Transfer of germinal vesicle to ooplasm of young mice could not rescue ageing-associated chromosome misalignment in meiosis of oocytes from aged mice

Long-Bo Cui1,2, Xiu-Ying Huang1 and Fang-Zhen Sun1,3

1 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080 and 2 Department of Biology, Yantai University, Yantai 264005, China

3 To whom correspondence should be addressed. Email: fzsun{at}genetics.ac.cn


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUD: Transferring a germinal vesicle (GV) from an aged woman's oocyte into ooplasm from a younger woman has been proposed as a possible way to overcome the problem of age-related decline in female fertility. Here we assessed this possibility by determining whether ooplasts derived from young mice could rescue ageing-associated chromosome misalignment in meiosis of oocytes from aged mice. METHODS: Three groups of reconstructed oocytes, young GV–young cytoplast (group YY), aged GV–young cytoplast (group AY), and young GV–aged cytoplast (group YA), were created by micromanipulation and electrofusion. RESULTS: Nuclear transplantation was successful in 89.8–94.4% of GV–ooplast complexes, and maturation rate of the reconstructed oocytes was 93.5–97.9%. Confocal microscopy analysis showed a significantly higher rate (49.2%) of chromosome misalignment in ageing mice than in young mice (16.9%), and 57.1% of oocytes in group AY exhibited chromosome misalignment, while the abnormality rate in groups YY and YA was 16.3 and 16.7% respectively. Calcium imaging showed that the three groups of reconstructed oocytes exhibited a similar pattern of calcium oscillations upon stimulation with bovine sperm extracts. Fertilization rate and developmental capacity to 2-cell embryos were also similar among the three groups of oocytes. CONCLUSIONS: Our findings suggest that: (i) the ooplasm from young mice could not rescue ageing-associated chromosome misalignment in meiosis of GV from aged mice; and (ii) behaviour of chromosome alignment over metaphase spindle is predominantly determined by GV material.

Key words: ageing/Ca2+ oscillations/chromosome/germinal vesicle transfer/oocyte


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Ageing-related decline in female fertility is a common phenomenon in older women (Tietze, 1957Go; Keefe, 1998Go). Maternal age has been shown to affect oocyte quality and early development (Navot et al., 1991Go). Aneuploidy in oocytes and embryos is significantly increased with advancing age in humans (Hassold and Chiu, 1985Go; Plachot et al., 1988Go). Most aneuplodies associated with maternal ageing are believed to derive from non-disjunction and meiotic errors initiated at meiosis I (Battaglia et al., 1996Go; Volarcik et al., 1998Go; Liu and Keefe, 2002Go). Abnormal oocyte spindle morphology is also related to female infertility. Spindle abnormalities, such as abnormal chromosome alignment and a microtubule matrix that compromises the meiotic spindle function, have been attributed to advanced maternal age (Battaglia et al., 1996Go). However, it is not clear whether abnormal distribution of chromosomes between oocytes and polar body always results from an error in spindle behaviour per se. Segregation of chromosomes appears to be controlled by the meiotic spindle, but its components are largely supplemented by the ooplasm. Therefore, it has been considered that dysfunctional cytoplasmic factor(s) is responsible for structural abnormalities of meiotic spindles, which lead to eventual chromosomal malsegregation (Gaulden, 1992Go; Battaglia et al., 1996Go).

