Nucleocytoplasmic ratio of fully grown germinal vesicle oocytes is essential for mouse meiotic chromosome segregation and alignment, spindle shape and early embryonic development

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, P.R.China

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: This study examined the effect of nucleocytoplasmic ratio of fully grown germinal vesicle (GV) oocytes on meiotic chromosome segregation and alignment, spindle shape, Ca2+ oscillations and capacity of early embryonic development in mouse. METHODS: GV oocytes with reduced volume (equal to 1/5 to 4/5 of an intact oocyte) were made by micromanipulation to remove different amounts of cytoplasm, and then matured and fertilized in vitro. RESULTS: When >1/2 of GV oocyte cytoplasm was removed, the time-course of GV breakdown (GVBD) was delayed and oocyte maturation rate decreased significantly. Abnormal chromosome segregation rate increased if >1/2 of the cytoplasm was removed from the oocyte. Length and structure of meiotic spindle and chromosome alignment were also impaired by the reduction of cytoplasmic volume. Once matured in vitro, the oocytes could undergo Sr2+-induced Ca2+ oscillations and form pronuclei in a manner independent of nucleocytoplasmic ratio, but their ability to develop to 2-cell embryos was affected if >1/2 of their cytoplasm was removed from the GV oocytes. CONCLUSIONS: These results suggest that nucleocytoplasmic ratio is essential for normal meiotic chromosome segregation, spindle formation and chromosome alignment over the metaphase spindle, and development to 2-cell stage, for which 1/2 of the volume of the GV oocyte appears to be a threshold.

Key words: Ca2+ oscillations/chromosome mouse/cytoplasm/nucleocytoplasmic ratio


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The interaction between nucleus and cytoplasm is of critical importance in determining the outcome of mammalian oocyte maturation and embryonic development (Fulka et al., 1998Go; Moor et al., 1998Go). It has been shown that GV material is not required for initiating protein synthesis reprogramming in ovine oocyte maturation (Sun and Moor, 1991Go); however, it is essential for the nuclear remodelling after nuclear transfer (Gao et al., 2002Go) and for decondensation and transformation of sperm nuclei into functional pronuclei (Balakier and Tarkowski, 1980)Go. Liu et al. (1999)Go have shown that maturity of host oocyte cytoplasm determines meiosis by reconstruction of mouse oocytes using germinal vesicle transfer. Therefore, nucleus and cytoplasm have complementary roles and both these components are necessary for the normal cell cycle (Iwashita et al., 1998Go).

The nucleocytoplasmic ratio of mammalian oocytes can be manipulated by removing or injecting cytoplasm microsurgically, or bisecting denuded oocytes (Petzoldt and Muggleton-Harris, 1987Go). The influence of modified nucleocytoplasmic ratio on competence of early embryonic development has been investigated in metaphase II (MII) oocytes (Northey et al., 1991Go; Westhusin et al., 1996Go; Zakhartchenko et al., 1997Go; Bordignon and Smith, 1998Go), fertilized oocytes (Petzoldt and Muggleton-Harris, 1987Go; Evsikov et al., 1990Go; Westhusin et al., 1996Go) and early embryonic cells (Howlett et al., 1987Go; Long et al., 1992Go). Kárníková et al. (1998)Go bisected mouse GV oocytes into 1/2, 1/3 and 1/4 oocytes and found that the decrease in cytoplasmic volume influenced the time-course of GVBD and the ability of oocytes to extrude the first polar body. Thus they postulated that a critical nucleocytoplasmic volume ratio is absolutely necessary for normal maturation in mammalian oocytes. However, little is known about the effect of modified nucleocytoplasmic ratio of GV oocytes on other key events of oocyte maturation and oocyte’s ability to initiate early development. In the present study, the nucleocytoplasmic ratio of mouse immature oocytes was modified by removing different amounts of cytoplasm, and the effects of nucleocytoplasmic ratio on oocyte maturation, segregation and alignment of meiotic chromosome, spindle shape, Ca2+ oscillations and development to 2-cell stage were evaluated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Collection of mouse GV oocytes
Female KM mice were injected i.p. with 5 IU pregnant mare’s serum gonadotrophin (PMSG; Sigma, St Louis, MO, USA). Fully grown GV oocytes were collected by puncturing the large antral follicles at 44–48 h post injection and attached cumulus cells were dissociated by repeated pipetting. Only those oocytes with clearly visible GV were used and cultured in human tubal fluid (HTF) medium (Irvine Scientific, Santa Anna, CA, USA) supplemented with 10% fetal calf serum (FCS; HyClone, Logan, UT, 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 GV oocytes with modified nucleocytoplasmic ratio
GV oocytes were incubated in modified HTF medium (Irvine Scientific) supplemented with 10% FCS, 50 µg/ml IBMX and 7.5 µg/ml cytochalasin B (Sigma) for 30 min at room temperature before micromanipulation. Following lancing of the zona pellucida with a sharp-tripped pipette, 1/5, 1/4, 1/3, or 1/2 of the cytoplasm was removed by a cylindrical micropipette with an inner diameter of 20 µm (Figure 1a) to prepare GV ooctyes with 4/5, 3/4, 2/3 or 1/2 of the original oocyte volume with zona pellucida (Figure 1c). Because it was difficult to remove much cytoplasm without the GV, the GV together with some cytoplasm were drawn to create GV oocytes with 1/3, 1/4 or 1/5 of the original oocyte volume and GV karyoplasts (GV surrounded by a thin layer of cytoplasm: Figure 1e). As a result, these oocytes (oocytes with 1/3, 1/4 or 1/5 of the original oocyte volume) and GV karyoplasts did not have the zona pellucida (Figure 1d and e). The oocytes with modified nucleocytoplasmic ratio were washed five times in modified HTF medium and then cultured in HTF medium with 10% FCS at 37°C, 5% CO2.



