Human Reproduction, Vol. 17, No. 10, 2678-2685,
October 2002
© 2002 European Society of Human Reproduction and Embryology
Ageing-associated aberration in meiosis of oocytes from senescence-accelerated mice
1 Department of Obstetrics and Gynecology, Brown University, Women and Infants Hospital, Providence, RI 02905 and 2 Laboratory for Reproductive Medicine, Marine Biological Laboratory, 7 MBL St, Woods Hole, MA 02543, USA
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BACKGROUND: The senescence-accelerated mouse (SAM) has been shown to exhibit ageing-associated mitochondrial dysfunction and oxidative stress, and early decline in fertility. METHODS: We compared meiotic progression of germinal vesicle oocytes between young (2–3 months) and old (10–14 months) SAM mice using triple immunostaining and fluorescence microscopy as well as Pol-Scope imaging. RESULTS: At 8–9 h of in-vitro maturation (IVM), most young SAM oocytes (86%, 32/37) were at meiosis I (MI) stage, with chromosomes aligned in the mid-region of MI spindles, whereas disrupted MI spindles and/or chromosome misalignments (45%, 18/40) and a few oocytes (20%, 8/40) with abnormal MII spindles were found in old SAM oocytes. At 15–17 h of IVM, old SAM oocytes, despite errors at MI stage, extruded a first polar body at an incidence of 88% (n = 85), which did not differ from that (92%, n = 106) of young SAM oocytes. However, oocytes from old SAM (64%, 32/50) showed aberrant MII, with chromosome misalignment and dispersal, in contrast to normal MII in most young SAM oocytes (87%, 65/75), showing chromosome alignment at the metaphase plate of MII spindles. Moreover, Pol-Scope imaging non-invasively detected disrupted or absent visible spindles and possibly aberrant chromosome alignment. CONCLUSIONS: Spindle disruption and/or chromosome misalignments at both MI and MII are associated with maternal ageing in the SAM mouse. Our findings also suggest that meiotic division lacks a competent checkpoint for spindle integrity and chromosome alignment during reproductive ageing-associated oocyte senescence.
Key words: ageing/meiosis/oocyte/Pol-Scope/senescence-accelerated mouse
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Introduction |
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Poor oocyte quality is a major cause for the ageing-related decline in female fertility (Navot et al., 1991
). It has been well recognized that oocyte and embryo aneuploidy is significantly increased with advancing age in humans (Hassold and Chiu, 1985; Plachot et al., 1988). Most aneuploidies associated with maternal ageing are believed to derive from non-disjunctions and meiotic errors initiated at meiosis I (MI) (Battaglia et al., 1996; Angell, 1997; Volarcik et al., 1998), although the underlying mechanism is not well understood (Nicolaidis and Petersen, 1998; Petersen and Mikkelsen, 2000). In addition, the number of oocytes obtained, even after hormone stimulation, progressively declines with ageing in women (Fitzgerald et al., 1998). By contrast, early work showed that the decline in ageing-associated fertility in most strains of mice was attributable to uterine rather than oocyte dysfunction (Finn, 1966; Talbert and Krohn, 1966). Moreover, non-disjunction has been found only rarely in mouse oocytes during reproductive ageing (Tease, 1982; Golbus, 1983; Sugawara and Mikamo, 1986; Eichenlaub-Ritter and Boll, 1989; Sakurada et al., 1996). Furthermore, age-related decrease in the numbers of ovulated oocytes is not observed in many laboratory mouse strains (Biggers et al., 1962; Harman and Talbert, 1970; Gosden, 1975; Ishikawa and Endo, 1996). These observations suggest that many mouse strains make unsuitable models for addressing the problem of ageing-associated oocyte infertility in humans.
