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BOR - Papers in Press, published online ahead of print July 21, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028985
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BIOLOGY OF REPRODUCTION 71, 1724–1729 (2004)
DOI: 10.1095/biolreprod.104.028985
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Nuclear Origin of Aging-Associated Meiotic Defects in Senescence-Accelerated Mice1

Lin Liu3,4,5, and David L. Keefe2,4,5

Department of Obstetrics and Gynecology,4 Brown Medical School and Women & Infants Hospital, Providence, Rhode Island 02905 Laboratory for Reproductive Medicine,5 Marine Biological Laboratory, Woods Hole, Massachusetts 02543


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Factors of both cytoplasmic and nuclear origin regulate metaphase chromosome alignment and spindle checkpoint during mitosis. Most aneuploidies associated with maternal aging are believed to derive from nondisjunction and meiotic errors, such as aberrations in spindle formation and chromosome alignment at meiosis I. Senescence-accelerated mice (SAM) exhibit aging-associated meiotic defects, specifically chromosome misalignments at meiosis I and II that resemble those found in human female aging. How maternal aging disrupts meiosis remains largely unexplained. Using germinal vesicle nuclear transfer, we found that aging-associated misalignment of metaphase chromosomes is predominately associated with the nuclear factors in the SAM model. Cytoplasm of young hybrid B6C3F1 mouse oocytes could partly rescue aging-associated meiotic chromosome misalignment, whereas cytoplasm of young SAM was ineffective in preventing the meiotic defects of old SAM oocytes, which is indicative of a deficiency of SAM oocyte cytoplasm. Our results demonstrate that both nuclear and cytoplasmic factors contribute to the meiotic defects of the old SAM oocytes and that the nuclear compartment plays the predominant role in the etiology of aging-related meiotic defects.

aging, developmental biology, gamete biology, meiosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Meiotic division is unique in that two successive metaphases occur without an intervening interphase to produce gametes (oocyte or sperm). During the first meiotic division (MI), homologous chromosomes segregate, but sister chromatids remain joined. Following arrest at metaphase of the second meiotic division (MII), fertilization causes completion of meiosis by sister chromatid separation. To ensure balanced chromosome segregation, each homologous pair at MI and each sister chromatid pair at MII must align on the metaphase plate of the spindle [1]. Misalignment of metaphase chromosomes could lead to missegregation of chromosomes and aneuploidy. Aneuploidy increases with advancing maternal age and is a major cause of the poor oocyte quality that is responsible for the aging-related decline in female fertility [2–4]. Most aneuploidies associated with maternal aging derive from nondisjunction, chromosome misalignment, or abnormal congression during MI [5–10].

Aberrant meiosis of oocytes from older females could originate from defects in the nucleus, the cytoplasm, or both. For example, reduced chiasmata or reduced cohesion between chromatids during the prolonged prophase I arrest with increasing maternal age may lead to premature chromatid separation, generating aneuploidy [3, 11]. On the other hand, dysfunctional cytoplasm could lead to formation of abnormal meiotic spindles and induce chromosomal malsegregation [8, 12]. Disturbances in mitochondrial distribution induced by diazepam were associated with congression failure of chromosomes and errors in chromosome segregation at meiosis [13].

In a series of elegant studies in the mouse, oocyte reconstruction at the germinal vesicle (GV) stage by nuclear transfer has been successfully employed in assessing the nuclear versus cytoplasmic compartment of oocyte quality during in vitro maturation (IVM), demonstrating that cytoplasmic deficiency is responsible for the poor oocyte quality of IVM and subsequent compromised embryo development [14–16]. By a similar approach, transplanting a nucleus from an older woman's oocyte into cytoplasm of a younger donor has been proposed as a corrective treatment that prevents age-related oocyte aneuploidy resulting from nondisjunction at MI [14, 17–19]. So far, however, no compelling evidence from appropriate animal models supports this approach to actually correct meiotic abnormalities in female aging.

Senescence-accelerated mice (SAM) exhibit early senescence compared to most laboratory strains of mice [20, 21]. Oxidative stress and mitochondrial dysfunction disrupt cellular metabolism in most aging cells [22–25] and have been shown to occur precociously in aging SAM [26–28]. Oxidative stress damages DNA and chromosomes [24, 29] as well as proteins during aging [30], and these could contribute to damage of microtubules or microtubule-based motor proteins that are important for chromosome alignment and checkpoint control [31].