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Figure 1. Preparation of GV oocytes with modified nucleocytoplasmic ratio. (a) Removing cytoplasm; (b) intact oocytes; (c) oocytes with 1/2 of the original oocyte volume; (d) oocytes with 1/3 of the original oocyte volume without zona pellucida; (e) GV-plasts (x400).

 

In our experiments, the volume of GV oocyte cytoplasm was calculated as follows. Under a x100 objective using an eyepiece fitted with a graticule: (i) the micropipette used for cytoplasm removal was arranged horizontally to a manipulation dish, which contained a microdrop (5 µl) of modified HTF medium containing GV oocytes; (ii) the oocyte was held by the holding pipette, and a defined amount of cytoplasm was sucked into the micropipette (always recording the length of cytoplasts inside the pipette) and the diameter of the manipulated oocyte outside the micropipette was measured using the eyepiece fitted with a graticule; (iii) the cytoplast was gently expelled into the manipulation medium, its diameter was measured and its volume was calculated based on the equation: V = 4/3x{pi}r3. In this way, each defined volume in the pipette corresponded to a defined length in the pipette determined by the eye graticule. Once a defined volume and its corresponding length in the pipette was determined, a range of removal volumes and their corresponding lengths against the graticule was defined following steps 1 to 3.

Maturation and IVF
GVBD and maturation of GV oocytes with modified nucleocytoplasmic ratio were evaluated at 1–3 and 16–18 h of culture respectively. Oocytes displaying a polar body were selected for further examination. Matured 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) (Sigma) for 1.5 h. Oocytes were incubated with the sperm in IVF medium with 15 mg/ml BSA for 6 h. The oocytes were then transferred to HTF medium with 10% FCS at 37°C, 5% CO2 for in vitro culture. The activation identified by the presence of pronuclei and the 2-cell embryos were monitored after 6 and 24 h culture respectively.

Cytogenetic evaluation of in vitro-matured oocytes
In order to assess whether meiosis progressed normally from GV to MII stage, oocytes with the first polar body were submitted to cytogenetic analysis according to Tarkowski (1966)Go. The oocytes were transferred into a 1% sodium citrate hypotonic solution for 10 min and then fixed with methanol:acetic acid (3:1) on a clean glass slide. Fixed oocytes were stained with Giemsa to determine the number of chromosomes.

Immunocytochemistry
Oocytes that had extruded first polar bodies were selected for the immunocytochemistry study of the meiotic spindle. The oocytes were fixed in 3.7% paraformaldehyde (Sigma) in PBS for 40 min, and then permeated 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. Afterwards, the oocytes were incubated with {beta}-tubulin mouse monoclonal antibody (1:150; Zymed Laboratories, Inc.) overnight at 4°C,washed, and then incubated with fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG (1:200; Sigma) at room temperature for 2 h. During one of the final washing steps, 5 µg/ml Hoechst 33258 (Sigma) in PBS was added to localize chromosomes.