The ideal animal model for ageing-associated oocyte dysfunction would enable investigation of the mechanisms underlying human reproductive ageing-associated non-disjunction and aneuploidy, and infertility. The senescence-accelerated mouse (SAM) is a pure strain developed as a model animal for ageing, as described previously, and has been shown to exhibit early senescence compared with normal mice (Takeda et al., 1981, 1997). SAM mice exhibit mitochondrial dysfunction and oxidative damage early during ageing (Mori et al., 1998; Nakahara et al., 1998). Mitochondrial dysfunction has become a leading theory to explain ageing (Adelman et al., 1988; Shigenaga et al., 1994; Sohal and Weindruch, 1996; De Grey, 1997).
Senescence-prone inbred strains of SAM (SAMP) exhibit precociously decreased litter sizes and reproductive dysfunction (Miyamoto et al., 1995). In our SAMP colony, originally provided by Dr M.Hosokawa at Kyoto University, Japan, SAM failed to breed after 8–9 months of age. However, the use of techniques of in-vivo development and breeding has been unable to distinguish whether depletion of the oocyte pool size versus uterine or male factors contribute to the decline in fertility of old SAM mice. To evaluate the importance of the oocyte and more specifically to investigate whether ageing-associated meiotic errors exist in SAM mice, we collected germinal vesicle (GV) oocytes from young and old SAM and compared their meiotic status in vitro using triple immunostaining and fluorescence microscopy, as well as Pol-Scope imaging. We report that meiotic spindle aberrations and/or chromosome misalignment are associated with ageing SAM. Furthermore, meiotic division lacks an efficient checkpoint for spindle integrity and chromosome alignment during reproductive ageing-associated oocyte senescence. SAM mice may provide a suitable rodent model for studying human age-associated infertility.
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Animals
SAMP mice, abbreviated as SAM, were kindly provided by Dr M.Hosokawa of Kyoto University, Japan. Mice were subjected to a 14 h light:10 h dark light cycle and cared for according to guidelines set by the National Institutes of Health and procedures approved by the Marine Biological Laboratory and Women and Infants Hospital Animal Care Committees.
In-vitro maturation of oocytes
The isolation and culture of immature oocytes were performed following previously described procedures (Eppig and Telfer, 1993). Cumulus–oocyte complexes were isolated from young or old SAM females 44–48 h after injection of 5 IU pregnant mare’s serum gonadotrophin (PMSG; Calbiochem, La Jolla, CA, USA) by puncturing ovarian follicles. Cumulus-intact oocytes at GV stage were cultured in minimum essential medium (MEM) (Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum and 5 IU PMSG/ml, under mineral oil at 37°C in an atmosphere of 7% CO2 in humidified air. The morphology of the oocytes was imaged and the number of oocytes recorded. Nude oocytes without cumulus cell layers were excluded from experiments. When collected for fixation or imaging, oocytes were striped of cumulus cells by brief incubation in 0.03% hyaluronidase and pipetting. All in-vitro manipulations were carried out at 36–37°C on heated stages or chambers.
Immunofluorescence microscopy
Tubulin, actin filament and chromatin were stained and observed by immunostaining and fluorescent microscopy, as described previously (Allworth and Albertini, 1993; Liu et al., 1998). Denuded oocytes were fixed and extracted for 30 min at 37°C in a microtubule-stabilizing buffer (Albertini and Clark, 1981; Allworth and Albertini, 1993). Oocytes were washed extensively and blocked overnight at 4°C in wash medium (phosphate-buffered saline, supplemented with 0.02% NaN3, 0.01% Triton X-100, 0.2% non-fat dry milk, 2% goat serum, 2% bovine serum albumin and 0.1 mol/l glycine). Afterwards, oocytes were incubated with ß-tubulin mouse monoclonal antibody (1:150; Sigma), washed, and then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (1:200; Molecular Probes, OR, USA) at 37°C for 2 h. After washing, samples were stained for actin filaments with Texas Red-conjugated Phalloidin (1:1000; Molecular Probes) for 30 min, washed again and mounted onto a slide under a coverslip in the Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA), with 0.5 µg/ml Hoechst 33342 added to stain DNA. The samples were observed using a Zeiss fluorescence microscope (Axioplan 2 imaging) and images were captured by an AxioCam using AxioVision 3.0 software.