Germinal vesicle (GV) can be effectively removed by micromanipulation and nuclear transfer is a useful technique for studying nuclear–cytoplasmic interaction in mammalian oocytes during meiotic maturation (Sun and Moor, 1991Go; Meng et al., 1996Go). Using micromanipulation and electrofusion procedures, it is possible to reconstruct an oocyte by transferring a GV from one oocyte into a cytoplast derived from an enucleated donor oocyte at the same developmental stage (Liu et al., 1999Go; Takeuchi et al., 1999Go, 2001Go; Zhang et al., 1999Go; Li et al., 2001Go; Moffa et al., 2002Go; Liu and Keefe, 2004Go). It has been proposed that transplanting a GV from an aged woman's oocyte into a younger ooplasm might be a way to reduce the incidence of oocyte aneuploidy (Zhang et al., 1999Go; Takeuchi et al., 1999Go; Palermo et al., 2002Go). In mouse, it has been demonstrated that nuclear transplantation can be accomplished efficiently, and this technique appears not to impair subsequent oocyte maturation or increase the incidence of chromosomal abnormalities (Liu et al., 1999Go; Takeuchi et al., 1999Go). Although maturation, fertilization, preimplantation and full term development in mouse have been established using GV transplantation (Liu et al., 2003Go; Takeuchi et al., 2004Go), it remains unclear whether transfer of GV from aged mice to cytoplasts derived from oocytes of young mice could reduce the incidence of age-associated aberration in meiosis.

Senescence-accelerated mice (SAM) exhibit ageing-associated meiotic defects (Liu and Keefe, 2002Go). A recent study shows that ageing-associated misalignment of metaphase chromosomes is predominately associated with the nuclear factors in the SAM model (Liu and Keefe, 2004Go). In the present study, conducted with a mouse strain which shows ageing-associated chromosome misalignments in meiotic oocytes, we attempted to create, by germinal vesicle transfer, different combinations of reconstructed GV oocytes derived from young and aged mice. We examined oocyte maturation, chromosome and spindle morphology, calcium signalling patterns and developmental capacity of these reconstituted oocytes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Collection of mouse GV oocytes
The mouse strain used throughout this study is the Kunming (KM) white albino (bred in the CAS Institute of Genetics and Developmental Biology, Beijing). Female KM mice of various ages were superovulated by a single i.p. injection with 5 IU pregnant mare serum gonadotrophin (Sigma, USA). Immature GV oocytes were collected by puncturing the ovarian follicles at 44–48 h post-injection and attached cumulus cells were dissociated by repeated pipetting. GV stage oocytes were cultured in human tubal fluid (HTF) medium (Irvine Scientific, USA) supplemented with 10% fetal calf serum (FCS; HyClone, USA) and 50 µg/ml 3-isobutyl-1-methylxanthine (IBMX; Sigma) for 2 h to prevent spontaneous GVBD and to develop a perivitelline space.

Micromanipulation: preparation of karyoplasts and cytoplasts for GV transfer
Preparation of karyoplasts and cytoplasts for GV transfer was conducted as described by Takeuchi et al. (1999)Go and Liu et al. (1999)Go. Briefly, GV stage oocytes were exposed to modified HTF medium supplemented with 10% FCS, 50 µg/ml IBMX and 7.5 µg/ml cytochalasin B for 30 min at room temperature before micromanipulation. Following lancing of the zona pellucida with a sharp-tripped pipette, the GV was gently aspirated into a cylindrical micropipette with an inner diameter of 20 µm. Each GV was surrounded by a small amount of cytoplasm (karyoplast), and appeared to be encapsulated by a membrane. Cytoplasts were obtained by enucleating GV stage oocytes with the same procedure. Karyoplasts were transferred individually into the perivitelline space of the previously prepared cytoplasts by microinjection, and the obtained GV–cytoplast complexes were incubated for 30 min in M2 medium (Sigma) at 37 °C, 5% CO2 prior to electrofusion.

Electrofusion of GV–cytoplast complexes
An Electro Cell Manipulator (BTX 200; BTX Inc., USA) was used for the fusion. Each GV–cytoplast complex was placed in M2 medium (fusion medium) between two platinum electrodes of a fusion chamber. The complex was manually aligned, and then fused with a direct current (d.c.) electrical pulse of 160 V/cm for 90 µs. The incorporation of GV into the cytoplast was monitored 30 min later.