Confocal fluorescence microscope 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. Paired images were digitally reproduced to examine the co-localization of tubulin and chromosomes.

Calcium measurement
Oocytes were loaded with 2 µmol/l fura-2/AM (Molecular Probes Inc., Eugene, OR, 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 Ca2+-free H6 medium and then transferred to a chamber containing 20 mmol/l SrCl2 in Ca2+-free 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, Garden City, NY, USA) for imaging, and maintained at 37°C by a thermostatic controller (Life Sciences Resources, Cambridge, UK). The calcium measurement was conducted using a MiraCal imaging system equipped with MiraCal Version 2.3 Software (Life Sciences Resources). The emitted fluorescence intensities at 510 nm were recorded with excitation at 340 nm and at 380 nm wavelengths by Mira-1000TE low-light-level CCD camera. The fluorescence signal is displayed as the ratio of the fluorescent intensity (510 nm) excited at the 340 nm over that excited at 380 nm. Calcium was calculated simultaneously by computer using the ratio equation described by Grynkiewicz et al. (1985)Go. Parameters used for the calculation were obtained according to Poenie et al. (1985)Go. The calcium image was recorded every 10 s for up to 2 h.

Data analysis
All experiments were repeated three to five times and the results were analysed using the {chi}2-test with significance determined at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Maturation of GV stage oocytes with modified nucleocytoplasmic ratio
As shown in Table I and Figure 2, 1/2 volume of oocytes appeared to be a threshold for rate of GVBD in 1–3 h of culture and for rate of maturation in 16–18 h of culture. The rates of GVBD and maturation were not significantly different between the intact (control) oocytes and oocytes with 4/5, 3/4, 2/3 or 1/2 of the original cell volume (i.e. oocytes with volume ≥1/2 of the intact cell), but significantly decreased in oocytes with only 1/3, 1/4 or 1/5 of the original cell volume (i.e. oocytes with volume <1/2 of the intact cell). In 16–18 h of culture, all oocytes whose volume was ≥1/4 of the intact cell underwent GVBD, while the GV-plasts remained intact and did not undergo GVBD even after 24 h of culture.


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Table I. Comparison of in vitro maturation of germinal vesicle (GV) oocytes with modified nucleocytoplasmic ratio

 


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Figure 2. Maturation of oocytes with intact (a), 1/2 (b) and 1/3 (c) of original oocyte volume (x400).

 

Nucleocytoplasmic ratio affects chromosome segregation
About 212 of 352 matured oocytes were successfully fixed for cytogenetic study. The results in Table II show that 1/2 volume of intact oocyte appears to be a demarcation line for normal rate of chromosome segregation during meiotic maturation. For intact oocytes and oocytes with ≥1/2 of original oocyte volume, the rate (82.6–90.5%) of oocytes displaying 20 chromosomes was similar. However, when the oocyte’s volume was further reduced to only 1/3 or 1/4 of intact oocyte size, the manipulated oocytes showed a significantly higher rate of abnormal chromosome construction than that in the control group. These results therefore indicate that ≥1/2 of the cytoplasm in the GV oocyte is essential for normal chromosome segregation during oocyte meiotic maturation.


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Table II. Chromosomal analysis of matured oocytes with modified nucleocytoplasmic ratio

 

Nucleocytoplasmic ratio affects spindle shape and chromosome alignment
In the intact oocytes the MII spindle was uniform in shape (Figure3a). In oocytes with 1/2 and 1/3 of original oocyte volume, however, the MII spindle was significantly shorter than that in intact oocytes, and was often abnormally shaped or disordered with regard to microtubule placement (Figure 3b, c). The chromosomes were evenly aligned at the spindle equator in most intact oocytes (83.1%). However, the chromosome spread was vast and irregularly aligned over the spindle in some oocytes with 1/2 (29.6%) and 1/3 (41.7%) of the original oocyte volume (Table III; Figure 3a'–c').



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Figure 3. Immunofluorescence images of spindles and chromosomes of oocytes with intact (a,a’), 1/2 (b, b’) and 1/3 (c,c’) of original oocyte volume. (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.

