Nuclear competence for maturation and pronuclear formation in mouse oocytes

Siqin Bao1,4, Yayoi Obata1,5, Yukiko Ono1,2, Nana Futatsumata3, Shueo Niimura3 and Tomohiro Kono1,2,6

1 Department of Animal Science and 2 Department of Bioscience, Tokyo University of Agriculture, Setagaya-ku, Tokyo and 3 Department of Agriculture, Niigata University, Niigata, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: In response to gonadotrophins, a fully grown mouse oocyte matures to the metaphase of the second meiotic division and becomes competent for the development of female and male pronuclei after fertilization. The present study was carried out to clarify when during the growth period an oocyte nucleus acquires the ability to promote pronuclei formation after fertilization. METHODS: Fully grown germinal vesicle (GV) oocytes were enucleated and fused with nuclei from growing oocytes from 1–20 day old mice by standard nuclear transfer technique. The reconstructed oocytes were matured and fertilized in vitro, and pronuclear formation was assessed. RESULTS: The oocytes whose nuclei were exchanged for those of the non-growing-stage oocytes matured to the metaphase of the second meiotic stage, but no normal female pronuclei were formed. Female pronuclei first formed in 27% of the oocytes reconstituted with the nuclei of oocytes from 8 day old pups after fertilization. Recondensed sperm chromatin was detected in 27% of the oocytes reconstructed with oocyte nuclei from 8 day old mice, and a male pronucleus was first formed in 6% of the oocytes that had been reconstructed with the nuclei of oocytes from 15 day old mice. The sizes of the female and male pronuclei increased with oocyte donor age, and reached normal size when the oocytes from 15 and 20 day old mice respectively were used. An electron microscopic study using oocytes that had received the oocyte nuclei of 8 day old mice confirmed these results. CONCLUSION: The factors required for pronuclear formation are derived from fully grown GV oocytes, and the transformation from decondensed sperm chromatin to a recondensed male pronucleus is governed by GV-derived factors.

Key words: germinal vesicle transfer/mouse/oocyte growth/oocyte maturation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The resumption of meiosis from the germinal vesicle (GV) stage is characterized by sequential events that include the disassembly of the nuclear membrane, chromatin condensation, assembly of the metaphase spindle, and the first meiotic division. After extrusion of the first polar body and formation of the metaphase plate, the oocyte is again arrested at the metaphase of the second meiotic division (MII) until fertilization.

In mice, primary follicle oocytes, which are 15–20 µm in diameter and arrested at the diplotene stage of the prophase of meiosis I, are surrounded by a single layer of thin, flattened follicle cells (Pedersen and Peters, 1968Go; Peters, 1969Go). Once oocytes enter their growth phase, their volume augments roughly 300 times in parallel with the follicular growth, during which an antral follicle is formed. During this period, the nucleus of the oocyte develops into a large GV with the specialized function of a storage organelle that includes histones, pore complexes, lamins, various small ribosomal nuclear proteins, and other components that are as yet undetermined (Wassarman and Josefowicz, 1978Go). In response to endogenous and exogenous gonadotrophins, fully grown oocytes of antral follicles resume meiosis, then reach and arrest at the metaphase of the second meiosis until fertilization (Hogan et al., 1994Go).

Oocytes first become competent to complete the maturation process as they near full size, which is 75 and 130 µm in diameter in mice (Szybek, 1972Go; Sorensen and Wassarman, 1976Go; Eppig et al., 1994Go), and in bovine (Motlik, 1989Go) respectively. However, our recent study in mice showed that the nuclei of non-growing-stage oocytes at the diplotene stage of the first meiosis are able to complete maturation when transferred into GV cytoplast (Kono et al., 1996Go). This finding revealed that the nuclei of non-growing-stage oocytes are already competent to accomplish the transition to the MII stage. The reconstituted oocytes can be fertilized in vitro and complete the second meiotic stage, emitting the second polar body. However, these oocytes lack the ability to form pronuclei, and this ability is essential for subsequent gene expression and development.

The nuclear membrane and the associated compounds, which are disassembled at the germinal vesicle breakdown (GVBD), diffuse into the ooplasm and are subsequently reassembled during the formation of pronuclei after fertilization (Szollosi, 1993Go). Vesicular components generated by the disassembling of the nuclear envelope and endoplasmic reticulum at the nuclear envelope breakdown (NEBD) are thought to be necessary for pronuclear development (Balakier and Tarkowski, 1980Go). However, the nature of the nuclear factors that directly or indirectly affect chromatin decondensation are still unclear.