Experimental design
Micromanipulation and electrofusion were used to create the following three groups of reconstructed oocytes: GV from oocytes of young mice (6–8 weeks old), cytoplast from oocytes of young mice (YY); GV from oocytes of young mice, cytoplast from oocytes of aged mice (12 months old) (YA); and GV from oocytes of aged mice, cytoplast from oocytes of young mice (AY).

Maturation, artificial activation and IVF of GV-transferred oocytes
Maturation of GV-transferred oocytes was evaluated after 16–18 h culture in vitro in HTF medium with 10% FCS at 37 °C, 5% CO2. Oocytes displaying a polar body were selected for further experiments. Matured reconstructed oocytes were activated artificially as described by Hagemann et al. (1995)Go. Oocytes were placed in phosphate-buffered saline (PBS) containing 3 µm A23187 (Sigma) for 5 min at room temperature, washed three times in modified HTF medium, and then cultured in HTF medium supplemented with 10% FCS and 7 µg/ml cycloheximide (Sigma) for 6–7 h. The reconstructed oocytes were then cultured in vitro in HTF medium with 10% FCS at 37 °C, 5% CO2, and monitored 4 h later for activation as indicated by the presence of a female pronucleus and 24 h later for 2-cell embryos.

Matured reconstructed oocytes were fertilized in vitro as described by Hogan et al. (1986)Go. Sperm were collected from the cauda epididymides of male mice and capacitated in IVF medium (Hogan et al., 1986Go) containing 15 mg/ml bovine serum albumin (BSA) for 1.5 h. Oocytes were incubated with the sperm in IVF medium with 15 mg/ml BSA for 6 h. The reconstituted oocytes were then cultured in vitro in HTF medium with 10% FCS at 37 °C, 5% CO2, and monitored 6 h later for activation as indicated by the presence of pronuclei and 24 h later for 2-cell embryos.

Microinjection
Preparation of bovine sperm extracts was conducted following the procedures described by Tang et al. (2000)Go. Briefly, the sperm were suspended in PBS and then centrifuged at 5000 g for 10 min. After three repeated washings in PBS, the sperm were then washed into an extracting buffer (120 mmol/l KCl, 20 mmol/l HEPES, 200 µmol/l EDTA, 0.05% Brij 35, 300 mmol/l phenylmethylsulphonyl fluoride, 10 mmol/l leupeptin, pH 7.3), and lysed by sonication using an ultrasonic homogenizer (Cole Parmer, USA). Final concentration was equivalent to two or three sperm per picolitre.

Microinjection was conducted following the procedures described by Tang et al. (2000)Go. Bovine sperm extracts were pressure-injected into the cytoplasm of metaphase II (MII) oocytes in Ca2+ -free H6 medium. The volume injected was 2 pl (total volume of oocytes was estimated at 200 pl). Immediately after injection, the oocytes were moved to an imaging system chamber to detect intracellular Ca2+ changes.

Calcium measurement
Oocytes were loaded with 2 µmol/l fura-2/AM (Molecular Probes Inc., USA) for 30 min in H6 medium at 37 °C immediately before measurement (Deng et al., 1998Go). After loading, the cells were washed three times in H6 and then transferred to a chamber containing H6 medium covered by light paraffin oil. The chamber was placed in a well on the stage of a Nikon Diaphot 200 inverted epifluorescence microscope (Nikon Instruments, USA) for imaging, and maintained at 37 °C by a thermostatic controller (Life Sciences Resources, UK). The system used for calcium measurements was a MiraCal imaging system equipped with MiraCal Version 2.3 Software (Life Sciences Resources, UK). The emitted fluorescence intensities at 510 nm were recorded at 340 and 380 nm excitation wavelengths by Mira-1000TE low-light-level CCD camera. The fluorescence signal is displayed as the ratio of fluorescence intensities for the 340 nm/380 nm excitation wavelengths. Calcium was estimated from the ratio equation described by Grynkiewicz et al. (1985)Go, which is calculated by computer simultaneously. Parameters required for the ratio equation were obtained according to Poenie et al. (1985)Go. The calcium image was recorded every 10 s for up to 4 h.