 

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Table III. Comparison of chromosome alignment in matured oocytes with modified nucleocytoplasmic ratio

 

Sr2+-induced Ca2+ oscillations occurred in the mature oocytes independent of nucleocytoplasmic ratio
Treating the matured oocytes with 20 mmol/l SrCl2 induced Ca2+ oscillation in most MII oocytes examined (Table IV). The Ca2+ oscillations had a mean interval of 386 ± 65 to 403 ± 62 s. The mean baseline was 88 ± 19 to 101 ± 20 nmol/l with an average peak of 354 ± 39 to 379 ± 42 nmol/l. The Ca2+ oscillation persisted for >4 h. All oocytes with modified nucleocytoplasmic ratioshowed a similar pattern of Ca2+ oscillation, and there was no observed difference in their Ca2+ amplitude, duration and spiking interval despite the varied cytoplasmic volume (Table IV; Figure 4).


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Table IV. Comparison of Sr2+-induced Ca2+ oscillations in matured oocytes with modified nucleocytoplasmic ratio

 


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Figure 4. Sr2+-induced Ca2+ oscillations in the oocytes matured from GV oocyte with intact (a), 1/2 (b) and 1/3 (c) of original oocyte volume. Oocytes were treated with 20 mmol/l SrCl2in Ca2+-free H6 medium at 37°C.

 

Nucleocytoplasmic ratio affects early cleavage but not pronuclear formation of the mature oocytes
After the GV oocytes with modified nucleocytoplasmic ratio had matured, they were fertilized in vitro. Although the rate of maturation was different among the GV oocytes with modified nucleocytoplasmic ratio, once matured, the rate of development to pronuclear stage from the GV oocytes with ≥1/4 of the original volume was comparable (83–91%; Table V, Figure 5a, b, c). However, the size of pronuclei formed was reduced with the reduction of oocyte cytoplasm.


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Table V. Comparison of development of matured oocytes with modified nucleocytoplasmic ratio after IVF

 


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Figure 5. Following IVF, the mature ooctyes developed to pronuclear (a, b, c) and 2-cell (d, e, f) stages. (a, d) From intact oocytes; (b, e) from oocytes with 1/2 of original oocyte volume; (c, f) from oocytes with 1/3 of original oocyte volume (x400).

 

Half volume of oocytes appeared to be a threshold for rate of their development to 2-cell stage. For intact oocytes and oocytes with ≥1/2 of the original cell volume, the rate of development to 2-cell stage was comparable (50–59%), and this rate was significantly higher than that of oocytes with ≤1/3 of the original cell volume (Figure 5d, e, f). No oocytes with ≤1/4 of the original volume developed to 2-cell stage (Table V).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present study extended previous findings (Kárníková et al., 1998Go; Takeuchi et al., 1999Go) by showing for the first time that nucleocytoplasmic ratio is essential for mouse normal meiotic chromosome segregation, spindle formation, chromosome alignment over the metaphase spindle, and mitotic divisions to 2-cell stage. We show that 1/2 volume of the GV oocyte appears to be a threshold for determining these important events.

When >1/2 of cytoplasm is removed, the time course of GVBD is delayed and maturation rate decreased with reduction of cytoplasmic volume. The influence, however, is not obvious when <1/2 of cytoplasm is removed. It is unclear why the process of oocyte maturation is influenced by the modified nucleocytoplasmic ratio. A possible explanation is seen in the analogy with incompetent oocytes which acquire the ability to undergo GVBD only after their volume increases to ≥80% of the fully grown oocytes (Wassarman, 1988Go). An insufficient complement of cell cycle proteins in incompetent oocytes might be responsible for this inability of maturation (Motlik and Kubelka, 1990Go). It has been shown that the basic cell cycle molecules are already present in fully grown oocytes. Maturation-promoting factor (MPF), which is composed of P34cdc2 and cyclin B, is an important factor for meiosis resumption, including the induction of GVBD, chromosome condensation and final nuclear maturation. The removal of a certain part of a cytoplasmic compartment may actually decrease the absolute amount of cell cycle regulators. The remaining molecules are thereafter unable to drive oocyte maturation as in the oocytes that are not manipulated (Kárníková et al., 1998Go).

How the modified nucleocytoplasmic ratio influences the developmental programme in MII oocytes is also poorly understood and the results are contradictory. Removing a large volume of cytoplasm in bovine MII oocytes influenced the developmental programme in manipulated cells (Bordignon and Smith, 1998Go). Zakhartchenko et al. (1997)Go also observed significantly impaired development after a substantial amount of cytoplasm was removed at enucleation. The present study shows that once GV oocytes with modified nucleocytoplasmic ratio have matured, they can form pronuclei following fertilization, suggesting that pronuclear formation occurred in those oocytes in a manner independent of nucleocytoplasmic ratio. Balakier and Tarkowski (1980)Go suggested that in mouse, GV material is essential for the transformation of sperm nucleus into functional male pronucleus. It is possible that although part of the GV cytoplasm had been removed, the presence of essential GV material enabled these matured oocytes with various nucleocytoplasmic ratio to form pronuclei after fertilization.