Although we saw no increased meiotic defects in inbred C57BL/6J or hybrid B6C3F1 mice from the age of 2–14 mo (data not shown), meiotic aberration, specifically chromosome misalignment at both metaphase I and II of meiosis, was associated with aging in SAM [32]. Here, we investigated whether nuclear, cytoplasmic, or both factors contribute to meiotic defects in aging SAM and whether cytoplasm from young mice can prevent chromosome misalignment during meiosis that is linked to reproductive aging.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and IVM of Oocytes

The SAM mice were kindly provided by Dr. M. Hosokawa at Kyoto University, Japan. The animal protocol used in the present study was approved by the Animal Care and Use Committees at the Marine Biological Laboratory and Women and Infants Hospital. The isolation and culture of immature oocytes were performed basically following procedures described previously [33]. Cumulus-oocyte complexes were isolated from females 44–48 h after injection of 5 IU of eCG (Calbiochem, La Jolla, CA) by puncturing ovarian follicles. Cumulus-intact oocytes at the GV stage were cultured in minimum essential medium (MEM) (Gibco BRL, Grand Island, NY) containing 10% fetal bovine serum (FBS) and 1 IU/ml of eCG under mineral oil at 37°C in an atmosphere of 7% CO2 in humidified air. Cumulus cells were removed by gentle pipetting to obtain denuded oocytes. The modified IVM medium for denuded oocytes was MEM supplemented with 10% FBS and 0.23 mM pyruvate [34].

Immunofluorescence Microscopy

Tubulin, actin filament, and chromatin were stained and observed by immunostaining and fluorescent microscopy as described previously [35, 36]. Denuded oocytes were fixed and extracted for 30 min at 37°C in microtubule-stabilizing buffer. Oocytes were washed extensively and blocked overnight at 4°C in wash medium (PBS supplemented with 0.02% NaN3, 0.01% Triton X-100, 0.2% nonfat dry milk, 2% goat serum, 2% BSA, and 0.1 M glycine). Afterward, oocytes were incubated with {alpha}- or ß-tubulin mouse monoclonal antibody (1:100; Sigma), washed, and then incubated with fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (1:200; Molecular Probes, OR) at 37°C for 2 h. Some oocytes also 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), which contained 0.5 µg/ml of Hoechst 33342 for DNA staining. The samples were observed using a Zeiss fluorescence microscope (Axioplan 2 imaging), and images were captured by an AxioCam using AxioVision 3.0 software.

Nuclear Transfer

Micromanipulation To facilitate micromanipulation at the GV stage, cumulus cells were removed from cumulus-oocyte complexes by gentle pipetting. Denuded oocytes matured to the MII stage at a rate similar to that of cumulus-oocyte complexes (unpublished data). Furthermore, chromosomes of MII oocytes aligned at the metaphase plate of spindles in both denuded and cumulus cell-enclosed oocytes, demonstrating that cumulus cells are dispensable, at least for nuclear meiotic maturation (see also Fig. 2g). At 20 min before micromanipulation, GV oocytes were incubated in Hepes (25 mM)-buffered IVM medium supplemented with 50 µg/ml of 3-isobutyl-1-methylxanthine (IBMX) and 2 µg/ml of cytochalasin D. This IBMX concentration prevents GV breakdown without affecting oocyte viability and progression to metaphase II [14]. Cytochalasin D increases the oolemmal elasticity necessary for micromanipulation. A beveled, polished micropipette with a spike was used to remove a GV surrounded by the smallest amount of cytoplasm (karyoplast) (see Fig. 2, a–c). As an alternative but similar effective approach, the zona pellucida was cut by pressing a glass microneedle tangentially into the perivitelline space against the holding pipette. Through this opening, the GV nucleus was removed using a polished micropipette with an inner diameter of 20 µm. The isolated GV karyoplast was inserted with the same tool into the perivitelline space of another previously enucleated GV oocyte (GV-cytoplast complex) (see Fig. 2d). The obtained GV-cytoplast complexes were incubated in IVM medium containing IBMX pending electrofusion.