Pol-Scope imaging
Pol-Scope imaging for metaphase spindles were carried out as described previously (Liu et al., 2000). Oocytes were imaged using a Zeiss Axiovert 100 inverted microscope, equipped with a Cohu analogue video camera and Pol-Scope hardware consisting of liquid crystals and electro-optical controller (Cambridge Research Instrumentation, Boston, MA, USA). Settings of the liquid crystals were computer-controlled through MetaMorph Pol-Scope imaging software (Universal Imaging Corp., PA, USA). Oocytes were imaged at 37°C in a plastic Petri dish with a cover glass bottom (MatTek Corp., Ashland, MA, USA).
Statistical analysis
Comparison of treatment means was carried out by analysis of variance and Fisher’s protected least-significant difference (PLSD) using StatView software (SAS Institute Inc., Cary, NC, USA). Percentages were transformed using an arcsin transformation. Significant difference was defined as P < 0.01.
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Results |
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On average, the number of cumulus–oocyte complexes collected from the ovaries of old SAM mice (10–14 months) was significantly less than that of young SAM mice (2–3 months) (13 ± 7, n = 11, and 34 ± 3, n = 6 respectively; P < 0.001), demonstrating decreased ovarian resource in the ageing SAM mouse. After in-vitro maturation (IVM), GV breakdown (GVBD) normally took place within 2–3 h of maturation and >90% maturation to telophase I–MII was observed by 12 h. At 8–9 h of IVM, most young SAM oocytes (86%, 32/37) were at the MI stage, with chromosomes aligned in the mid-region of barrel-shaped MI spindles (Table I
, Figure 1A). Only very few oocytes remained at GV–GVBD stage. Many oocytes (75%, 30/40) from old SAM mice also were at MI stage. However, 18 out of 30 MI oocytes (60%) showed chromosome misalignments over their MI spindles (Figure 1B,C). Some oocytes exhibited disrupted MI spindles. A few oocytes (20%, 8/40) prematurely reached MII stage with abnormal spindles (Table I). Consistent with fluorescence microscopic imaging, Pol-Scope imaging detected the characteristic, barrel-shaped spindles of MI stage in young SAM oocytes (n = 13), and chromosome alignments over the mid-spindles (Figure 2A). In 10 old SAM oocytes imaged by the Pol-Scope, four oocytes showed MI spindles and three exhibited misalignment of at least one chromosome and associated aberrant spindles (Figure 2B,C). Five oocytes were at Telophase I–MII stage with one polar body and premature spindles. The other oocyte showed no spindle. Interestingly, chromosome misalignments in oocytes from old SAM mice were found over both intact and disrupted MI spindles, as identified by fluorescence microscopy or Pol-Scope imaging (Figures 1B,C and 2B,C), suggesting that MI spindles can be formed without alignment of chromosomes. So, chromosome abnormality alone cannot determine the integrity of spindles at MI. On the other hand, disrupted spindles, detected by Pol-Scope imaging, generally indicated chromosome misalignments or defects.
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At 15–17 h of IVM, the majority of oocytes (92%, n = 106) from young SAM mice extruded a first polar body and progressed to MII stage. Most young SAM oocytes (87%, 65/75) showed chromosome alignment at the metaphase plate of MII spindles (Table I
, Figure 3A,A’). Similarly, oocytes (86%, n = 50) from old SAM also reached MII stage with extrusion of a first polar body, the rate of which did not differ (P > 0.05) from that of young SAM oocytes. Nevertheless, old SAM oocytes (64%, 32/50) exhibited aberrant MII, with chromosome misalignment and dispersal along spindles, or with completely disrupted spindles (Figure 3B–D). Again, chromosome misalignment and spreading could be found over disrupted spindles (Figure 3B,B’), elongated abnormal spindles (Figure 3C,C’) or intact spindles in appearance (Figure 3D,D’). When the normal configuration of meiotic spindles was observed, the pole-to-pole spindle length varied among individual oocytes. It appears that abnormalities at meiosis do not prevent meiotic progression from MI to MII, but rather meiosis seems to proceed by default, suggesting that this unique cell cycle lacks a checkpoint for chromosome alignment and spindle integrity, or repair machinery during ageing-associated oocyte senescence.
