Mitotic arrest-deficient-2 (MAD2) is one of 6 yeast genes that are
required for execution of the mitotic checkpoint. Dysfunction of MAD2 may lead to malignancy or degeneration of cells.
Li and Benezra (1996) isolated a human homolog of MAD2 (MAD2L1) in a screen for high copy-number suppressors
of thiabendazole sensitivity in yeast lacking CBF1, a component of the kinetochore. (Thiabendazole is a mitotic spindle
assembly inhibitor.) The gene encodes a 205-amino acid polypeptide. DNA sequence determination revealed that the
open reading frame of the human clone is 60% identical to the yeast MAD2 gene. They used antibody electroporation
experiments to demonstrate that the human MAD2 gene was a necessary component of the mitotic checkpoint in HeLa
cells. Through immunofluorescence studies they demonstrated that the human MAD2 protein is localized at the
kinetochore after chromosome condensation but that it is no longer observed at the kinetochore in metaphase. Based on
this observation they proposed that MAD2 monitors the completeness of the spindle kinetochore attachment.
NCBI Summary:
MAD2L1 is a component of the mitotic spindle assembly checkpoint that prevents the onset of anaphase until all chromosomes are properly aligned at the metaphase plate. MAD2L1 is related to the MAD2L2 gene located on chromosome 1. A MAD2 pseudogene has been mapped to chromosome 14. [provided by RefSeq]
Kallio M et al 2000 reported differences in spindle association of the mitotic checkpoint
protein Mad2 in mammalian spermatogenesis and oogenesis.
They investigated expression and subcellular localization of the spindle
checkpoint protein Mad2 during rat and mouse spermatogenesis and in
superovulated mouse oocytes. Immunofluorescence studies demonstrate
substantial differences in the localization patterns of kinetochore-associated
Mad2 in these meiotic systems compared with previous studies of mitosis. In
addition, the association of Mad2 with second-division-metaphase kinetochores
differed significantly in male versus female meiosis.
Mad2 persisted at the kinetochores of normal, second-division oocytes at
metaphase. These findings suggest that the role of the kinetochore in
signaling in the spindle checkpoint may differ markedly between mammalian
mitosis and meiosis, between the two meiotic divisions, and between male and
female meiosis.
Cellular localization
Nuclear
Comment
2-methoxyestradiol induces spindle aberrations, chromosome congression failure, and nondisjunction in mouse oocytes. Eichenlaub-Ritter U et al. 2-Methoxyestradiol (2-ME) is a metabolite of 17beta-estradiol and a natural component of follicular fluid. Local concentrations of 2-ME may be increased by exposure to environmental pollutants that activate the expression of enzymes in the metabolic pathway from 17beta-estradiol to 2-ME. It has been suspected that this may have adverse effects on spindle formation in maturing oocytes, which would affect embryo quality. To study the dose-response patterns, we exposed denuded mouse oocytes to 2-ME during in vitro maturation. Meiotic progression, spindle morphology, centrosome integrity, and chromosome congression were examined by immunofluorescence and noninvasive polarizing microscopy (PolScope). Chromosomal constituents were assessed after spreading and C-banding. 2-ME sustained MAD2L1 expression at the centromeres and increased the number of meiosis I-blocked oocytes in a dose-dependent manner. 2-ME also caused dramatic dose-dependent increases in the hyperploidy of metaphase II oocytes. Some of these meiosis II oocytes contained anaphase I-like chromosomes, which suggests that high concentrations of the catecholestradiol interfere with the physical separation of chromosomes. Noninvasive PolScope analysis and tubulin immunofluorescence revealed that perturbations in spindle organization, which resulted in severe disturbances of the chromosome alignment at the spindle equator (congression failure), were caused by 2-ME at meiosis I and II. Pericentrin-positive centrosomes failed to align at the spindle poles, and multipolar spindles and prominent arrays of cytoplasmic microtubule asters were induced in 2-ME-exposed metaphase II oocytes. In conclusion, a micromolar level of 2-ME is aneugenic for mammalian oocytes. Therefore, exposure to 2-ME and conditions that increase the intrinsic local concentration of 2-ME in the ovary may affect fertility and increase risks for chromosomal aberrations in the oocyte and embryo.
Gene expression profiles of single human mature oocytes in relation to age. Gr?l ML et al. BACKGROUND The development competence of human oocytes declines with increasing age. The objective of this study was to investigate the effect of age on gene expression profile in mature human oocytes. METHODS mRNA was isolated for whole genome gene expression microarray analysis from metaphase II (MII) oocytes donated by IVF or ICSI patients, 10 women aged <36 years (younger) and five women aged 37-39 years (both inclusive) (older), undergoing controlled ovarian stimulation. The oocytes were donated and prepared immediately after recovery from the follicle. RT-PCR on additional four younger and two older oocytes confirmed the array analysis. RESULTS On the basis of 15 independent replicates of single MII oocytes, 7470 genes (10 428 transcripts) were identified as present in the MII oocytes. Of these, 342 genes showed a significantly different expression level between the two age groups; notably, genes annotated to be involved in cell cycle regulation, chromosome alignment (e.g. MAD2L1 binding protein), sister chromatid separation (e.g. separase), oxidative stress and ubiquitination. The top signaling network affected by age was 'cell cycle and organism development' (e.g. SMAD2 and activin B1 receptor). CONCLUSION There is a substantial difference between younger and older oocytes in the transcriptional level of genes involved in central biological functions of the oocytes, thus providing information on processes that may be associated with the ageing phenomenon and possibly contributing to decreased fertility.