The question we wanted to answer was: at what point in their growth cycle do oocytes become competent to form pronuclei after fertilization? To address this point, we reconstructed oocytes by enucleating GV oocytes, and then introducing the nuclei of oocytes from 1–20 day old mice. This allowed us to assess the nuclear competence for pronuclear formation. We show here that GV oocytes reconstructed with non- and early-growing-stage oocytes are unable to decondense the chromatin after resuming meiosis. This suggests that materials and factors derived from fully grown germinal vesicles are needed in order to reassemble the nuclear membrane, and also for the decondensation and recondensation of sperm chromatin.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Collection of oocytes
B6CB F1 (C57BL/6NCrjxCBA/JNCrj) hybrid mice were used as oocyte donors. The ovaries of 1, 8, 13, 15, 17 and 20 day old females were immersed in 3.5 ml of M2 medium (Fulton and Whittingham, 1978Go) containing 1.5 mg/ml crude collagenase (Wako Pure Chemical Industries Ltd, Tokyo, Japan) for 1 h. Oocyte–granulosa cell complexes from the pre-antral follicles were transferred to 0.05% trypsin (Sigma, MO, USA). After 15 min, the complexes were washed and the granulosa cells were removed by pipetting. The oocytes were then placed in phosphate-buffered saline containing 0.5% polyvinylpyrrolidone (Sigma) and 0.5% pronase (Kaken Pharmaceutical Co. Ltd., Tokyo, Japan) to remove the zona pellucida. The oocytes from 1, 8, 13, 15, 17 and 20 day old females, which were 15–20, 35–45, 55–65, 60–70, 65–75 and 70–75 µm in diameter respectively were selected and used as nuclear donor for GV transfer. Oocytes collected from 1 day old mice were defined as `non-growing-stage oocytes'.

Fully grown GV oocytes were collected from adult ovaries 46–48 h after the i.p. injection of 5 IU of equine chorionic gonadotrophin (eCG, Peamex; Sankyo Ltd, Tokyo, Japan). The fully grown GV oocytes were released from the antral follicles using a sterile needle, and the cumulus-intact oocytes were collected. The cumulus cells were removed from the oocytes by pipetting through a fine-bore pipette. To prevent germinal vesicle breakdown (GVBD), all of the manipulation was carried out in an M2 medium containing 240 µmol/l dbcAMP (Sigma), and 5% fetal calf serum (FCS) (Gibco BRL, NY, USA).

GV transfer
GV transfer was carried out by standard micromanipulation techniques (Kono et al., 1996Go; Bao et al., 2000Go). Before GV transfers, the zona pellucida of recipient fully grown GV oocytes were slit with a glass needle along 10–20% of their circumference, and the oocytes were then placed in a small drop of M2 medium containing cytochalasin B (10 µg/ml) and colcemid (0.1 µg/ml). The fully grown GV was removed along with a minimum volume of the cytoplasm using an enucleation pipette 25 µm in diameter. Non-growing oocytes from 1 day old pups and nuclei collected with minimal cytoplasm from growing oocytes of 8, 13, 15, 17 and 20 day old mice were introduced with Sendai virus into the perivitelline space of GV cytoplast. The manipulated oocytes were cultured in Waymouth (Gibco BRL) medium containing dbcAMP to induce cell fusion. The reconstituted oocytes were washed and cultured in Waymouth medium for 17 h in an atmosphere of 5% CO2, 5% O2 and 90% N2 at 37°C.

IVF
The reconstituted oocytes that emitted the first polar bodies after culture in vitro for 17 h were selected for use in experiments. Sperm was collected from known fertile mice and capacitated for 1 h in T6 medium at a concentration of 0.5–1x106 sperm/ml. For IVF, the matured oocytes were incubated with capacitated sperm in T6 medium for up to 3 h. After they were washed, the oocytes were cultured in CZB medium for 5 h.

Effect of DTT on pronuclear development
We used MII oocytes reconstructed with non-growing oocytes at the GV stage to study the effect of dithiothreitol (DTT) on pronuclear development. Oocytes that emitted the second polar bodies were collected 3 h after insemination and placed in a drop of CZB medium containing either 1, 5 or 10 mmol/l DTT for 1 h. After being washed several times with M2 medium, these embryos were cultured in a drop of CZB medium for 4 h, and then processed for assessment of pronuclear development by whole-mount preparation.