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We compared images captured by the Pol-Scope of oocytes from young and old SAM after 15–17 h of IVM. In 11 oocytes from young SAM, all oocytes arrested at the MII stage and exhibited spindles with normal configurations (Figure 4A,B). Ten oocytes showed visible alignment of chromosomes at the metaphase plate of the meiotic spindles (Figure 4A’,B’
). Only one oocyte showed poorly aligned chromosomes over the spindles. By contrast, spindle morphology differed in 10 imaged oocytes from old SAM. One oocyte showed no microtubule or spindle structure. Four oocytes showed disrupted spindles (Figure 4C), or slim and slightly elongated spindles (Figure 4D). Another five oocytes showed spindles with normal configuration (Figure 4E). However, seven of 10 imaged oocytes did not show visible alignment of chromosomes at the metaphase plate (Figure 4C’–E’), probably indicating chromosome misalignment or spreading, which was barely seen by the original Pol-Scope images. Again, fluorescence imaging after Hoechst staining for DNA appeared to be the more sensitive assay for chromosome misalignment (Figure 3), although the Pol-Scope is a specific test when disrupted spindles or spindles with abnormal morphology are detected. Without requirement of fixation and staining, Pol-Scope provides near real-time imaging of live oocytes.
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The progression of MI to MII and extrusion of a polar body suggests that cytokinesis was not compromised in ageing SAM mice. Not surprisingly, actin filaments were stained at either MI or MII stages in both young and old SAM oocytes (Figure 5
). In merged images, actin filaments preferentially localized to regions above chromosomes and spindles and around the first polar body. Consistently, chromosome alignments were observed at the metaphase plate in young SAM oocytes (Figure 5A,C), but chromosome misalignments were observed at both MI and MII stages in old SAM oocytes regardless of spindle morphology (Figure 5B,D). At telophase I, an obvious function of actin filaments was evidenced by formation of a contractile ring, resulting in a separation of telophase chromosomes (Figure 5E). In addition, cytofragmentation, including shrunken oocytes that subsequently underwent cytofragmentation, occurred after in-vitro maturation (Table I). The fragmentation described here resembles the classical morphological hallmarks of apoptosis, i.e. apoptotic bodies (Kerr et al., 1972). Obviously, oocyte fragmentation contained chromosome fragmentation, and required organization of microtubules and contraction of actin filaments (Figure 5F).
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Discussion |
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Old SAM produces much fewer cumulus–GV oocyte complexes available for meiotic maturation compared with young SAM. Furthermore, oocytes of old SAM exhibit meiotic aberrations, as evidenced by chromosome misalignment over both MI and MII spindles, and in some cases gross disruption of spindle morphology.
Our findings also suggest that meiotic division lacks an effective checkpoint for chromosome alignment and spindle integrity during reproductive ageing-associated oocyte senescence. In mitosis, the spindle checkpoint arrests cells in response to defects in the assembly of the mitotic spindle or errors in chromosome alignment (Scolnick and Halazonetis, 2000). In ageing SAM, progression from MI to MII occurs, despite chromosome misalignment, further confirming that mammalian female meiosis lacks an efficient metaphase checkpoint control (LeMaire-Adkins et al., 1997; Roeder, 1997), in contrast to mitotic cells (Taylor, 1999; Dobles et al., 2000). The fragile meiotic checkpoint may be a major factor related to ageing-associated infertility in old SAM, similar to ageing humans, in which meiotic chromosomes frequently missegregate, leading to aneuploidies and a high frequency of failed implantation and miscarriage.