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  		FIG. 2. Validation and efficiency of GV nuclear transfer by micromanipulation and fusion in oocytes of young hybrid B6C3F1 or CD-1 mouse. a–c) GV removal. Shown are a GV oocyte before GV removal (a), insertion of a beveled pipette close to the GV (b), and removal of the GV of a donor oocyte by the beveled pipette (c). Arrow indicates GV. d–f) Reconstitution of GV oocytes. Shown are the GVs placed in close contact with other recipient oocytes whose GVs have been removed (d), 30–60 min during fusion (e), and 1 h after fusion (f). g) Comparison of percentages of oocytes reaching MII (black) and chromosome misalignment (gray; mean ± SEM, three replicates). Bar = 25 µm

Electrofusion The GV-cytoplast complexes were placed in fusion medium (0.28 M mannitol, 0.1 mM CaCl2, and 0.1 mM MgSO4) between the platinum electrodes of a fusion chamber. They were first aligned with an AC pulse of 5 V for 4–5 sec and then fused with a DC electrical pulse of 1.8–2 kV/cm for 50 µsec generated by a BTX 2001 Electro Cell Manipulator (Genetronics, Inc., San Diego, CA). The fusion procedure was repeated three times at an interval of 30 min between pulses. Incorporation of a GV nucleus into the cytoplast was monitored 30 min after each electropulse. Successfully fused, reconstituted GV oocytes were then washed and cultured in IVM medium without IBMX.

Localization of Mitochondria

Mitochondria were identified by loading oocytes with MitoTracker Red (Molecular Probes). Oocytes were stained with 400 nM MitoTracker for 15 min at 37°C and washed, and MitoTracker fluorescence was imaged with a rhodamine filter using the Zeiss inverted fluorescence microscope. Proportion of mitochondria was estimated by measuring relative mitochondrial fluorescence intensity after thresholding the images and subtracting the background using MetaMorph Image Analysis software (Universal Imaging Corporation, West Chester, PA).

Chromosome Analysis

Ovulated oocytes enclosed in cumulus masses were collected from oviduct ampullae at 14 h post-hCG injection of mice primed with 5 IU of eCG. Cumulus cells were removed by gentle pipetting after brief incubation of oocyte-cumulus masses in 0.03% hyaluronidase. Chromosome spreads were prepared as previously described [37]. Briefly, oocytes were transferred to 1% sodium citrate for 20 min, fixed in methanol:glacial acetic acid (3:1), and air-dried. Chromosomes were stained with 0.5 µg/ ml of Hoechst 33342 or 4',6'-diamidino-2-phenylindole, mounted in Vectashield mounting medium, and imaged using a Zeiss fluorescence microscope (Axioplan 2).

Statistical Analysis

Comparison of treatment means was carried out by one-way ANOVA and Fisher protected least-significant difference using StatView software (SAS Institute, Inc., Cary, NC). Percentages were transformed using an arcsin transformation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
At 15–17 h of IVM, most oocytes enclosed in cumulus complexes (93%, n = 89) from young SAM extruded a first polar body and progressed to MII. Furthermore, most MII oocytes (90%, 75/83) of young SAM manifested proper chromosome alignment at the metaphase plate (Fig. 1a). Similarly, most oocytes (88%, n = 85) from old SAM reached MII, with extrusion of a first polar body, to the same extent as young SAM oocytes (P > 0.05). Nevertheless, a majority (74%) of old SAM oocytes exhibited aberrant MII, with chromosome misalignment or dispersion along spindles. Chromosome misalignment could be found over elongated abnormal spindles as well as over intact spindles.



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  		FIG. 1. Immunofluorescence images of spindles and chromosomes, showing metaphase chromosome alignment of meiosis II oocytes. a) Chromosome alignment on the metaphase plate in most oocytes from young SAM but chromosome misalignment in most oocytes from old SAM. b) MII spindles and chromosome alignment of oocytes in vivo matured and ovulated, reconstituted by nuclear transfer (GV NT), IBMX treated for 8 h and of denuded CD-1 mouse oocytes as IVM control. Chromosomes aligned at the metaphase plates of MII spindles. c) Meiosis and chromosome alignment or misalignment on the MII spindles resulting from nuclear transfers of GVs between oocytes of young (y) and old (o) SAM. Controls were oocytes in IBMX for the same period as in nuclear transfer, followed by IVM in normal medium. d) Meiosis resulting from nuclear transfers of GVs between oocytes of old SAM and young hybrid B6C3F1 mice. Chromosome alignment in most oocytes of young B6 or misalignment on the MII spindles in most oocytes of old SAM are shown. Green indicates tubulin, blue chromosomes, and red actin. C, Cytoplasm; N, GV nucleus. Bar = 5 µm (a) and 7.5 µm (b–d)

We also collected in vivo-matured oocytes from ovaries of old SAM before ovulation, 10.5 h after hCG injection of mice primed 44–48 h previously with eCG. The in vivo-matured oocytes also exhibited misalignment of chromosomes at a rate of 65% (n = 23), similar to that of old SAM oocytes matured in vitro, demonstrating that the increased meiotic defects in old SAM oocytes were intrinsic to aging rather than to conditions of the IVM. Thus, GV oocytes of old SAM can mature and progress to MII, but they exhibit increased meiotic aberrations, specifically chromosome misalignment and spindle disruption, in contrast to oocytes of young SAM.