Evsikov et al. (1990)Go reported that the cleavage rate of the fertilized oocytes with cytoplasm content reduced by 1/3 was significantly lower than that in the control oocytes. The zygote karyoplasts whose nucleocytoplasmic ratio is maximal, arrested at the 2-cell stage. In the present experiment, the ability to develop to 2-cell stage was affected when >1/2 of the cytoplasm content was removed. These results illustrate an important function of the cytoplasm in maintaining the nucleus viability (Evsikov et al., 1990Go).

Mammalian MII oocytes are activated by sustained Ca2+oscillation following sperm penetration (see reviewed by Swann and Ozil, 1994Go). The sperm factor mobilizes Ca2+ release from the intracellular stores (Swann, 1994Go) and the maintenance of Ca2+ oscillation is regulated by a maternal machinery which functions only once in mammalian oocytes (Tang et al., 2000Go). Parthenogenetic agents, such as Sr2+, are also able to mimic sperm penetration to trigger oocyte activation and embryonic development by increasing intracellular free Ca2+ level in the oocytes (Bos-Mikich et al., 1995Go; Tang et al., 1998). In the present study, our results demonstrate that although the oocyte volume reduces even to 1/4 of normal volume, Sr2+-induced Ca2+ oscillation pattern is not affected compared with that of the intact oocytes. Thus, the Sr2+-induced Ca2+oscillation is independent of nucleocytoplasmic ratio. The observation that the oocytes of various sizes produced similar Ca2+ oscillation patterns may also explain why the oocytes with modified nucleocytoplasmic ratio, once matured, can be activated to form pronuclei.

The interactions between nuclear and cytoplasmic factors influence the reduction in chromosome number from 4N (GV stage) to 2N (MII stage) in mammalian oocytes (Hashimoto and Kishimoto, 1998). These interactions ensure that normal meiotic spindles are constructed from microtubules at each meiotic division. Numerous microtubule-organizing centres, the centrosomes, can be found adjacent to GV in mouse oocytes (Messinger and Albertini, 1991Go). They appear to be recruited for spindle assembly as the transition from prophase to metaphase begins. These domains eventually give rise to spindles (Battaglia et al., 1996Go). At MII stage, the normal metaphase plate within the spindle is distinct, with the chromosomes in compact alignment within it (Liu and Keefe, 2002Go). In the present study, cytogenetic analysis indicates that aneuploidy increases significantly when the oocytes are reduced to <1/2 of their normal volume. Spindle shape, length and chromosome alignment in the oocytes were indicators of a predisposition of non-disjunction and aneuploidy (Eichenlaub-Ritter et al., 1986Go, 1988Go; Liu and Keefe, 2002Go). We found that spindle length, size and chromosome alignment were apparently altered in the oocytes with 1/3 or 1/2 of the normal oocyte volume. It is logical to assume that, with reduction of cytoplasm in the GV oocytes, centrosomes, microtubules and some regulatory factors in the cell decrease or some relative protein synthesis is impaired. Such effects could be responsible for structural abnormalities of meiotic spindles and/or alteration of the timing of the phase of meiosis resulting in eventual chromosome malsegregation. Disorder of chromosomes in the spindle of the oocytes with modified nucleocytoplasmic ratio may reflect a disturbance in chromosome segregation during meiosis I, or indicate that these oocytes are predisposed to errors in chromatid segregation during meiosis II (Eichenlaub-Ritter, 1998Go), which may therefore influence further development of the cells.

Transferring a germinal vesicle (GV) from an aged woman’s oocyte into ooplasm from a younger woman has been proposed as a potential way to overcome the problem of age-related decline in female fertility (Zhang et al., 1999Go). The concept is that the aneuploidy rate in the first meiotic division may be reduced by transferring the GV from an older woman into the donated ooplasm from a young woman. Using a mouse model, we have shown very recently 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 the metaphase spindle is predominantly determined by GV material (Cui et al., 2005Go). Clearly, modified procedures for GV transfer should be developed before it is applied for practical medical therapy. The findings in the present study show that nucleocytoplasmic ratio is essential for normal meiotic chromosome segregation, spindle formation and chromosome alignment over the metaphase spindle, and development to 2-cell stage, implying that when performing human GV exchange experiments a balanced nucleocytoplasmic ratio may be another determinant factor that regulates chromosome behaviour and developmental ability.

