase and prometaphase; whereas, a lesser amount remains at kinetochores until anaphase onset. Proteasomes translocated to meiotic spindles of rat oocytes and MG-132 induced-inhibition of proteasomal activity resulted in partial segregation of chromosomes during meiosis I 42. Moreover, defective proteasomal activity in fission yeast impaired chromosome segregation 38.

To test the hypothesis that transient inhibition of proteasomal activity during mouse meiosis I was associated with chromosome missegregation, mouse oocytes were exposed in vitro to the reversible proteasome inhibitor MG-132 and metaphase II (MII) oocytes were analyzed for structural and numerical chromosome aberrations. This transient arrest of proteasomal activity represents a perturbation during the normal temporal sequence of events during OM.

Transient exposure of mouse oocytes to MG-132 for 6 h followed by washout of the compound and an additional 17 h culture in vitro enabled exposure of cells during meiosis I and sufficient time for them to progress to metaphase II. Although the majority of MII oocytes were classified as normal (Fig. 1A), the data indicated that MG-132 induced a dose-response perturbation or delay in the rate of OM and an increase of single, unpaired chromatids and hyperploidy in MII oocytes (Table I). The significant (P < 0.01) increase in the proportion of MI oocytes (Fig. 1B) in the 10.0 mcg/ml group relative to the other groups and the significant (P < 0.01) decrease of oocytes exhibiting premature centromere separation (PCS) (Fig. 1C) in the 10.0 mcg/ml group relative to the controls suggest that proteasomal inhibition retarded the rate of OM. An inverse relationship has been shown to exist between the proportions of arrested MI oocytes and MII oocytes exhibiting PCS 43. The significant (P < 0.01) dose responses of higher frequencies of MII oocytes with single chromatids (Figs. 1C,1E) and of hyperploidy (Figs. 1D,1E) suggest that the MG-132-induced transient inhibition of proteasomal and calpain activities during meiosis I predisposed oocytes to abnormal chromosome segregation.

When oocytes were continuously exposed to 0.0, 0.5, 5.0, 25.0 or 50.0 μg/ml MG-132 from one h after initiating the culture until 17 h later, the proportions of MI arrested oocytes were 5.7% (5/87), 12.3% (14/114), 39.3% (44/112), 100% (98/98), and 100% (91/91), respectively. Of the MI oocytes in the 25.0 and 50.0 μg/ml MG-132 groups, 68.2% (30/44) and 100% exhibited diffuse chromatin (Fig. 1F); whereas, those oocytes continuously exposed to 0.0, 0.5 or 5.0 μg/mlMG-132 did not display diffuse chromatin. Additionally, the proportions of MI oocytes displaying homologue separation (Figs. 1G,1H) in oocytes transiently exposed to MG-132 for 6 h were 0% (0/13), 0% (0/19), 11.5% (3/26), 15.1% (8/53), respectively for 0.0, 5.0, 7.5, and 10.0 μg/mlMG-132. Oocytes with premature anaphase, polyploidy, or structural chromosome aberrations were not found.

Proteasome activity is required for germinal vesicle breakdown (GVBD) in starfish and toad oocytes 4445 and for activating Xenopus oocytes 4546. Also, proteasomes are needed for regulating centrosome segregation 3847, kinetochore structure 39, and sister chromatid cohesion and separation 48. When rat oocytes were exposed to the proteasome inhibitor MG132, proteasomes translocated to the meiotic spindle, MPF activity was sustained, and oocytes were arrested in MI with partially segregated chromosomes 42.

Besides inhibiting proteasome activity, the peptide aldehyde proteasome inhibitors have been also reported to repress certain lysosomal cysteine proteases and calpains 49. The finding that MG-132 can obstruct the casein degrading activity of the calcium-dependent protease calpain 50 appears relevant to oocyte maturation. Calpain activity is needed for proteolysis of the c-mos proto-oncogene product pp39 mos in Xenopus oocytes 51, certain cytoskeletal proteins (alpha-tubulin, actin, dynamin, lamin B) in starfish oocytes 52, and of cyclin in Xenopus egg extracts 53.