Electron microscopic study
The oocytes were fixed in a 0.1 mol/l cacodylate buffer solution (pH 7.4) containing 4% glutaraldehyde and 2% paraformaldehyde at 4°C for 3 h. After being rinsed thoroughly with a 0.1 mol/l cacodylate buffer solution (pH 7.4), the oocytes were post-fixed in a 0.1 mol/l cacodylate buffer solution (pH 7.4) containing 1% osmium tetroxide. The oocytes thus fixed were dehydrated through an acetone series and then embedded in Quetol 812. These samples were cut using an ultramicrotome stained with uranium acetate and lead nitrate, and then photographed under an EM-208 electron microscope (Philips Electron Optics, Eindhoven, The Netherlands).

Statistical analysis
Data was analysed by the {chi}2-test and Student's t-test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Maturation of reconstructed oocytes
After micromanipulation, 90–99% of the oocytes were successfully fused with a non-growing-stage or a karyoplast from the growing-stage oocytes within 20 min. Following culture for maturation, 84–96% of the reconstructed oocytes extruded from the first polar body and formed a normal metaphase plate (Table IGo). These results show that nuclei from oocytes at the diplotene stage of the first meiosis are competent to transform to the MII stage when fused with fully grown GV oocytes.


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Table I. Maturation of the reconstructed oocytes in vitro
 
Pronuclear formation
After fertilization in vitro, the demeanour of the chromatin in the reconstructed oocytes varied significantly depending on the stage of the donor oocytes (Tables II and IIIGoGo; Figure 1Go). The ability of the reconstituted oocytes to develop their pronuclei was examined by fertilization in vitro. A high proportion (>95%) of the reconstructed oocytes resumed meiosis after fertilization, extruding the second polar body. The development of pronuclei, however, was restricted in the oocytes receiving a nucleus from non-growing- and early-growing-stage oocytes. In the oocytes reconstructed with non-growing oocytes, the female chromosomes were arrested as a dense clump of chromatin (13%), and at the reticular nucleus stage (87%) (Table IIGo), while the sperm nuclei were arrested at the decondensed stages, except for one case (6%) that progressed to the swollen nucleus state (Table IIIGo). A pronucleus consisting of swollen chromatin with a clearly defined nuclear envelope was first seen in 27% of the oocytes that were constituted with the nuclei of oocytes from 8 day old pups (Table IIGo). The proportion of oocytes that formed female pronuclei increased with donor oocyte age, and reached 71% when oocytes from 17 day old mice were used as nuclear donors (Table IIGo).


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Table II. Development of female pronuclei in the reconstructed oocytes after IVF
 

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Table III. Development of male pronuclei in the reconstructed oocytes after IVF
 


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Figure 1. Nuclear remodelling in reconstructed oocytes was examined at 8 h after fertilization in vitro. Arrowheads and arrows indicate the female and male nuclei respectively. (a) The female nucleus is in reticular stage 1, and the male is arrested as a decondensed spermhead. (b) The female nucleus is in reticular stage 2, and the male is in the swollen sperm head stage. (c) Both the female and male nuclei are in the pronuclear stage.

 
The progress of the transient sperm nucleus to a male pronucleus is classified into four steps: decondensation, swelling, recondensation, and pronuclear formation (see Figure 1Go). The recondensed sperm chromatin was formed in the oocytes reconstructed with oocytes from 8 day old pups, and a male pronucleus was found in 6 and 29% of the oocytes that were reconstructed with the nuclei of oocytes from 15 and 17 day old pups respectively (Table IIIGo). The sizes of both female and male pronuclei increased with donor age, and the pronuclei reached normal size when oocytes from 17 and 20 day old pups respectively were used (Table IVGo). These results show that the materials necessary for the transition of male and female chromatin to pronuclei accumulate in the nuclei of oocytes during the growth period.


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Table IV. The size of female and male pronuclei (PN) in the reconstructed oocytes
 
Effect of DTT on chromosome decondensation
To test whether or not the reduction of disulphide bonds of chromatin induces the swelling of a sperm nucleus, oocytes containing non-growing oocyte nuclei were treated with DTT after IVF (Table VGo). The DTT treatment induced swelling and recondensation of the sperm nuclei, but not the development of the nuclei to male pronuclei. This suggests that the materials needed for nuclear membrane reassembly and pronuclear development are insufficient in the oocytes.


