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
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Abstract |
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Key words: ageing/Ca2+ oscillations/chromosome/germinal vesicle transfer/oocyte
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Introduction |
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Germinal vesicle (GV) can be effectively removed by micromanipulation and nuclear transfer is a useful technique for studying nuclearcytoplasmic interaction in mammalian oocytes during meiotic maturation (Sun and Moor, 1991; Meng et al., 1996
). 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., 1999
; Takeuchi et al., 1999
, 2001
; Zhang et al., 1999
; Li et al., 2001
; Moffa et al., 2002
; Liu and Keefe, 2004
). 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., 1999
; Takeuchi et al., 1999
; Palermo et al., 2002
). 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., 1999
; Takeuchi et al., 1999
). Although maturation, fertilization, preimplantation and full term development in mouse have been established using GV transplantation (Liu et al., 2003
; Takeuchi et al., 2004
), 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, 2002). 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, 2004
). 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.
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Materials and methods |
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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) and Liu et al. (1999)
. 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 GVcytoplast complexes were incubated for 30 min in M2 medium (Sigma) at 37 °C, 5% CO2 prior to electrofusion.
Electrofusion of GVcytoplast complexes
An Electro Cell Manipulator (BTX 200; BTX Inc., USA) was used for the fusion. Each GVcytoplast 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 (68 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 1618 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). 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 67 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). Sperm were collected from the cauda epididymides of male mice and capacitated in IVF medium (Hogan et al., 1986
) 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). 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). 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., 1998). 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)
, which is calculated by computer simultaneously. Parameters required for the ratio equation were obtained according to Poenie et al. (1985)
. 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--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 2-test and t-test. P<0.05 was considered siginificant.
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Results |
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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 23 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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Discussion |
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Ageing-associated chromosome misalignment in meiosis of oocytes
Ageing-associated aberration in meiosis of oocyes has been observed in humans (Battaglia et al., 1996) and SAM (Liu and Keefe, 2002
, 2004
). 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, 2003
; 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., 1991). GV transplantation has been proposed as an approach for improving oocyte quality of aged women (Takeuchi et al., 1999
; Zhang et al., 1999
), 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., 2000
) and even full term development (Liu et al., 2003
; Takeuchi et al., 2004
) 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 GVcytoplast complexes can be effectively induced to undergo cell fusion (89.894.4%) and a majority (93.597.9%) 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., 1999; Takeuchi et al., 1999
; Zhang et al., 1999
). Various cell fusion efficiencies ranging from 31 to 43% (Moffa et al., 2002
), to 73% (Liu et al., 1999
) and to 87% (Takeuchi et al., 1999
) 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, 1989
). 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, 1992
; Takeuchi et al., 1999
).
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 GVyoung ooplasm group and young GVaged 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., 1999), 2/2 (Takeuchi et al., 2001
) or 5/7 (Palermo et al., 2002
) reconstructed oocytes. Conversely, 2/3 karyotypes of younger GV maturing in older ooplasm showed abnormal karyotypes (Takeuchi et al., 2001
). 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., 1999
; Takeuchi et al., 2001
; Palermo et al., 2002
). 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., 2002); 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)
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., 1992; Kline and Kline, 1992
; Sun et al., 1992
; Miyazaki et al., 1993
). These oscillations are initiated by a sperm-derived protein factor (Swann, 1990
; Saunders et al., 2002
), and their maintenance is determined by a maternal machinery which functions only once in mammalian oocytes (Tang et al., 2000
). Calcium oscillations in mouse can be induced by injection of mammalian sperm extracts (Swann, 1990
; Wu et al., 1998
; Tang et al., 2000
; Saunders et al., 2002
), and the activity of sperm factor in triggering calcium oscillations is not species specific (Dong et al., 2000
; Li et al., 2001
). 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 45 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., 1998
), the developmental competence of the transferred GV genome to blastocyst stage was achieved through sequential nuclear transfers (Liu et al., 2000
). 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.
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Acknowledgements |
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References |
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Cui LB (2003) Effects of germinal vesicle exchange between young and old mice and changed nucleocytoplasmic ratio on oocyte maturation and embryo development. PhD thesis, Northeast Agricultural University, China.
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, 807881.[Abstract]
Dong JB, Tang TS and Sun FZ (2000) Xenopus and chicken sperm contain a cytosolic soluble protein factor which can trigger calcium oscillations in mouse eggs. Biochem Biophys Res Commun 268, 947951.[CrossRef][ISI][Medline]
Fissore RA, Dobrinsky JR, Balise JJ, Duby RT and Robl JM (1992) Patterns of intracellular Ca2+ concentrations in fertilized bovine eggs. Biol Reprod 47, 960969.
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, 928934.
Gaulden ME (1992) Maternal age effect: the enigma of Down syndrome and other trisomic conditions. Mutat Res 296, 6988.[ISI][Medline]
Grynkiewicz G, Poenie M and Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260, 34403450.[Abstract]
Hagemann LT, Hillery-Weinhold FL, Leibfried Rutledge ML and First NL (1995) Activation of murine oocytes with Ca2+ ionophore and cycloheximide. J Exp Zool 271, 5761.[ISI][Medline]
Hassold T and Chiu D (1985) Maternal age-specific rates of numerical chromosome abnormalities with special reference to trisomy. Hum Genet 70, 1117.[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. 107109.