To dissect the relative contributions of nucleus or cytoplasm in the misalignment of metaphase chromosome in aging SAM, we performed reciprocal GV nuclear transfer between old and young SAM oocytes. First, we ensured that the GV nuclear transfer procedure itself does not compromise meiotic division or cause abnormalities in chromosome alignment at meiosis by transferring GVs among oocytes from the commonly used hybrid mouse strains B6C3F1 or outbred CD-1 (Fig. 2, a–f). The efficiency for reconstitution of GV-transferred CD-1 mouse oocytes by electrofusion was 79% ± 4% (n = 159, six replicates, mean ± SD). Reconstituted GV oocytes were matured to MII, and the MII spindles of GV-transferred and IBMX-treated oocytes were comparable to those of in vivo-matured MII oocytes (Fig. 1b). Rates of maturation to MII did not differ between GV-reconstituted, IBMX-treated, and control denuded oocytes matured in vitro without IBMX treatment. Most important, chromosomes aligned properly on metaphase plates of MII spindles after nuclear transfer. Fewer than 9% of reconstituted oocytes showed chromosome misalignment, with no differences among groups (Fig. 2g), demonstrating that removal of cumulus cells, IBMX treatment, and GV transfer do not cause abnormalities in meiotic progression.

To assess the extent to which mitochondria were carried over during GV removal, we also labeled active mitochondria with MitoTracker Red of intact GV oocytes, GV karyoplasts, and enucleated cytoplasts. As a result, the contaminated mitochondria in GV karyoplasts (Fig. 3, arrow) were minimal (~5%).



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  		FIG. 3. Active mitochondrial distribution in intact GV oocytes, GV karyoplasts, and enucleated cytoplasts detected by MitoTracker Red staining. Because the focus was on the GV karyoplast, the mitochondrial stain appeared blurred in the bottom panel. Arrows indicate GV. Bar = 20 µm

Transfer of GV nuclei between oocytes of young and old SAM showed that oocytes from all groups underwent meiotic maturation to MII, with polar body extrusion occurring at a similar frequency (93–98%), regardless of age or nuclear transfer. Most control MII oocytes of young SAM (n = 42) exhibited proper alignment of metaphase chromosomes, with only 20% showing chromosome misalignment and 26% showing abnormal meiosis, including both chromosome misalignment and meiotic arrest before the MII stage (Figs. 2c and 4). By contrast, most oocytes of old SAM (82%, n = 26, P < 0.0001) manifested chromosome misalignment, and some exhibited spindle aberrations. Reconstituted oocytes containing nuclei of old SAM and cytoplasm of young SAM showed pronounced chromosome misalignment (82%, n = 34) and meiotic abnormalities similar to those of old SAM controls (P = 0.94) and significantly higher rates than those of young SAM controls (P < 0.0001). Oocytes reconstituted with young SAM nuclei and old cytoplasm, however, exhibited significantly lower rates (39%, n = 35, P = 0.005) of chromosome misalignment and meiotic abnormality than those of old SAM as controls and of cytoplasm from young SAM reconstituted with nucleus of old SAM, but they also showed higher rates than those of young SAM controls (P = 0.032). It appears that cytoplasm of young SAM is minimally effective in rescuing the meiotic abnormality in old SAM. Moreover, cytoplasm of old SAM also exhibits defects, to some degree, in supporting normal meiosis, including proper chromosome alignment and spindle morphology.



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  		FIG. 4. Nuclear transfers of GVs between oocytes of young (y) and old (o) SAM. Comparison of rates of meiotic maturation to MII (white bar), metaphase chromosome misalignment based on MII oocytes (gray bar), and meiotic defects (black bar), including both chromosome misalignment and arrest before the MII stage, based on total number of successfully reconstituted oocytes examined (mean ± SEM, three replicates). C, Cytoplasm; N, GV nucleus

It is noteworthy that after removal of cumulus cells and treatment with IBMX for 6–8 h to simulate conditions for oocytes used for nuclear transfer, denuded control SAM oocytes exhibited slightly increased meiotic abnormalities compared to intact cumulus-oocyte complexes not subjected to IBMX treatment. This increase may stem from increased sensitivity of denuded SAM oocytes to those treatments. However, as previously shown, oocytes of young B6C3F1 reconstituted by nuclear transfer did not display noticeable differences in meiosis compared to IBMX-treated and denuded control oocytes.