In conclusion, our results demonstrate that nucleocytoplasmic ratio is essential for mouse normal meiotic chromosome segregation, spindle formation and chromosome alignment over the metaphase spindle, and development to 2-cell stage, for which 1/2 volume of the GV oocyte appears to be a demarcation line. In addition, our observations indicate that although GV material is present in oocytes with modified nucleocytoplasmic ratio, events of mammalian oocyte meiotic maturation and embryonic development is readily compromised by imperfection of nucleocytoplasmic ratio at the GV stage.


    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 30430390) and Cambridge Bay Biomedical Institute’s research fund.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Balakier H and Tarkowski AK (1980) The role of germinal vesicle karyoplasm in the development of male pronucleus in the mouse. Exp Cell Res 128,79–85[CrossRef][ISI][Medline]

Battaglia DE, Goodwin P, Klein NA and Soules MR (1996) Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum Reprod 11, 2217–2222.[Abstract]

Bordignon V and Smith LC (1998) Telophase enucleation: an improved method to prepare recipient cytoplasts for use in bovine nuclear transfer. Biol Reprod 49,29–36.

Bos-Mikich A, Swann S and Whittingham DG (1995) Calcium oscillations and protein synthesis inhibition synergistically activate mouse oocytes. Mol Reprod Dev 41,84–90.[CrossRef][ISI][Medline]

Cui LB, Huang XY and Sun FZ (2005) Transfer of germinal vesicle to ooplasm of young mice could not rescue ageing-associated chromosome misalignment in meiosis of oocytes from aged mice. Hum Reprod, in press.

Deng MQ, Huang XY, Tang TS and Sun FZ (1998) Spontaneous and fertilization-induced Ca2+ oscillations in mouse immature germinal vesicle-stage oocytes. Biol Reprod 58,807–813.[Abstract]

Eichenlaub-Ritter U (1998) Genetics of oocyte ageing. Maturitas 30,143–169.[CrossRef][ISI][Medline]

Eichenlaub-Ritter U, Chandley AC and Gosden RG (1986) Alterations to the microtubular cytoskeleton and increased disorder of chromosome alignment in spontaneously ovulated mouse oocytes aged in vivo: an immunofluorescent study. Chromosoma 94,337–345.[CrossRef][ISI][Medline]

Eichenlaub-Ritter U, Chandley AC and Gosden RG (1988) The CBA mouse as a model for age-related aneuploidy in man: studies of oocyte maturation, spindle formation and chromosome alignment during meiosis. Chromosoma 96,220–226.[CrossRef][ISI][Medline]

Evsikov SV, Morozoval LM and Solomko AP (1990) The role of the nucleocytoplasmic ratio in development regulation of the early mouse embryo. Development 109,323–328.[Abstract]

Fulka J Jr, First NL and Moor RM (1998) Nuclear and cytoplasmic determinants involved in the regulation of mammalian oocyte maturation. Mol Hum Reprod 4,41–49.[Abstract]

Gao S, Gasparrini B, McGarry M, Ferrier T, Fletcher J, Harkness L, Sousa PD and Wilmut I (2002) Germinal vesicle material is essential for nucleus remodeling after nuclear transfer. Biol Reprod 67,928–934.[Abstract/Free Full Text]

Grykiewicz G, Poenie M and Tsien RY (1985) A new gereration of Ca2+ indictors with greatly improved fluorescence properties. J Biol Chem 260,3440–3450.[Abstract]

Hashimoto N and Kishimoto T (1988) Regulation of meiotic metaphase by a cytoplasmic maturation-promoting factor during mouse oocte maturation. Dev Biol 126,242–252.[CrossRef][ISI][Medline]

Hogan B, Costantini F and Lacy E (1986) Manipulating the mouse embryo, a laboratory manual. Cold Spring Harbor Laboratory, New York, pp 107–109.

