Molecular Human Reproduction, Vol. 7, No. 1, 49-55,
January 2001
© 2001 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
Association between spindle assembly checkpoint expression and maternal age in human oocytes
1 Gamete and Embryo Research Laboratory, Institute for Reproductive Medicine and Science of Saint Barnabas, West Orange, NJ 07052, 2 Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223, and 3 Department of Biological Sciences, Florida International University, Miami, FL 33199, USA
Abstract
The spindle assembly checkpoint modulates the timing of anaphase initiation in response to the improper alignment of chromosomes at the metaphase plate. If defects are detected, a signal is transduced to halt further progression of the cell cycle until correct bipolar attachment to the spindle is achieved. The mitotic arrest deficient (MAD2) and budding uninhibited by benomyl (BUB1) genes encode conserved kinetochore-associated proteins believed to be components of the checkpoint regulatory pathway. A failure in this surveillance system could lead to genomic instability that may underlie the increased incidence of aneuploidy in the gametes of older women. To explore this possibility, the concentrations of these transcripts in human oocytes at various stages of maturation were determined by real-time rapid cycle fluorescent reverse transcription–polymerase chain reaction (RT–PCR). The results obtained following quantitative analysis suggest that these messages degrade as oocytes age. Potentially, this may impair checkpoint function in older oocytes and may be a contributing factor in age-related aneuploidy.
BUBI gene/MAD2 gene/oocyte/RT-PCR/spindle assembly checkpoint
Introduction
The proper segregation of chromosomes is a fundamental prerequisite for the orderly completion of cell division. Inappropriate chromosome separation can result in the production of aneuploid cells and abnormal gametes. While aneuploidy in somatic cells is likely to be responsible for the development of various cancers (Hartwell, 1992
), the incidence of aneuploidy in gametes contributes to birth defects and pregnancy loss (Bauld et al., 1974). It has been established that non-disjunction of bivalent chromosomes during oogenesis increases significantly with increasing maternal age (Dailey et al., 1996; Hassold and Sherman, 2000). In order to determine the aetiology of age-related aneuploidy, it will be necessary to identify the molecular signals orchestrating chromosome distribution during human oocyte maturation.
The current model of eukaryotic cell cycle regulation suggests that it is driven by an oscillating biochemical clock which in turn is regulated by surveillance systems called checkpoints (Hartwell and Weinert, 1989). The checkpoints are intended to guard against malfunctions, e.g. DNA damage, monitor spindle integrity and to determine whether the chromosomes are evenly segregated. The probability of preventing aberrant chromosome distribution is increased by delaying anaphase onset when defects are detected. Unattached or unstably attached kinetochores have been implicated as the most likely source of the wait signals produced upon checkpoint activation (Rieder et al., 1995; for review, see Gorbsky, 1997). The kinetochore binds an evolutionarily conserved set of proteins believed to be components of the checkpoint signalling apparatus (Jablonski et al., 1998; reviewed in Straight, 1997). The checkpoint is able to discern whether chromosomes have been appropriately distributed by monitoring kinetochores, whose biochemical composition is believed to change as they migrate toward the spindle equator (Nicklas et al., 1998).
The characterization of two human genes, mitotic arrest deficient (MAD) and budding uninhibited by benomyl (BUB), each encoding kinetochore-associated proteins, has engendered consideration of roles for these genes as regulatory components in the cell cycle pathway (Li and Benezra, 1996; Ouyang et al., 1998). The existence of homologues in organisms as diverse as yeast (Hoyt et al., 1991; Li and Murray, 1991), Xenopus (Chen et al., 1996) and mouse (Taylor and McKeon, 1997) suggests that these genes have been conserved through evolution. These proteins concentrate at unattached kinetochores prior to metaphase but dissipate upon proper chromosome alignment (Li and Benezra, 1996; Jablonski et al., 1998; Ouyang et al., 1998). This pattern of localization together with results from antibody electroporation experiments suggests that these proteins constitute part of the checkpoint signalling apparatus and that they are involved in monitoring the level of kinetochore tension and/or microtubule occupancy (Li and Benezra, 1996; Ouyang et al., 1998; Brady and Hardwick, 2000; Luo et al., 2000; for review, see Straight, 1997).