To further assess the nuclear versus cytoplasmic contribution to the meiotic defects associated with aging in SAM, we performed reciprocal nuclear transfer of GV nuclei between old SAM and young B6C3F1 (B6) mice, and vice versa. Oocytes reconstituted with nuclei of old SAM and cytoplasm of young B6 (n = 35) or cytoplasm of old SAM reconstituted with nucleus of young B6 (n = 34) manifested rates of maturation to the MII stage similar (P > 0.05) to those of B6 and SAM control oocytes matured after similar duration of incubation in IBMX (91% and 98%, respectively; n = 49 and 36, respectively; four replicates) (Figs. 2d and 5), again demonstrating that neither IBMX nor nuclear transfer affect meiotic nuclear maturation. Consistently, 84% (30/35) of MII oocytes from old SAM exhibited severe chromosome misalignments, and some exhibited chromosome dispersal and spindle disruption. In contrast, only 2% (1/45, P < 0.0001) of B6 (control) MII oocytes displayed metaphase chromosome misalignment. Under the same conditions of micromanipulation and IVM, oocytes reconstituted from nuclei of old SAM and cytoplasm of young B6 exhibited a lower incidence (63%, P = 0.0436) of severe chromosome misalignment compared to old SAM controls but significantly higher (P < 0.0001) rates than young B6 controls. Oocytes reconstituted from nuclei of young B6 and cytoplasm of old SAM manifested significantly lower rates (27%, P < 0.0001) of less severe chromosome misalignments (Fig. 5) compared to oocytes of old SAM but still higher rates (P = 0.0233) than those of young B6 controls. Interestingly, the combined incidence (90%) of chromosome misalignments in reconstituted oocytes in those two groups was close to that of old SAM controls. Moreover, 18% of reconstituted oocytes containing cytoplasm of old SAM and nucleus of young B6 arrested at MI to telophase I before MII, compared to only 3% in reconstituted oocytes of young B6 cytoplasm with SAM nucleus. Overall meiotic defects, including both those before MII arrest and chromosome misalignments of MII oocytes, exhibited similar trends, as described for those four groups in chromosome misalignments alone (Fig. 5).



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  		FIG. 5. Nuclear transfers of GVs between oocytes of old SAM and young hybrid B6C3F1 mice. Comparison of rates of meiotic maturation to MII (white bar), chromosome misalignment based on MII oocytes (gray bar), and meiotic defects (black bar), including both chromosome misalignment and arrest before the MII stage, based on total number of successfully reconstituted oocytes examined (mean ± SEM, four replicates). C, Cytoplasm; N, GV nucleus

By chromosome analysis, we found that the number of single chromatids (7.0 ± 6.3, n = 12, P < 0.01) was significantly increased in MII oocytes of old SAM (Fig. 6, arrows), compared to that of young SAM (0.7 ± 1.4, n = 11). Furthermore, 11 of 12 metaphase spreads obtained from old SAM exhibited single chromatid separation, whereas only 4 of 11 oocytes from young SAM showed single chromatids. The increased single chromatids in these limited number of analyzed oocytes seemed to coincide with prominent chromosome misalignment, as previously described.



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  		FIG. 6. Representative chromosome spreads of MII oocytes from young and old SAM. Single chromatids (arrows) were frequently seen in oocytes of old SAM. Original magnification x400


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many factors of both cytoplasmic and nuclear origin could influence spindle formation and chromosome alignment. Animal studies support a role for mitochondria and mtDNA in the meiotic apparatus. For instance, the Dip1 mutation in mice, which produces a high incidence of ovulated diploid oocytes, is carried by mitochondria [38]. Mitochondrial dysfunction has been implicated in the pathogenesis and etiology of Down syndrome [39]. Evidence also suggests that chromosomes can control meiotic spindle formation and instructions for proper spindle behavior at MI reside within the chromosomes, not in the spindles (for review, see [40]). Our findings obtained from reciprocal GV nuclear transfer in oocytes between young and old SAM suggest that aging-associated misalignment of metaphase chromosomes and related meiotic abnormalities mostly arise from nuclear defects. Cytoplasm of old SAM also partly contributes to the meiotic aberrations. Experiments with GV nuclear transfer between oocytes of young B6C3F1 and old SAM further support the notion that chromosome misalignments are derived from nuclear defects in the majority of oocytes and from cytoplasmic defects in a small percentage of oocytes of old SAM. However, it does not exclude the possibility that a limited amount of healthy B6 cytoplasm carried over by transfer of B6 GV nuclei may benefit meiosis in oocytes reconstituted with cytoplasm of old SAM.