Metaphase I arrest upon activation of the Mad2-dependent spindle checkpoint in mouse oocytes
Wassmann K, Niault T, Maro B .
BACKGROUND: The importance of mitotic spindle checkpoint control has been well established during somatic cell divisions. The metaphase-to-anaphase transition takes place only when all sister chromatids have been properly attached to the bipolar spindle and are aligned at the metaphase plate. Failure of this checkpoint may lead to unequal separation of sister chromatids. On the contrary, the existence of such a checkpoint during the first meiotic division in mammalian oocytes when homologous chromosomes are segregated has remained controversial. RESULTS: Here, we show that mouse oocytes respond to spindle damage by a transient and reversible cell cycle arrest in metaphase I with high Maturation Promoting Factor (MPF) activity. Furthermore, the mitotic checkpoint protein Mad2 is present throughout meiotic maturation and is recruited to unattached kinetochores. Overexpression of Mad2 in meiosis I leads to a cell cycle arrest in metaphase I. Expression of a dominant-negative Mad2 protein interferes with proper spindle checkpoint arrest. CONCLUSIONS: Errors in meiosis I cause missegregation of chromosomes and can result in the generation of aneuploid embryos with severe birth defects. In human oocytes, failures in spindle checkpoint control may be responsible for the generation of trisomies (e.g., Down Syndrome) due to chromosome missegregation in meiosis I. Up to now, the mechanisms ensuring correct separation of chromosomes in meiosis I remained unknown. Our study shows for the first time that a functional Mad2-dependent spindle checkpoint exists during the first meiotic division in mammalian oocytes.
Mad2 is required for inhibiting securin and cyclin B degradation following spindle depolymerisation in meiosis I mouse oocytes Homer HA, et al .
Mad2 is a pivotal component of the spindle assembly checkpoint (SAC) which inhibits anaphase promoting complex/cyclo-some (APC/C) activity by sequestering Cdc20 thereby regulating the destruction of securin and cyclin B. During mitosis, spindle depolymerisation induces a robust Mad2-dependent arrest due to inhibition of securin and cyclin B destruction. In contrast to mitosis, the molecular details underpinning the meiosis I arrest experienced by mouse oocytes exposed to spindle depolymerisation remain incompletely characterised. Notably, the role of Mad2 and the fate of the anaphase-marker, securin, are unexplored. As shown previously, we find that spindle depolymerisation by nocodazole inhibits first polar body extrusion (PBE) and stabilises cyclin B and cyclin-dependent kinase 1 activity in mouse oocytes. Here we show that stabilisation of cyclin B in nocodazole can be sustained for several hours and is associated with stabilisation of securin. These effects are SAC-mediated as, in oocytes depleted of the majority of Mad2 by morpholino antisense, securin and cyclin B are destabilised and 15% of oocytes undergo PBE. This reflects premature APC/C activation as a mutant form of cyclin B lacking its APC/C degradation signal is stable in Mad2-depleted oocytes. Moreover, homologues do not disjoin during the prolonged meiosis I arrest (> 18 h) induced by nocodaozole indicating that a non-cleavage mechanism is insufficient on its own for resolution of arm cohesion in mammalian oocytes. In conclusion, when all kinetochores lack attachment and tension, mouse oocytes mount a robust Mad2-dependent meiosis I arrest which inhibits the destruction of securin and cyclin B.
RNA Interference as a tool to study the function of MAD2 in mouse oocyte meiotic maturation. Wang JY et al. Spindle checkpoint proteins control entry into anaphase and chromosome segregation. As a member of spindle checkpoint proteins, MAD2 takes a central role in the regulation of anaphase onset and genome integrity. Here, we used MAD2 siRNA transfection approach to study MAD2 functions during mouse oocyte meiotic maturation in vitro. Real-time PCR and laser scanning confocal microscopy showed that we successfully downregulated MAD2 transcript and protein expression. We further demonstrated that MAD2 downregulation resulted in a shortened duration of meiosis I and meiotic spindle abnormality, suggesting the function of MAD2 in mouse oocyte meiotic maturation. We also showed that MAD2 interference to some extent decreased GVBD rate, but increased apoptosis in mouse oocytes. In conclusion, our study shows that siRNA transfection is an effective tool to study MAD2 functions, and our results provide further evidence for the role of MAD2 as a spindle checkpoint protein in mouse oocytes. Mol. Reprod. Dev. (c) 2006 Wiley-Liss, Inc.