It remains to be determined how the kinetochore–microtubule interactions are transformed into biochemical information that is transduced to the cell cycle machinery. The unattached kinetochore is believed to broadcast a wait signal, at least in part, by the products of MAD and BUB genes. Potentially, upon briefly binding to kinetochores, checkpoint proteins could become modified and then released to act as soluble inhibitors (Rieder et al., 1997; reviewed in Hardwick, 1998). The cyclosome/anaphase promoting complex (APC), the enzyme that ubiquitinates cyclins targeting them for destruction, is the most likely target of their inhibitory signal (Li et al., 1997). As is customary in many signalling cascades, reversible phosphorylation probably operates at various points of the checkpoint signalling pathway. Phosphorylation of diverse sites and numerous elements of the cyclosome/APC machinery may serve to either activate or inhibit its activity (reviewed in Gorbsky, 1997). Thus, progression through the cell cycle is blocked until bipolar attachment to the spindle is achieved. Upon proper attachment to spindle microtubules, the checkpoint is abrogated and chromosome segregation proceeds (Figure 1).
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Assuming such surveillance systems are operational during oogenesis, it is possible that a breakdown in this signalling mechanism in ageing oocytes could be responsible for the increased incidence in aneuploidy. Accordingly, we have undertaken this study in order to investigate the level of expression of these genes during oocyte maturation. The presence of MAD2 and BUB1 transcripts were examined and quantified by real-time rapid cycle fluorescent reverse transcription–polymerase chain reaction (RT–PCR). The suitability of this technique for the analysis of gene expression in individual oocytes and embryos has been established (Steuerwald et al., 1999
). In addition, the effect of maternal age on the concentration of these transcripts at various stages of maturation was evaluated. Materials and methods
Oocytes
Human oocytes deemed non-viable material were obtained from patients undergoing assisted reproduction at The Institute for Reproductive Medicine and Science of Saint Barnabas, West Orange, USA, following written consent and Institutional Review Board approval. A total of 80 oocytes was used in this study. These consisted of 35 discarded immature oocytes [germinal vesicle (GV) or meiosis I following germinal vesicle breakdown (MI)] and 45 mature oocytes [meiosis II (MII)] that failed to fertilize following insemination. The oocytes from younger (maternal age <36 years) versus older patients (maternal age >36 years) were evenly distributed in all categories analysed.
Sample collection
GV oocytes unsuitable for assisted fertilization procedures were collected on the day of retrieval since additional time in culture might promote in-vitro maturation. MI and MII oocytes were collected the next day following fertilization check. Individual oocytes were washed through three successive drops of phosphate-buffered saline (PBS; Sigma, St Louis, MO, USA) supplemented with 0.1% polyvinylpyrrolidone (PVP; Sigma), collected in 1 µl of media and frozen at –70°C pending further analysis.
Primer and probe design
Complementary DNA PCR primers were designed using Oligo primer analysis software (National Biosciences Inc, Plymouth, MN, USA) from DNA and RNA sequences obtained from GenBank (Benson et al., 1998) for human MAD2 (Accession number AJ000186), BUB1 (Accession number AF047471) and ß-actin (Ponte et al., 1984). Primer (Gibco BRL, Grand Island, NY, USA) sequences are presented in Table I.
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Quantitative RT–PCR
Protocols used for RNA isolation, first-strand cDNA synthesis and quantitative RT–PCR are detailed elsewhere (Steuerwald et al., 1999
, 2000). PCR was performed using a Light-cyclerTM (Wittwer et al., 1997a), a combination microvolume fluorimeter and rapid temperature cycler (Idaho Technology Inc, Idaho Falls, ID, USA). Template from single oocytes was amplified by 40 cycles of denaturation for 0 s at 95°C, annealing of primers at 50°C (for MAD2) or 48°C (for BUB1) for 0 s and extension at 72°C for 10 s. Fluorescence was acquired at each cycle in order to determine the threshold cycle or the cycle during the log-linear phase of the reaction at which fluorescence rises above background for each sample (Figure 2). The LightcyclerTM quantification software generates a best-fit line and determines unknown concentrations by interpolating the noise-band intercept of an unknown sample against the standard curve of known concentrations (Wittwer et al., 1997b).
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Statistical analysis
To examine the linear relationship between mRNA concentrations and maternal age, simple linear regression was used. Statistical analysis was performed using WINKS Basic Edition, version 4.5 (TexaSoft, Cedar Hill, TX, USA).