Keefe DL (1998) Reproductive aging is an evolutionarily programmed strategy that no longer provides adaptive value. Fertil Steril 70, 204206.[CrossRef][ISI][Medline]
Kline D and Kline JT (1992) Repetitive calcium transients and the role of calcium in exocytosis and cell division in mouse eggs. Dev Biol 149, 8089.[CrossRef][ISI][Medline]
Li GP, Chen DY, Lian L, Sun QY, Wang MK, Liu JL, Li JS and Han ZM (2001) Viable rabbit derived from reconstituted oocytes by germinal vesicle transfer after intracytoplasmic sperm injection (ICSI). Mol Reprod Dev 58, 180185.[CrossRef][ISI][Medline]
Li ST, Huang XY and Sun FZ (2001) Flowering plant sperm contains a cytosolic soluble protein factor which can trigger calcium oscillations in mouse eggs. Biochem Biophys Res Commun 287, 5659.[CrossRef][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, 23572361.
Liu H, Zhang J, Krey LC and Grifo JA (2000) In-vitro development of mouse zygotes following reconstruction by sequential transfer of germinal vesicles and haploid pronuclei. Hum Reprod 15, 19972002.
Liu H, Chang HC, Zhang J, Grifo J and Krey LC (2003) Metaphase II nuclei generated by germinal vesicle transfer in mouse oocytes support embryonic development to term. Hum Reprod 18, 19031907.
Liu L and Keefe DL (2004) Nuclear origin of aging-associated meiotic defects in senescence-accelerated mice. Biol Reprod 71, 17241729.
Liu L and Keefe DL (2002) Ageing-associated aberration in meiosis of oocytes from sensecence-accelerated mice. Hum Reprod 17, 26782685.
Meng L, Rutledge J, Zhu Y, Kidder GM, Khamsi F and Armstrong DT (1996) Role of germinal vesicle on protein synthesis in rat oocyte during in vitro maturation. Mol Reprod Dev 43, 228235.[CrossRef][ISI][Medline]
Miyazaki S, Shirakawa H, Nakada K and Honda Y (1993) Essential role of the inositol 1,4,5-trisphosphate/Ca2+ release channel in Ca2+ waves and Ca2+ oscillations at fertilization of mammalian eggs. Dev Biol 158, 6278.[CrossRef][ISI][Medline]
Moffa F, Comoylio F, Krey LC, Grifo JA, Revelli A, Massobrio M and Zhang J (2002) Germinal vesicle transfer between fresh and cryopreserved immature mouse oocytes. Hum Reprod 17, 178183.
Moor RM, Dai Y, Lee C and Fulka J (1998) Oocyte maturation and embryonic failure. Hum Reprod Update 4, 223236.
Navot D, Bergh PA, Williams MA, Garrisi GJ, Guzman I, Sandler B and Grunfeld L (1991) Poor oocytes quality rather than implantation failure as a cause of age-related decline in female fertility. Lancet 337, 13751377.[CrossRef][ISI][Medline]
Palermo GD, Takeuchi T and Rosenwaks Z (2002) Technical approaches to correction of oocyte aneuploidy. Hum Reprod 17, 21652173.
Plachot M, Veiga A, Montagut J, de Grouchy J, Calderon G, Lepretre S, Junca AM, Santalo J, Charles E and Mandelbaum J (1988) Are clinical and biological IVF parameters correlated with chromosomal disorders in early life: a multicentric study. Hum Reprod 3, 627635.[Abstract]
Poenie M, Alderton J, Tsien RY and Steinhardt RA (1985) Changes in free calcium with stages of the cell cycle. Nature 315, 147149.[CrossRef][ISI][Medline]
Rickord LF and White KL (1992) Effect of electrofusion pulse in either electrolyte or non-electrolyte fusion medium on subsequent murine embryonic development. Mol Reprod Dev 32, 259264.[CrossRef][ISI][Medline]
Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Swann K and Lai A (2002) PLC: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development. Development 129, 35333544.
Sun FZ and Moor RM (1989) Factors controlling the electrofusion of murine embryonic cells. Bioelectrochem Bioenerg 21, 149160.[CrossRef]
Sun FZ and Moor RM (1991) Nuclear-cytoplasmic interactions during ovine oocyte maturation. Development 111, 171180.[Abstract]
Sun FZ, Hoyland J, Huang X, Mason W and Moor RM (1992) A comparison of intracellular calcium changes in porcine eggs of fertilization and electroactivation. Development 115, 947956.
Swann K (1990) A cytosolic sperm factor stimulates repetitive calcium increase and mimics fertilization in hamster eggs. Development 110, 12951302.[Abstract]
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, 13121317.
Takeuchi T, Gong J, Veeck LL, Rosenwaks Z and Palermo GD (2001) Preliminary finding in germinal vesicle transplantation of immature human oocytes. Hum Reprod 16, 730736.
Takeuchi T, Rosenwaks Z and Palermo GD (2004) A successful model o assess embryo development after transplantation of prophase nuclei. Hum Reprod 19, 975981.
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, 11411150.
Tietze C (1957) Reproductive span and rate of reproduction among Hutterite women. Fertil Steril 8, 8997.[ISI][Medline]
Volarcik K, Sheean L, Goldfarb J, Woods L, Abdul-karim FW and Hunt P (1998) The meiotic competence of in-vitro matured human oocytes is influenced by donor age: evidence that folliculogenesis is compromised in the reproductively aged ovary. Hum Reprod 13, 154160.[Abstract]
Wu H, He CL, Jehn B, Black SJ and Fissore RA (1998) Partial characterization of the calcium-releasing activity of porcine sperm cytosolic extracts. Dev Biol 203, 369381.[CrossRef][ISI][Medline]
Zhang J, Wang CW, Krey L, Liu H, Meng L, Blaszczky A, Adler A and Grifo J (1999) In vitro maturation of human preovulatory oocytes reconstructed by germinal vesicle transfer. Fertil Steril 71, 726731.[CrossRef][ISI][Medline]
Submitted on December 1, 2004; resubmitted on January 26, 2005; accepted on January 31, 2005.
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