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Table V. The effect of dithiothreitol (DTT) on transformation of sperm nuclei in the reconstructed oocytes
 
Electron microscopic observation
We examined the in-vitro remodelling of female and male chromatin in oocytes reconstructed with the nuclei of oocytes from 8 day old mice using electron microscopy (Figure 2Go). The female chromatin was decondensed with a reassembly of the nuclear membrane components; however, the membrane was not enriched or completed with break points (Figure 2bGo). While the sperm chromatin started to decondense, the transition of chromatin from decondensation to recondensation did not take place (Figure 2aGo). The swollen sperm head was left in the peripheral region of the oocyte even 8 h after fertilization, at which time the female pronucleus migrated into the centre of the oocyte. These observations show that the components necessary for nuclear membrane reassembly after fertilization are not all present in the reconstructed oocytes, and that the factors that induce the decondensation and recondensation of sperm chromatin and their migration into the centre of the oocyte are lacking.



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Figure 2. Electron micrographs of nuclear remodelling in oocytes reconstructed with nuclei from day 8 oocytes at 8 h after fertilization. (a) The arrowhead shows a swollen sperm head left in the peripheral region of a reconstructed oocyte (x13 000). (b) A female pronucleus in the reconstituted oocyte (x4600). Arrowheads show the border of the female pronucleus. Note the unclear and incomplete reassembly of the nuclear envelope. (c) A pronucleus in a control oocyte (x5800). Arrowheads show the pronuclear envelope.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Meiotic competence
Only mouse oocytes >60 µm in diameter can mature and reach the metaphase of the second meiotic division in vitro through the GVBD and first polar body extrusion; the smaller oocytes remain at the diplotene stage (Szybek, 1972Go; Wassarman and Josefowicz, 1978Go). Earlier studies have shown that meiotic maturation in mitotically incompetent small oocytes can be induced by fusion with mitotically competent oocytes (Balakier, 1978Go). This was further confirmed and clarified in our previous study, which showed that GV-stage oocytes that were enucleated and then received non-growing-stage oocytes matured to the MII stage (Kono et al., 1996Go; Obata et al., 1998Go; Bao et al., 2000Go). When oocytes collected from day 14.5 and 16.5 fetuses were used as nuclear donors, only 0 and 33% respectively formed a normal metaphase (Y.Obata and T.Kono, unpublished data). Therefore, the ability of oocyte nuclei to mature develops independently of the cytoplasm, and this property is present when the nuclei reach the diplotene stage of the first meiotic division. However, the oocytes are unable to form pronuclei after fertilization due to a lack of components, which are derived from GV. This defect, therefore, can be conquered by transferring the MII plate into enucleated, freshly ovulated MII oocytes (Kono et al., 1996Go; Obata et al., 1998Go; Bao et al., 2000Go).

Transformation of male chromatin
Sperm nucleus decondensation is independent of oocyte activation, but the transformation of a decondensed sperm nucleus to a pronucleus is dependent on oocyte activation (Clarke and Masui, 1986Go, 1987Go; Borsuk and Manka, 1988Go). After entry into ooplasm, sperm nuclei must undergo a series of transformations, such as nuclear membrane disintegration, replacement of the sperm-specific protamines with ooplasmic histones, chromatin decondensation and recondensation, and pronuclear development.

The above transformations of the sperm nuclear membrane and chromatin do not take place in the immature GV oocytes. The sperm nuclear membrane is retained when it fuses with immature GV oocytes in mice (Szollosi et al., 1990Go) and rabbits (Berrios and Bedford, 1979Go), but the envelope is disassembled quickly in cattle (Crozet, 1984Go) and hamsters (Usui and Yanagimachi, 1976Go). These results suggest that materials derived from GVBD are required for a series of transformations of sperm nuclei. It has also been reported that the replacement of protamines with histones is necessary for sperm nuclear decondensation and swelling (Yanagimachi, 1994Go). Nucleoplasmine, which is a DNA binding protein, is the most abundant protein in the nuclear matrix. In the present study, the failure of sperm nuclear transformation may be due to a lack of sufficient quantities of nucleoplasmine in the reconstructed oocytes. In oocytes whose nuclei had been replaced with non-growing oocytes at the GV stage and matured in vitro, the swelling of sperm nuclei was restricted, and the nuclei failed to transform to recondensed chromatin. This activity appeared in oocytes that had been reconstituted with the nucleus of a day 8 oocyte, suggesting that this activity is localized in the oocyte nucleus during the early phase of oocyte growth.