Howlett SK, Barton SC and Surani MA (1987) Nuclear cytoplasmic interactions following nuclear transplantation in the mouse embryos. Development 101,915–925.[Abstract]

Iwashita J, Hayano Y and Sagata N (1998) Essential role of germinal vesicle material in the meiotic cell cycle of Xenopus oocytes. Proc Natl Acad Sci USA 95,4392–4397.[Abstract/Free Full Text]

Kárníková L, Urban F, Moor R and Fulka Jr J (1998) Mouse oocyte maturation: the effect of modified nucleocytoplasmic ratio. Reprod Nutr Dev 38,665–670.[ISI][Medline]

Liu H, Wang CW, Grifo JA, Krey LC and Zhang J (1999) Reconstruction of mouse oocytes by germinal vesicle transfer: maturity of host oocyte cytoplasm determines meiosis. Hum Reprod 14,2357–2361.[Abstract/Free Full Text]

Liu L and Keefe DL (2002) Ageing-associated aberration in meiosis of oocytes from sensecence-accelerated mice. Hum Reprod 17,2678–2685.[Abstract/Free Full Text]

Long CR, Westhusin ME and Looney CR (1992) Manipulation of early bovine embryos: effect on subsequent development and embryo quality in vitro. Theriogenology 37,249.[CrossRef]

Messinger SM and Albertini DF (1991) Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte. J Cell Sci 100,289–298.[Abstract]

Moor RM, Dai Y, Lee C and Fulka J Jr (1998) Oocyte maturation and embryonic failure. Hum Reprod Update 4,223–236.[Abstract/Free Full Text]

Motlik J and Kubelka M (1990) Cell-cycle aspects of growth and maturation of mammalian oocytes. Mol Reprod Dev 27,366–375.[CrossRef][ISI][Medline]

Northey DL, Nuttleman PR and Rosenkrans CF (1991) Removal of bovine oocyte cytoplasm prior to fertilization reduces cell number in embryos. Biol Reprod 44,156.

Petzoldt U and Muggelton-Harris A (1987) The effect of the nucleocytoplasmic ratio of protein synthesis and expression of a stage-specific antigen in early cleaving mouse embryos. Development 99,481–491.[Abstract]

Poenie M, Alderton J and Tsien RY (1985) Changes of free calcium levels with stages of the cell division cycle. Nature 315,147–149.[CrossRef][ISI][Medline]

Sun FZ and Moor RM (1991) Nuclear-cytoplasmic interactions during ovine oocyte maturation. Development 111,171–180.[Abstract]

Swann K (1994) Ca2+ oscillations and sensitization of Ca2+ release in unfertilized mouse eggs injected with a sperm factor. Cell Calcium 15,331–339.[CrossRef][ISI][Medline]

Swann K and Ozil JP (1994) Dynamic of the calcium signal that triggers mammalian oocyte activation. Int Rev Cytol 152,183–222.[ISI][Medline]

Takeuchi T, Ergün B, Huang TH, Rosenwaks Z and Palermo GD (1999) A reliable technique of nuclear transplantation for immature mammalian oocytes. Hum Reprod 14,1312–1317.[Abstract/Free Full Text]

Tang TS, Dong JB, Huang XY and Sun FZ (2000) Ca2+ oscillations induced by a cytosolic sperm protein factor are mediated by a maternal machinery that functions only once in mammalian eggs. Development 127,1141–1150.[Abstract/Free Full Text]

Tarkowski AK (1966) An air-drying method for chromosome preparations from mouse eggs. Cytogenetics 5,394–400.[ISI]

Wassarman PM (1988) The mammalian ovum. In Knobil E and Neill JD (eds) The Physiology of Reproduction. Vol 1, New York, Raven Press, pp 69–102.

Westhusin ME, Collas P, Marek D, Sullivan E, Stepp P, Pryor J and Barnes F (1996) Reducing the amount of cytoplasm available for early embryonic development decreases the quality but not quantity of embryos produced by in vitro fertilization and nuclear transplantation. Theriogenology 46,243–252.[CrossRef][ISI]

Zakhartchenko V, Stoikovic M, Brem G and Wolf E (1997) Karyoplast-cytoplast volume ratio in bovine nuclear transfer embryos: effect on developmental potential. Mol Reprod Dev 48,332–338.[CrossRef][ISI][Medline]

Zhang J, Wang CW, Krey L, Liu H, Meng L, Blaszczy A, Adler A and Grifo J (1999) In vitro maturation of human preovulatory oocytes reconstructed by germinal vesicle transfer. Fertil Steril 71,726–731.[CrossRef][ISI][Medline]

Submitted on December 1, 2004; resubmitted on May 1, 2005; accepted on May 11, 2005.





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