Results
Successful amplification from control-derived cDNA was confirmed prior to the inclusion of each sample in the study. In addition, the extent of material loss during RNA isolation was evaluated by comparison to a `reverse transcription only' control containing the identical copy number of the exogenous template added to each sample. The standard curves and best-fit lines were generated from four points run in triplicate spanning the anticipated unknown values. The threshold values of the triplicate standards were very consistent both within and between reactions. Every unknown sample was run in triplicate.
Results following the quantitative analysis of the human MAD2 gene are presented in Table II. Oocytes were examined at the GV, MI and MII stages of maturation. The numbers reflect the overall average value calculated from triplicate samples grouped by maternal age and stage of maturation. The numbers computed for individual replicate values were very reproducible. However, some variation was observed between individual human samples. Nevertheless, when the mRNA concentration for individual oocytes was plotted against maternal age by stage of maturation, a negative relationship became apparent. Regression analysis confirmed that a linear correlation exists between these two variables for all stages examined (Figure 3). The slopes of the regression lines were significantly different from zero (P = 0.044 for GV; P < 0.001 for MI; P = 0.017 for MII). Residual plots were inspected for the existence of patterns.
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Copy numbers obtained following examination of the human BUB1 gene are presented in Table III
. Quantitative analysis was performed on oocytes at the GV, MI and MII stages of maturation. Again, data were averaged by age and stage of maturation. Likewise, a trend emerged when the transcript copy numbers for each oocyte were plotted versus maternal age but only for GV and MI stages. For these stages, a significant (P = 0.043 for GV, P = 0.005 for MI) negative linear relationship was shown to exist (Figure 4).
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In order to determine whether the correlation between the checkpoint genes and maternal age also existed for a housekeeping gene within the GV, MI or MII stage, ß-actin copy numbers versus maternal age were plotted at all three stages of maturation for every individual human oocyte examined (Figure 5
). Contrary to results obtained following the analysis of checkpoint transcripts, no statistically significant association was discernible between maternal age and the concentration of ß-actin mRNA at any stage of maturation. Nonetheless, it should be noted that a decline in ß-actin expression irrespective of maternal age was observed between the GV and MII stage, as predicted by experiments with mouse and human oocytes (Paynton et al., 1988; Bachvarova et al., 1989; Taylor and Pikó, 1990; Steuerwald et al., 2000).
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Discussion
The results obtained following our quantitative analysis of checkpoint transcripts in human oocytes suggest that these messages either remain relatively stable (as is the case for MAD2) or are dramatically down-regulated (as with BUB1) through oocyte maturation. Interestingly, it appears that the steady-state levels of these transcripts at a particular stage of maturation may decrease as the oocyte ages potentially due to degradation over time (Figures 2 and 3
). As expected based on our previous findings (Steuerwald et al., 2000) and consistent with the relevant literature (Paynton et al., 1988; Bachvarova et al., 1989; Taylor and Pikó, 1990), we detected a reduction in ß-actin mRNA content between GV and MII oocytes. However, a correlation between maternal age and ß-actin transcript copy number was not evident at any stage (Figure 4). That is to say, we did not observe a statistically significant age-related decline in copy number at any stage of maturation for ß-actin. Since the concentration of ß-actin mRNA remained fairly stable within each stage of maturation examined with respect to age, it seems that the phenomenon observed with the checkpoint transcripts is not universal of all transcripts in oocytes from older women. Given the role that these checkpoint messages are believed to play in the signalling pathway, it is tempting to conclude that surveillance systems in oocytes may become impaired with advanced maternal age. If so, this is likely to contribute to age-related aneuploidy. However, the existence of such monitoring mechanisms in human oocytes has yet to be confirmed. Also, the link between the decreased expression of these genes and the increased incidence of aneuploidy needs to be established. Nonetheless, the possibility certainly warrants further consideration.