We have not yet characterized specific nuclear or nuclear-associated factors that are responsible for the meiotic defects, but we speculate that increased premature separation of chromatids in oocytes of old SAM may contribute, in part, to misalignment of metaphase chromosomes, presumably arising from loss of cohesion or centromere damage, because cohesion is concentrated in the centromere region at the MII stage. Single chromatids are frequently seen in MII oocytes of old SAM, in contrast to young SAM or B6 oocytes. Reduction or loss of cohesion between chromosomes and chromatids in bivalents could result in increased premature chromatid separation. Cohesion components play an important role in coordinating reductional division and chromosome fidelity [41]. In MI, homologous chromosomes segregate from each other. Cohesion along chromosome arms must dissolve to allow homologues to separate. In contrast, at centromeres, cohesion must persist until MII. Angell [7] found incorrect distribution of chromosome materials in different patterns of free chromatids and no evidence of extra whole chromosomes in abnormal MII metaphases from human oocytes. It also has been proposed that the loss or reduction of cohesion between chromatids during the prolonged prophase I arrest with increasing maternal age leads to premature chromatid separation, thus generating aneuploidy [11]. In addition, the anaphase-promoting complex activity, securin destruction, and separase activation that are functionally associated with cohesion may have been defective and, thus, resolved centromere cohesion prematurely [42, 43].

Ninety-five percent of children with Down syndrome, a condition associated with advanced maternal age, receive their extra chromosome from their mother, and in 80% or more of these, the nondisjunction occurred in the MI division [12]. Oocytes of old SAM also showed misalignment of single chromosome at MI in addition to marked spindle abnormalities and chromosome misalignments [32]. The disturbance in chromosome alignment at MI could indicate a predisposition to nondisjunction, thus supporting the hypothesis that explains maternal age effects for human aneuploidy based on a reduced number of chiasmata or premature separation of univalents in oocytes of aged females [44, 45]. A clear relationship exists between aging and abnormalities of chromosome/chromatid segregation because of the nondisjunction of bivalents during meiosis [6].

Checkpoints for meiotic chromosome behavior at the metaphase-to-anaphase transition are less efficient in female meiosis [46, 47]. By contrast, during mitosis, the spindle checkpoint arrests cells in response to defects in the assembly of mitotic spindle or to errors in chromosome alignment [48–50]. Many components associated with spindles or chromosomes, such as Bub1, BubR1, STAG3, and SCP3, also play an important role in coordinating meiotic division [41, 51, 52]. For instance, absence of SCP3 promotes aneuploidy in female oocytes by inducing defective meiotic chromosome segregation [53].

We have shown that nuclear or nuclear-associated defects appear to be a major factor that contributes to metaphase chromosome misalignment and meiotic abnormality in reproductive aging of older SAM. The GV nuclear transfer into cytoplasts of young donors in the SAM model proved to be minimally effective in rescuing meiotic defects encountered in older females. However, cytoplasm of young B6C3F1 could rescue meiotic defects that originate from some oocytes of old SAM. Furthermore, cytoplasm of old SAM also contributes to meiotic defects in some oocytes. The present study also demonstrates the utility and significance of nuclear transfer as a potential tool in dissecting nuclear versus cytoplasmic function in normal development as well as in the aging process. It remains to be determined whether nuclear structure, chromosomes, or their associated factors are altered and, thus, directly responsible for the meiotic defects. It is also equally critical to address how cytoplasmic factors and mitochondria may have contributed to aging of the nucleus.


   ACKNOWLEDGMENTS
 
We thank Dr. M. Hosokawa for providing SAM mice and Prof. Ryuzo Yanagimachi, Prof. David Albertini, and Dr. Dongbao Chen for critical reading of the manuscript.


   FOOTNOTES
 
1 Supported in part by Women and Infants Hospital/Brown Faculty Research Fund. Back

2 Correspondence: David Keefe, Laboratory for Reproductive Medicine, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543. FAX: 508 540 6902; dkeefe{at}wihri.org Back

3 Correspondence: Lin Liu, Laboratory for Reproductive Medicine, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543. FAX: 508 540 6902; lliu{at}mbl.edu Back

Received: 25 February 2004.

First decision: 17 March 2004.

Accepted: 1 July 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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