Immunocytochemistry
Reconstructed oocytes extruded first polar bodies were selected for immunocytochemistry of the meiotic spindle. Oocytes were fixed in 3.7% paraformaldehyde (Sigma) in 0.1 mol/l PBS for 40 min, and then permeabilized in PBS containing 0.1% Triton X-100 (Sigma) for 30 min at room temperature. They were subsequently washed for 1 h in PBS containing 5% BSA. Oocytes were incubated with anti-{beta}-tubulin mouse monoclonal antibody (1:150; Sigma) overnight at 4 °C, washed, and then incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (1:200; Molecular Probes, USA) at room temperature for 2 h. Hoechst 33258 (Sigma) 5 µg/ml in PBS was included in one of the final washing steps to localize chromosomes.

Confocal fluorescence microscopy was used to obtain the FITC localization patterns using a Nikon Labphot Microscope coupled to a Bio-Rad confocal laser. Hoechst 33258 fluorescence was obtained simultaneously, and optical sections were collected and reproduced on a SPARC workstation. Paired images were digitally reproduced to examine the co-localization of tubulin and chromosomes.

Data analysis
Data were analysed using {chi}2-test and t-test. P<0.05 was considered siginificant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Nuclear transfer efficiency and oocyte maturation rate
Table I summaries efficiency of nuclear transfer and maturation rate of reconstructed and control oocytes. Fusion rate of GV–cytoplast complexes after electrical stimulation showed no significant difference between the three groups (in the range of 89.8–94.4%). Following in vitro culture for 17 h, the maturation rate revealed by first polar body emission was 96.2, 93.5 and 97.9% for groups YY, YA and AY respectively, which was not significantly different from that of the controls (98.0% for the young control, and 99.1% for the aged control).


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Table I. Efficiency of nuclear transfer and maturation rate of reconstructed oocytes

 
Influence of ageing and ooplasm on chromosome and spindle organization
Confocal microscopy analysis (Table II) showed that only 16.9% (11/65) MII oocytes derived from young mice exhibited chromosome misalignment and dispersal over the metaphase plate, whereas MII oocytes derived from aged mice showed a significantly high abnormality rate (49.2%; 32/65) in chromosome alignment. These abnormalities were found over both intact and abnormal spindles (Figure 1).


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Table II. Influence of ageing and ooplasm on chromosome alignment in mouse metaphase II (MII) oocytes

 


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Figure 1. Immunofluorescence images of spindles and chromosomes of (a) oocyte from young GV–aged cytoplast (group YA), (b and c) oocytes from group aged GV–young cytoplast (group AY). (ac) Meiotic II spindles stained by anti-{beta}-tubulin and FITC-conjugated second antibody; (a'c') chromosomes stained by Hoechst 33258. Arrows indicate chromosome accumulation at the plate. Arrowheads indicate misalignment of chromosomes (x400).

 
When GV exchanges were conducted between oocytes from young mice, abnormal chromosome and spindle organization occurred at a rate of 16.3% (8/49), which is similar to that in the young control group. When GV derived from oocytes of young mice were fused with cytoplasts from aged mice, the rate (16.7%; 12/72) of abnormal chromosome and spindle organization was similar to that in MII oocytes of young control mice. When GV derived from oocytes of aged mice were fused with cytoplasts from young mice, 57.1% (36/63) of reconstructed oocytes following maturation exhibited chromosome organization abnormality over both intact and abnormal spindles (Figure 1).

Ca2+ oscillations in reconstructed oocytes induced by bovine sperm extracts
Responses of oocytes to the sperm extract injection were summarized in Table III. Injecting the extracts into matured oocytes induced Ca2+ oscillations in majority of oocytes examined. These oscillations in the injected oocytes persisted for 2–3 h with a mean amplitude of 485±90 to 533±71 nmol/l and a spiking interval of 99±64 to 110±49 s. Moreover, reconstructed oocytes in the three experimental groups and oocytes in the two control groups exhibited a similar pattern of Ca2+ oscillations in their amplitude, spike duration and interval (Table III and Figure 2).