While the requirement for a spindle assembly checkpoint for accurate chromosome segregation in mitosis and for embryonic viability has been demonstrated (Dobles et al., 2000), conflicting results have been reported regarding the existence of checkpoint mechanisms in oocytes. Indeed, their existence has been questioned due to the high rate of chromosomal aberrations routinely observed during preimplantation development (Handyside and Delhanty, 1997). However, recently, the activation of a meiotic checkpoint in response to the persistence of double-stranded DNA breaks during Drosophila oogenesis has been demonstrated (Ghabrial and Schupbach, 1999). Evidence for the existence of meiotic checkpoint controls has also been presented based on experiments with mouse oocytes subjected to combinations of inhibitors or fused at different stages of maturation (Fulka et al., 1994, 1995a). Later, these researchers concluded that such controls were absent due to the failure of damaged or replicating chromatin to halt meiosis in the giant oocytes that they constructed (Fulka et al., 1995b, 1997). Experiments that analysed the effect of chemical mutagens on mouse oocytes have similarly produced contradictory results (Eichenlaub-Ritter and Betzendahl, 1995; Yin et al., 1998). Potentially, surveillance mechanisms in oocytes are not as robust as those present in somatic cells and consequently may be more vulnerable to error, environmental assault or cytoplasmic manipulations. In fact, it has been demonstrated that nucleocytoplasmic density is critical in order to successfully activate the spindle assembly checkpoint when incubating Xenopus oocyte extracts with sperm nuclei (Chen and Murray, 1997). Nonetheless, the lack of an observable delay in anaphase onset in the gametes of XO female mice has lead to the suggestion that oocytes lack a metaphase/anaphase checkpoint altogether (LeMaire-Adkins et al., 1997). However, the incidence of age-related aneuploidy argues strongly against this hypothesis, as such a trend is unlikely to be possible in the complete absence of a checkpoint. Subsequently, these investigators concluded, based on their work with Mlh1 mutant mice, that this checkpoint functions only to detect spindle aberrations during female meiosis (Woods et al., 1999).
More recently, evidence for the existence of a functional spindle checkpoint during meiosis in yeast oocytes has been reported (Shonn et al., 2000; commentary by Sluder and McCollum, 2000). Moreover, these researchers found that a defective checkpoint caused by a mutation in MAD2 reduced the accuracy of chromosome segregation in meiosis I to a far greater extent than in meiosis II. This difference may be a consequence of the specialized manner by which chromosome segregation is accomplished during the first meiotic division. They further proposed that checkpoint defects may be a contributing factor in the incidence of birth defects, e.g. Down's syndrome, which is mostly caused by non-disjunction in meiosis I and is strongly correlated with increased maternal age. They suggested that the loss of the spindle checkpoint in an age-dependent manner might underlie the higher incidence of aneuploidy in the gametes of older women. This theory is certainly consistent with the results presented in our study.
The one caveat worth mentioning is that the quality of the material available for analysis may influence quantitative outcome. Since the human samples examined in this study are non-viable discarded material, they are potentially compromised. Patient aetiology may also play a role. The disparity observed between individual samples may well be attributed to these factors. However, this stipulation is not unique to our analysis, as this is generally the case in any investigation involving spare human material from IVF clinics. Moreover, it should be noted that the expression of ß-actin in our human samples parallels results obtained in a study performed using viable mouse oocytes (Steuerwald et al., 2000) indicating that the material used in the present investigation is suitable for quantitative analysis.
Due to the characterization of checkpoint genes and their transcripts, researchers are beginning to decipher the molecular choreography staged at the kinetochore that mediates the spindle checkpoint. The understanding of this phenomenon may have profound consequences for clinical IVF. The observations reported here provide evidence that the MAD2 and BUB1 transcripts may degrade as the oocyte ages. However, these results do not establish that reduced checkpoint gene expression is responsible for the increased incidence of aneuploidy in older women. Verification of such a hypothesis awaits further investigation, which is currently underway.
Acknowledgments
The authors gratefully acknowledge the efforts of the team of embryologists at the Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center; and Doctors David Sable, Benjamin Sandler, Larry Grunfelt and Patricia Hughes for their support of this study. The authors would also like to thank Dagan Wells for his critical review of the manuscript.
Notes
4 To whom correspondence should be addressed at: Gamete and Embryo Research Laboratory, Institute for Reproductive Medicine and Science of Saint Barnabas, West Orange, New Jersey, 07052, USA. E-mail: nury.steuerwald{at}embryos.net 
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Submitted on July 18, 2000; accepted on October 27, 2000.
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M. Sandalinas, C. Marquez, and S. Munne Spectral karyotyping of fresh, non-inseminated oocytes Mol. Hum. Reprod., June 1, 2002; 8(6): 580 - 585. [Abstract] [Full Text] [PDF]
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 F. Sun, H. Yin, and U. Eichenlaub-Ritter Differential chromosome behaviour in mammalian oocytes exposed to the tranquilizer diazepam in vitro Mutagenesis, September 1, 2001; 16(5): 407 - 417. [Abstract] [Full Text] [PDF]
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