In oocytes, because of its stronger affinity, nucleoplasmine that is disassembled into ooplasm at GVBD binds protamine in the presence of glutathione (GSH), which is abundant in the cytoplasm (Boerjan and de Boer, 1990Go; Yoshida et al., 1993Go). DTT, which is a disulphide bond-reducing agent, improves male pronuclear development by reducing the disulphide bonds of DNA-associated protamines (Sutovsky and Schatten, 1997Go). Therefore, we tested the effect of DTT on male pronuclear formation in oocytes that had received non-growing oocyte nuclei. DTT treatment induced swelling and recondensation of sperm nuclei in the oocytes, but no development of the pronucleus was shown. This suggests that the factor necessary for the transition from condensed male chromatin to pronucleus is also lacking in the reconstructed oocytes. Perhaps the factor is localized into the GV during oocyte growth, and disassembled into ooplasm at GVBD, and this may be necessary for both male and female pronuclear development.

Pronuclear development
The ooplasmic material necessary to support sperm and oocyte pronuclei formation is limited, since polyspermic fertilization resulted in imperfect pronuclear development (Yanagimachi, 1994Go). The vesicular components of MII oocytes derived from the nuclear membrane and endoplasmic reticulum are the major sources of the pronuclear membrane (Wilson and Newport, 1988Go). One possible explanation for the failure of complete female pronuclear formation is the insufficient amount of nuclear formative materials in the reconstructed oocytes, because the GV, as a major source of pronuclear membrane components, had been removed. In the meantime, the nuclear laminas, which is a main component of the inner layer of the nuclear envelope and supports the nuclear structure as a reinforcement (Borsuk and Tarkowski, 1989Go; Kubiak et al., 1991Go; Meier et al., 1991Go), was also removed with the GV.

In conclusion, the factors that induce the transformation of sperm nuclei and the formation of male and female pronuclei develop and accumulate in the germinal vesicle during oocyte growth. However, it remains unclear whether the ooplasmic factors that control the development of male pronuclei are identical to or different from those controlling the development of female pronuclei.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank Dr Azim Surani for his critical reading of the manuscript. This work was supported by grants from the Ministry of Education, Science, Culture and Sports of Japan, the Ministry of Agriculture of Japan, and the Japanese Society for the Promotion of Science.


    Notes
 
4 Present address: Wellcome/CRC Institute, University of Cambridge, Tennis Court Road, Cambridge, UK Back

5 Present address: Gene Research Center, Gunma University, Maebashi, Gunma 371-8511, Japan Back

6 To whom correspondence should be addressed at: Department of Bioscience, Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan. E-mail: tomohiro{at}nodai.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Balakier, H. (1978) Induction of maturation in small oocytes from sexually immature mice by fusion with meiotic or mitotic cells. Exp. Cell Res., 112, 137–141.[ISI][Medline]

Balakier, H. and Tarkowski, A.K. (1980) The role of germinal vesicle karyoplasm in the development of male pronucleus in the mouse. Exp. Cell Res., 128, 79–85.[ISI][Medline]

Bao, S., Obata, Y., Carroll, J. and Kono, T. (2000) Epigenetic modifications necessary for normal development are established during oocyte growth in mice. Biol. Reprod., 62, 616–621.[Abstract/Free Full Text]

Berrios, M. and Bedford, J.M. (1979) Oocyte maturation: aberrant post-fusion responses of the rabbit primary oocyte to penetrating spermatozoa. J. Cell Sci., 39, 1–12.[Abstract]

Boerjan, M.L. and de Boer, P. (1990) First cell cycle of zygotes of the mouse derived from oocytes aged postovulation in vivo and fertilized in vivo. Mol. Reprod. Dev., 25, 155–163.[ISI][Medline]

Borsuk, E. and Manka, R. (1988) Behavior of sperm nuclei in intact and bisected metaphase II mouse oocytes fertilized in the presence of colcemid. Gamete Res., 20, 365–376.[ISI][Medline]

Borsuk, E. and Tarkowski, A.K. (1989) Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs. Gamete Res., 24, 471–481.[ISI][Medline]

Clarke, H.J. and Masui, Y. (1986) Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse. J. Cell Biol., 102, 1039–1046.[Abstract]

Clarke, H.J. and Masui, Y. (1987) Dose-dependent relationship between oocyte cytoplasmic volume and transformation of sperm nuclei to metaphase chromosomes. J. Cell Biol., 104, 831–840.[Abstract]

Crozet, N. (1984) Ultrastructural aspects of in vitro fertilization in the cow. Gamete Res., 10, 241–251.[ISI]

Eppig, J.J., Schultz, R.M., O'Brien, M. and Chesnel, F. (1994) Relationship between the developmental programs controlling nuclear and cytoplasmic maturation of mouse oocytes. Dev. Biol., 164, 1–9.[ISI][Medline]

Fulton, B.P. and Whittingham, D.G. (1978) Activation of mammalian oocytes by intracellular injection of calcium. Nature, 273, 149–151.[ISI][Medline]

Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994) Manipulating the Mouse Embryo. CSHL Press, New York.