The frequency of chromosome abnormalities in human oocytes increases with maternal age (Hassold and Chiu, 1985; Plachot et al., 1988). Increased aberrations in spindle formation and chromosome alignment are also associated with ageing in humans (Battaglia et al., 1996; Volarcik et al., 1998). The frequency of errors in chromosome segregation at the first meiotic division is influenced by donor age in IVM oocytes, as in oocytes undergoing meiotic maturation in vivo (Volarcik et al., 1998). Although ageing-associated decreases in fertility have also been found in mice, age-related abnormality in meiotic spindle organization and chromosome alignment at metaphase of MI has not been recognized. Moreover, the aneuploidy rate of ovulated oocytes does not increase significantly during female ageing in the majority of mice (Zuccotti et al., 1998). Low but significant increases in aneuploidy and premature chromatid segregation have been observed in aged oocytes and embryos of some strains (Brook et al., 1984; Eichenlaub-Ritter and Boll, 1989; Sakurada et al., 1996). Much higher rates of aneuploidy could be found in mice with genetic defects, which contain consistently one or many univalent chromosomes, such as XO mice (Brook, 1983) and Mlh1 mutant mice (Woods et al., 1999). It has been demonstrated that 95% of Down’s syndrome children receive their extra chromosome from their mother, and in 
80% of these the non-disjunction occurred in the first meiotic division, which is completed in the ovary (Gaulden, 1992). In old SAM mice, we did find misalignment of a single chromosome at MI, in addition to marked spindle abnormalities and chromosome misalignments, possibly resulting in aneuploidy. The disturbance in chromosome alignment at MI could indicate a predisposition to non-disjunction, supporting the hypothesis that explains the maternal age effect for human aneuploidy on the basis of depleted pool size and fewer chiasmata or premature separation of univalents in oocytes of aged females (Henderson and Edwards, 1968; Crowley et al., 1979). CBA/Ca mice do have increased aneuploidy in embryos (Brook et al., 1984) and oocytes (Eichenlaub-Ritter and Boll, 1989) although to a much lower extent as compared with the human (Eichenlaub-Ritter, 2000). The severe disturbances seen in old SAM oocytes may mimic better the human situation. Thus, depletion of pool size as well as oxidative damage, which may contribute significantly to the decade-long ageing of the human oocyte, are associated with ageing in this mutant. SAM may be a valuable model to study ageing-associated infertility.
The observation that characteristic features of the first meiotic spindle and chromosome alignment were virtually identical in oocytes between young and old CBA/Ca mice argues against the CBA/Ca mouse as a model for age-related aneuploidy in humans (Eichenlaub-Ritter et al., 1988). Young CBA mouse oocytes maturated in M2 medium exhibited displacement of chromosome alignment and obvious variations in spindle configuration at MI stage and lagging chromosomes at anaphase I, probably indicating asynchronous disjunction of individual chromosomes (Eichenlaub-Ritter et al., 1988). In contrast, young SAM exhibited normal MI spindles when their oocytes were matured in the MEM-based medium used in our study. Moreover, while inbred C57Bl mice or CBA mice exhibit the earliest significant decline in fertility at 10–12 months of age (Harman and Talbert, 1970; Gosden, 1975), SAM showed a dramatic decline in fertility at 8 months of age. In our SAM colony, SAM at the age of 2–6 months breed normally, but by 10 months they no longer produce live offspring. The significantly reduced number of GV oocytes, indicative of physiological ageing of the ovaries, and aberration in spindle morphology and metaphase chromosome alignment after meiotic maturation of oocyte presumably contribute to the infertility of old SAM.