We report that exposure of mouse oocytes to 10.0 mcg/ml MG-132 during meiosis I led to significant (P < 0.01) increases in the proportions of MI arrested oocytes and of MII oocytes with SC and hyperploidy. Coupled with these findings was a significant (P < 0.01) decrease in the incidence of MII oocytes exhibiting PCS (Table 1). Chemical induction of MI arrest in mammalian oocytes and spermatocytes indicates cell cycle retardation which is often associated with aneuploidy 4354. Although 10.0 mcg/ml MG-132 increased the frequency of MI arrested oocytes, this cell-cycle retardation may not be entirely attributed to proteasome inhibition. MG-132 can also inhibit calpains 50 which are needed for cell cycle progression 52 and cyclin degradation 53. Previous studies 555 have shown that an inverse relationship usually exists between the proportions of arrested MI oocytes and MII oocytes exhibiting PCS. Oocytes residing in MII for longer periods of time tend to display higher frequencies of PCS; whereas, those deferred in MI spend less time in MII prior to fixation. The significant (P < 0.01) increase in the proportions of MII oocytes exhibiting PCS in controls relative to the 10.0 μg/ml group reflects the greater length of time that control oocytes spent in MII prior to fixation. We suggest that the PCS detected in MII oocytes mainly resulted from in vitro aging. As such, the frequencies of PCS noted in Table 1 would represent independent events relative to the other cytogenetic abnormalities reported.

On the other hand, PCS during meiosis I can result in aneuploid MII oocytes. PCS of homologous chromosomes and sister chromatids prior to establishing proper kinetochore-spindle attachment during meiosis I increases the probabilities of both homologues going to the same pole and of equational division of sister chromatids during anaphase I. Upon normal fertilization, MII oocytes with an additional chromosome result in trisomic zygotes; whereas oocytes containing an extra chromatid would be expected to produce 50% trisomic zygotes due to random segregation during anaphase II. This situation also applies to oocytes missing chromosomes and chromatids. Experimental data support PCS as a prelude to aneuploidy. PCS of homologues during meiosis I and equational division of sister chromatids during anaphase I 5657 and premature separation of chromatids during meiosis II 5859 and mitosis 6061 have been shown to increase the incidence of aneuploidy in subsequent divisions. The partially segregated chromosomes found in rat oocytes exposed to MG-132 may have represented homologous chromosomes and/or sister chromatids 42.

The origin of single, unpaired chromatids in MII oocytes has been proposed to result from premature separation of sister chromatids prior to anaphase I 5657. The possibility exists that both SC and aneuploidy may have originated from PCS of homologues and sister chromatids during the MAT of meiosis I. Upon removal of MG-132 from the culture media during meiosis I and the subsequent resumption of OM and proteasomal activity, faulty kinetochore-microtubule attachment accompanied by degradation of cohesion proteins could conceivably lead to PCS of homologues and sister chromatids. Following anaphase I, a probability exists that both homologues and sister chromatids could randomly assort to the same pole and result in SC and aneuploid MII oocytes.

Although the frequency of homologue separation increased with MG-132 dose, the sample size of 11 oocytes is much too low to comment about their possible origin or their influence on other abnormalities detected in MII oocytes. The possibility exists that the observed homologue separation may actually reflect anaphase onset. The finding of diffuse chromatin in MI oocytes continuously exposed to 25.0 and 50.0 μg/mlMG-132 may be related to the sustained elevation of MPF activity noted in rat oocytes exposed to MG-132 42 and the stabilization of polyubiquitinated proteins and enhanced deubiquitination of nucleosomal histones in chromatin following proteasomal inhibition 62.

The findings of the present study indicate that transient in vitro exposure of mouse oocytes to MG-132 is associated with abnormal chromosome segregation. However, additional data are clearly needed to further investigate the roles of calpain proteolysis and proteasome-mediated proteolysis on chromosome segregation as well as the numerous other potential molecular mechanisms of aneuploidy.

Each author contributed substantially to the various aspects required for the timely completion of this study. Their respective efforts are reflected by the order of authorship.