Role of Filia, a maternal effect gene, in maintaining euploidy during cleavage-stage mouse embryogenesis. Zheng P et al. During oogenesis, mammalian eggs accumulate proteins required for early embryogenesis. Although limited data suggest a vital role of these maternal factors in chromatin reprogramming and embryonic genome activation, the full range of their functions in preimplantation development remains largely unknown. Here we report a role for maternal proteins in maintaining chromosome stability and euploidy in early-cleavage mouse embryogenesis. Filia, expressed in growing oocytes, encodes a protein that binds to MATER and participates in a subcortical maternal complex essential for cleavage-stage embryogenesis. The depletion of maternal stores of Filia impairs preimplantation embryo development with a high incidence of aneuploidy that results from abnormal spindle assembly, chromosome misalignment, and spindle assembly checkpoint (SAC) inactivation. In helping to ensure normal spindle morphogenesis, Filia regulates the proper allocation of the key spindle assembly regulators (i.e., AURKA, PLK1, and gamma-tubulin) to the microtubule-organizing center via the RhoA signaling pathway. Concurrently, Filia is required for the placement of MAD2, an essential component of the SAC, to kinetochores to enable SAC function. Thus, Filia is central to integrating the spatiotemporal localization of regulators that helps ensure euploidy and high-quality cell cycle progression in preimplantation mouse development. Defects in the well-conserved human homologue could play a similar role and account for recurrent human fetal wastage.
Expression regulated by
Comment
Ovarian localization
Oocyte
Comment
Intra-oocyte Localization of MAD2 and Its Relationship with Kinetochores, Microtubules, and Chromosomes in Rat Oocytes During Meiosis Zhang D, et al .
The present study was designed to investigate subcellular localization of MAD2 in rat oocytes during meiotic maturation and its relationship with kinetochores, chromosomes, and microtubules. Oocytes at germinal vesicle (GV), prometaphase I (ProM-I), metaphase I (M-I), anaphase I (A-I), telophase I (T-I), and metaphase II (M-II) were fixed and immunostained for MAD2, kinetochores, microtubules and chromosomes. The stained oocytes were examined by confocal microscopy. Some oocytes from GV to M-II stages were treated by a microtubule disassembly drug, nocodazole, or treated by a microtubule stabilizer, Taxol, before examination. Anti-MAD2 antibody was also injected into the oocytes at GV stage and the injected oocytes were cultured for 6 h for examination of chromosome alignment and spindle formation. It was found that MAD2 was at the kinetochores in the oocytes at GV and ProM-I stages. Once the oocytes reached M-I stage in which an intact spindle was formed and all chromosomes were aligned at the equator of the spindle, MAD2 disappeared. However, when oocytes from GV to M-II stages were treated by nocodazole, spindles were destroyed and MAD2 was observed in all treated oocytes. When nocodazole-treated oocytes at M-I and M-II stages were washed and cultured for spindle recovery, it was found that, once the relationship between microtubules and chromosomes was established, MAD2 disappeared in the oocytes even though some chromosomes were not aligned at the equator of the spindle. On the other hand, when oocytes were treated with Taxol, MAD2 localization was not changed and was the same as that in the control. However, immunoblotting of MAD2 indicated that MAD2 was present in the oocytes at all stages; nocodazole and Taxol treatment did not influence the quantity of MAD2 in the cytoplasm. Significantly higher proportions of anti-MAD2 antibody-injected oocytes proceeded to premature A-I stage and more oocytes had misaligned chromosomes in the spindles. The present study indicates that MAD2 is a spindle checkpoint protein in rat oocytes during meiosis. When the spindle was destroyed by nocodazole, MAD2 was reactivated in the oocytes to overlook the attachment between chromosomes and microtubules. However, in this case, MAD2 could not check unaligned chromosomes in the recovered spindles, suggesting that a normal chromosome alignment is maintained only in the oocytes without any microtubule damages during maturation.
Follicle stages
Comment
Post-ovulatory aging of mouse oocytes leads to decreased MAD2 transcripts and increased frequencies of premature centromere separation and anaphase.
Steuerwald NM, et al .
Numerous cytological and biochemical alterations occur as mammalian oocytes age post-ovulation. Some of these changes can predispose cells to aneuploidy. The objective of this study was to test the hypothesis that the level of MAD2 spindle assembly checkpoint (SAC) transcripts decrease as mouse oocytes age post-ovulation and that this decrease was associated with chromosome missegregation. Female Institute of Cancer Research (ICR) mice were superovulated and oocytes collected at 14 h, 19 h and 24 h post-HCG for cytogenetic and quantitative real-time rapid cycle fluorescent RT-PCR analyses. Premature centromere separation (PCS) is now generally recognized as a predisposition to aneuploidy. The data showed that the frequencies of PCS-incomplete (PCS-I) did not significantly (P > 0.05) increase with time post-ovulation; whereas the proportions of oocytes displaying PCS-complete (PCS-C) and premature anaphase (PA) were significantly (P < 0.01) greater at 19 h and 24 h post-HCG, respectively. The higher frequencies of PCS-C and PA found at 19 h and 24 h coincided with decreased levels of MAD2 transcripts at these same times. Although the decline in MAD 2 transcripts with oocyte aging represents only one of many potential mechanisms responsible for aneuploidy, a compromised SAC appears to have a role in the unfavourable reproductive outcome associated with post-ovulatory aged oocytes.