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Table III. Bovine sperm extract-induced Ca2 + oscillations in mouse metaphase II oocytes

 


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Figure 2. Bovine sperm extract-induced Ca2 + oscillations in the matured oocytes from (a) young GV–young cytoplast (group YY) and (b) young GV–aged cytoplast (group YA). Each oocytes was injected with 2 pl of bovine sperm extracts.

 
Early cleavage of reconstructed ooctyes after artificial activation and IVF
After artificial activation with A23187 + cycloheximide, 78.4–85.4% of the reconstructed oocytes reached pronuclear stage and 55.4–56.5% of them developed to 2-cell stage embryos. Table IV shows that the rates of oocyte activation and cleavage were not significantly different from those of the two control groups (82.9–86.3% for pronuclear formation; 63.4–66.4% for cleavage to 2-cell stage).


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Table IV. Development of matured reconstructed oocyts after artificial activation

 
After IVF, 80.2–84.4% reconstructed oocytes in the three reconstructed oocyte groups formed normal pronuclei and 43.8–54.3% of them cleaved to 2-cell stage (Table V; Figure 3). In the control groups, 83.6–92.0% MII oocytes formed normal pronuclei and 47.8–51.8% cleaved to 2-cell stage (Table V). Neither pronuclear formation nor cleavage rate to the 2-cell stage was significantly different between the reconstructed groups and the controls.


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Table V. Development of matured reconstructed oocyts after IVF

 


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Figure 3. Following IVF, the matured reconstructed ooctyes formed pronuclear stage (a) and developed to 2-cell stgage (b) (x400).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present study was undertaken to determine whether transfer of GV from aged mice to cytoplasts derived from GV oocytes of young mice could reduce ageing-associated aberrations in meiosis of oocytes. Our major finding is that although oocytes exchanged with GV between young and aged mice could undergo maturation, calcium oscillations and early cleavage, the young mice ooplasm could not rescue ageing-associated chromosome misalignment in meiosis of GV from aged mice. Our observation that behaviour of chromosome alignment over the metaphase spindle is predominantly determined by GV material is consistent with a recent paper (Liu and Keefe, 2004Go) showing that the nuclear compartment plays the predominant role in the aetiology of ageing-related meiotic defects in SAM.

Ageing-associated chromosome misalignment in meiosis of oocytes
Ageing-associated aberration in meiosis of oocyes has been observed in humans (Battaglia et al., 1996Go) and SAM (Liu and Keefe, 2002Go, 2004Go). In the present study, we have chosen to use the KM mice as the animal model, because we have observed in this mouse strain ageing-associated aberrations in meiosis of oocytes. The aberrations were characterized by an increase in the rate of abnormal chromosomal alignment and dispersal at the metaphase plate of MII spindles from 16.9% in oocytes of young mice to 49.2% in oocytes from aged mice (Table II). In addition, we have observed that aged KM mice exhibited a significant decrease in the number of both follicles in their ovaries and ovulated oocytes, and that incidence of single chromatid in MII oocytes increased from 9.1% (6 week old mice) to 38.1% in the aged 12 month old mice (Cui, 2003Go; our unpublished results). These characteristics suggest that this mouse strain is a suitable model for addressing the problem of ageing-associated chromosome misalignment in meiosis of oocytes.