Kono, T., Obata, Y., Yoshimizu, T., Nakahara, T. and Carroll, J. (1996) Epigenetic modifications during oocyte growth correlates with extended parthenogenetic development in the mouse. Nature Genet., 13, 91–94.[ISI][Medline]

Kubiak, J.Z., Prather, R.S., Maul, G.G. and Schatten, G. (1991) Cytoplasmic modification of the nuclear lamina during pronuclear-like transformation of mouse blastomere nuclei. Mech. Dev., 35, 103–111.[ISI][Medline]

Meier, J., Campbell, K.H., Ford, C.C., Stick, R. and Hutchison, C.J. (1991) The role of lamin LIII in nuclear assembly and DNA replication, in cell-free extracts of Xenopus eggs. J. Cell Sci., 98, 271–279.[Abstract]

Motlik, J. (1989) Cytoplasmic aspects of oocyte growth and maturation in mammals. J. Reprod. Fertil., 38 (Suppl.), 17–25.

Obata, Y., Kaneko-Ishino, T., Koide, T., Takai, Y., Ueda, T., Domeki, I., Shiroishi, T., Ishino, F. and Kono, T. (1998) Disruption of primary imprinting during oocyte growth leads to the modified expression of imprinted genes during embryogenesis. Development, 125, 1553–1560.[Abstract/Free Full Text]

Pedersen, T. and Peters, H. (1968) Proposal for classification of oocytes and follicles in the mouse ovary. J. Reprod. Fertil., 17, 555–557.[Medline]

Peters, H. (1969) The development of the mouse ovary from birth to maturity. Acta Endocrinol. (Copenh.), 62, 98–116.[Medline]

Sorensen, R.A. and Wassarman, P.M. (1976) Relationship between growth and meiotic maturation of the mouse oocytes. Dev. Biol., 50, 531–536.[ISI][Medline]

Sutovsky, P. and Schatten, G. (1997) Depletion of glutathione during bovine oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization. Biol. Reprod., 56, 1503–1512.[Abstract]

Szollosi, D. (1993) Oocyte maturation. In Thibault, C., Leavasseur, M.C. and Hunter, R.H.F. (eds), Reproduction in Mammals and Man. Ellipses, Paris, pp. 307–325.

Szollosi, D., Szollosi, M.S., Czolowska, R. and Tarkowski, A.K. (1990) Sperm penetration into immature mouse oocytes and nuclear changes during maturation: an EM study. Biol. Cell, 69, 53–64.[ISI][Medline]

Szybek, K. (1972) In-vitro maturation of oocytes from sexually immature mice. J. Endocrinol., 54, 527–528.[ISI][Medline]

Usui, N. and Yanagimachi, R. (1976) Behavior of hamster sperm nuclei incorporated into eggs at various stages of maturation, fertilization, and early development. The appearance and disappearance of factors involved in sperm chromatin decondensation in egg cytoplasm. J. Ultrastruct. Res., 57, 276–288.[ISI][Medline]

Wassarman, P.M. and Josefowicz, W.J. (1978) Oocyte development in the mouse: an ultrastructural comparison of oocytes isolated at various stage of growth and meiotic competence. J. Morphol., 156, 209–235.[ISI][Medline]

Wilson, K.L. and Newport, J. (1988) A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro. J. Cell Biol., 107, 57–68.[Abstract]

Yanagimachi, R. (1994) Mammalian fertilization. In Knobil, E. and Neil, J.D. (eds), The Physiology of Reproduction, 2nd edn., New York, pp. 189–317.

Yoshida, M., Ishigaki, K., Nagai, T., Chikyu, M. and Pursel, V.G. (1993) Glutathione concentration during maturation and after fertilization in pig oocytes: relevance to the ability of oocytes to form male pronucleus. Biol. Reprod., 49, 89–94.[Abstract]

Submitted on July 26, 2001; resubmitted on November 7, 2001; accepted on January 13, 2002.