The mechanism underlying aberrant meiosis observed in ageing SAM is not yet clear. Mitochondrial dysfunction and oxidative stress are associated with accelerated ageing in SAM (Butterfield et al., 1998; Mori et al., 1998; Nakahara et al., 1998; Nishikawa et al., 1998). Furthermore, four unique mutations in mitochondrial DNA have been found in SAM, but not in other laboratory strains of inbred mice (Mizutani et al., 2001). Oxidative stress and mitochondrial dysfunction are likely contributors to reproductive ageing as well as somatic cell ageing (Adelman et al., 1988; Shigenaga et al., 1994; Sohal and Weindruch, 1996; Beckman and Ames, 1998; Tarín et al., 1998a,b; Cadenas and Davies, 2000; Finkel and Holbrook, 2000; Liu and Keefe, 2000). Oxidative stress damages DNA and chromosomes (Beckman and Ames, 1998; Limoli et al., 1998) as well as proteins during ageing (Stadtman, 1992; Berlett and Stadtman, 1997), which may involve microtubules and small molecules important for spindle organization. Many chromosome- or kinetochore-associated proteins, such as Mps1, Mad2 and Xkid, have been shown to play a role in chromosome alignment at the metaphase plate of spindles (Antonio et al., 2000; Dobles et al., 2000; Funabiki and Murray, 2000; Abrieu et al., 2001). Oxidative stress has been shown to induce disturbances in chromosomal distribution in the metaphase II spindle of mouse oocytes in vitro (Tarín et al., 1998b). Disturbances in mitochondrial distribution by diazepam were associated with congression failure of chromosomes at MI (Sun et al., 2001) and errors in chromosome segregation at anaphase I (Yin et al., 1998a). Although it cannot be decided from the present study what is at the basis of the aged phenotype in SAM oocytes, we may speculate that spindle disruption and/or chromosome misalignments found in old SAM meiosis could result from mitochondrial dysfunction and cytoplasmic deficiency and/or oxidative stress damages directly or indirectly to DNA and spindles.
We found chromosome misalignments over both disrupted meiotic spindles and intact spindles in ageing SAM. This observation demonstrates that meiotic spindles can form in the absence of normal chromosome alignment. Progression to metaphase II and permissive anaphase checkpoint was also noted in chemically-exposed oocytes failing to congress chromosomes at the equatorial plane during MI in response to pesticide (Yin et al., 1998b). The observations of abnormal MI in female Mlh1 mutant mice suggest that chromosomes can influence meiotic spindle assembly (Woods et al., 1999). DNA damage and chromosome aberration alone could explain chromosome misalignment associated with disrupted spindles, but not the chromosome misalignment associated with normal spindles. The misalignment of chromosomes may rather signal congression failure, although it also seems likely that chromosome aberrations disrupt the alignments of chromosomes over the meiotic spindles. This might also result from mitochondrial dysfunction, reduced energy supply and compromised activity of microtubule motor proteins. The observed spindle disruption does not necessarily imply that chromosome aberration prevents the formation of intact spindles. It is possible that oxidative stress occurring during ageing may have damaged both DNA and microtubules. However, DNA damage may be more susceptible than microtubules to oxidative stress.
By taking advantage of the Pol-Scope’s ability non-invasively to observe birefringent structures in living cells, including mammalian oocytes (Oldenbourg, 1996; Keefe et al., 1997; Liu et al., 2000), we imaged spindles from both young and old SAM oocytes of meiotic maturation. We found not only disruption of spindles and chromosome misalignment at MI stage, but also spindle disruption in MII oocytes from old SAM. This observation provides us with the possibility that certain abnormalities in DNA or chromosomes at metaphase will be able to be diagnosed by non-invasively observing spindle morphology with Pol-Scope imaging. Human oocytes with visible spindles produced fertilization at a higher rate than those without spindles, imaged by Pol-Scope (Wang et al., 2001). Considering the fact that the Pol-Scope has successfully differentiated oocytes with visible or no spindles, and that oocytes with disrupted spindles show misalignments of metaphase chromosomes or chromosome dispersal, Pol-Scope may prove useful for the selection of healthy oocytes for human IVF. We now are trying to improve the resolution of Pol-Scope images to be able to distinguish normal chromosome alignment from misalignment, even with a normal-appearing MII spindle.
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We thank Dr M.Hosokawa at Kyoto University, Japan for providing SAM mice. This work was supported in part by the National Institutes of Health (NIH K081099) and Women and Infants Hospital/Brown Faculty Research Fund.
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3 To whom correspondence should be addressed. E-mail: dkeefe{at}wihri.org
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Submitted on March 11, 2002; accepted on May 23, 2002.
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