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
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Abstract |
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Key words: germinal vesicle transfer/mouse/oocyte growth/oocyte maturation
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Introduction |
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In mice, primary follicle oocytes, which are 1520 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, 1968; Peters, 1969
). 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, 1978
). 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., 1994
).
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, 1972; Sorensen and Wassarman, 1976
; Eppig et al., 1994
), and in bovine (Motlik, 1989
) 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., 1996
). 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, 1993). 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, 1980
). 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 120 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.
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Materials and methods |
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Fully grown GV oocytes were collected from adult ovaries 4648 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., 1996; Bao et al., 2000
). Before GV transfers, the zona pellucida of recipient fully grown GV oocytes were slit with a glass needle along 1020% 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 0.51x106 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 2-test and Student's t-test.
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Results |
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Discussion |
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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, 1986, 1987
; Borsuk and Manka, 1988
). 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., 1990) and rabbits (Berrios and Bedford, 1979
), but the envelope is disassembled quickly in cattle (Crozet, 1984
) and hamsters (Usui and Yanagimachi, 1976
). 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, 1994
). 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, 1990; Yoshida et al., 1993
). DTT, which is a disulphide bond-reducing agent, improves male pronuclear development by reducing the disulphide bonds of DNA-associated protamines (Sutovsky and Schatten, 1997
). 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, 1994). 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, 1988
). 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, 1989
; Kubiak et al., 1991
; Meier et al., 1991
), 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.
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Acknowledgements |
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Notes |
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5 Present address: Gene Research Center, Gunma University, Maebashi, Gunma 371-8511, Japan
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
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References |
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Submitted on July 26, 2001; resubmitted on November 7, 2001; accepted on January 13, 2002.