Intact GV transplantation did not rescue ageing-associated chromosome misalignment
Poor oocyte quality is a major cause for the ageing-related decline in female fertility (Navot et al., 1991Go). GV transplantation has been proposed as an approach for improving oocyte quality of aged women (Takeuchi et al., 1999Go; Zhang et al., 1999Go), assuming that cytoplasmic factor(s) in ooplasm derived from younger women could reduce the incidence of oocyte abnormalities of older women. Although in the mouse, maturation, fertilization, preimplantation development (Liu et al., 2000Go) and even full term development (Liu et al., 2003Go; Takeuchi et al., 2004Go) has been established using GV transplantation, the question of whether young ooplasm could rescue abnormalities in meiosis of aged GV remains unclear.

In the present study, we show that GV–cytoplast complexes can be effectively induced to undergo cell fusion (89.8–94.4%) and a majority (93.5–97.9%) of them can be matured in vitro, in a manner independent of mouse age. These results support previous findings that GV transplantation procedures itself do not impair subsequent oocyte maturation (Liu et al., 1999Go; Takeuchi et al., 1999Go; Zhang et al., 1999Go). Various cell fusion efficiencies ranging from 31 to 43% (Moffa et al., 2002Go), to 73% (Liu et al., 1999Go) and to 87% (Takeuchi et al., 1999Go) have been reported. Such variation between different laboratories is probably due to difference in fusion medium compositions as well as electrofusion parameters used, because electrostimulation conditions may alter cell fusion efficiency (Sun and Moor, 1989Go). We used M2 as fusion medium and adopted manual alignment to avoid cell exposure to stress of alternating current, thus minimizing any unpredictable and undesirable harmful effects during electronfusion (Rickord and White, 1992Go; Takeuchi et al., 1999Go).

In the present study, we have observed by confocal microscope a significantly higher rate (49.2%) of chromosome misalignment and dispersal in ageing mice than in young mice (16.7%), and 57.1% mature oocytes reconstructed with GV from aged mice and ooplasm from young mice exhibited chromosome misalignment and dispersal, whereas the abnormality rate in young GV–young ooplasm group and young GV–aged ooplasm group was 16.3 and 16.7% respectively. These results demonstrate that: (i) GV transplantation to ooplasm derived from young mice did not rescue ageing-associated chromosome misalignment in meiosis of aged mice, and (ii) behaviour of chromosome alignment and dispersal over the metaphase spindle is predominantly determined by the GV, not the ooplasm. Our observations are contrary to those reported in the humans. It was reported that when GV from oocytes of older women were transferred into enucleated immature oocytes of younger women, a normal, second meiotic metaphase chromosome complement was observed in 4/5 (Zhang et al., 1999Go), 2/2 (Takeuchi et al., 2001Go) or 5/7 (Palermo et al., 2002Go) reconstructed oocytes. Conversely, 2/3 karyotypes of younger GV maturing in older ooplasm showed abnormal karyotypes (Takeuchi et al., 2001Go). These findings in humans seem to support the idea that young ooplasm has a rescuing role in GV from older women. However, because the number of reconstructed oocytes examined was so small, it is hard to draw any definite conclusions (Zhang et al., 1999Go; Takeuchi et al., 2001Go; Palermo et al., 2002Go). Whether there is a species difference of young ooplasm on aged GV between mouse and humans remains to be revealed.

Why is ooplasm of young oocytes unable to rescue ageing-associated chromosome misalignment in meiosis of aged mice?
There are at least two possibilities for explaining why transfer of aged GV to ooplasm derived from young mice failed to rescue ageing-associated chromosome misalignment in meiosis. First, this may be due to absence of GV material derived from oocytes of young mice, because GV material of young oocytes was completely removed by intact GV enucleation. GV material has been shown to be essential for nuclear remodelling (Gao et al., 2002Go); the lack of functional nuclear factors from the young oocyte may restrict the ooplasm's capacity for correcting ageing-associated chromosome misalignment during meiotic maturation. This possibility can be tested by transferring GV or GVBD karyoplasts from aged mice to ooplasm which are derived from GVBD oocytes of young mice, assuming that essential GV material is distributed throughout the ooplasm. Secondly, dysfunctional factors derived from GV of aged mice may remain by binding to the chromosomes and spindles, thus restricting the rescuing effect of ooplasm factors (including GV material) derived from the young mice. Our observation that transfer of GV from oocytes of young mice to ooplasm derived from aged mice does not increase the rate of chromosome misalignment and dispersal over the metaphase spindle, indicating that GV material, not cytoplasm, is essential for determining chromosome and spindle integrity. This is in agreement with the finding by Liu and Keefe (2004)Go that oocyte cytoplasm of young SAM was ineffective in preventing the meiotic defects in oocytes derived from old SAM mice.

Signalling the oocytes for activation and cleavage
In mammals, MII oocytes are released from the metaphase block when a fertilizing sperm activates the oocyte by triggering repetitive calcium oscillations in oocyte cytosol (Fissore et al., 1992Go; Kline and Kline, 1992Go; Sun et al., 1992Go; Miyazaki et al., 1993Go). These oscillations are initiated by a sperm-derived protein factor (Swann, 1990Go; Saunders et al., 2002Go), and their maintenance is determined by a maternal machinery which functions only once in mammalian oocytes (Tang et al., 2000Go). Calcium oscillations in mouse can be induced by injection of mammalian sperm extracts (Swann, 1990Go; Wu et al., 1998Go; Tang et al., 2000Go; Saunders et al., 2002Go), and the activity of sperm factor in triggering calcium oscillations is not species specific (Dong et al., 2000Go; Li et al., 2001Go). In the present study, we chose to use microinjection of sperm extracts to test the response of various types of oocytes to sperm factor stimulation, because under given experimental conditions the volume of sperm extracts injected was identical for all cells, enabling an identical stimulus to be applied to all oocytes examined. We show that when injected with a physiological dosage of bovine sperm extracts, all groups of reconstituted oocytes following maturation exhibited a similar pattern of calcium oscillations in their amplitude, frequency and spike duration, suggesting that neither the transplantation process itself nor maternal ageing in the mice alter the capacity of sperm factor-induced calcium oscillations in mouse oocytes. In addition, we show that when the reconstructed oocytes and control oocytes from young or aged mice were fertilized, they exhibited a similar rate of fertilization and cleavage to 2-cell embryos, suggesting that capacity for fertilization and initiation of early cleavage up to the 2-cell stage is not affected by maternal ageing and status of chromosome and spindle organization. We should also point out that the GV oocytes used in the present study were completely stripped of cumulus and arrested in IBMX for 4–5 h. High rates of maturation, fertilization and cleavage to the 2-cell stage also reflect that these treatments do not affect the early events of development. However, since oocyte maturation is critically important for normal embryonic development (Moor et al., 1998Go), the developmental competence of the transferred GV genome to blastocyst stage was achieved through sequential nuclear transfers (Liu et al., 2000Go). Since GV exchange between aged and young mice did not reduce ageing-associated aberrations in meiosis of oocytes (Figure 1 and Table II), we consider that developing new approaches for reducing the aberrations is one of the most important tasks of current research.

In conclusion, our results suggest that: (i) ageing-associated chromosome misalignment originating from GV of aged mice is not rescued by transplanting to ooplasm derived from oocytes of young mice; (ii) chromosome and spindle organization in mouse reconstructed oocytes is predominantly determined by GV material. Further efforts should be directed to determine species specificity of ooplasm influence on ageing-associated aberration in meiosis of oocytes from aged animals and humans, and to identify the impact of GV material on rescuing chromosome and spindle abnormalities. In addition, it will be interesting to determine whether injection of oocyte factors responsible for chromosome and spindle organization will rescue ageing-associated abnormalities in oocytes from aged animals and humans.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Our research was supported by State Key Basic Research Program of China (TG1999055902), the National Natural Science Foundation of China (Grant No. 30370699) and Cambridge Bay Biomedical Institute research fund.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on December 1, 2004; resubmitted on January 26, 2005; accepted on January 31, 2005.





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