1 UMR1198 Biologie du Développement et Reproduction, INRA, Jouy-en-Josas Cedex, 2 Centre FIV Pierre Cherest, Neuilly-sur-Seine Cedex and 3 Laboratoire dEylau, Paris, France
4 To whom correspondence should be addressed at: UMR1198 Biologie du Développement et Reproduction, INRA, 78352, Jouy-en-Josas Cedex, France. e-mail: debey{at}mnhn.fr
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Abstract |
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Key words: cortical granules/human metaphase II oocytes/IVF failure/metaphase-promoting factor/nuclear and cytoplasmic competence
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Introduction |
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The success of clinical IVF is based largely on the ability to retrieve good quality metaphase II (MII) oocytes at a high frequency. The quality of oocytes is characterized by inter-related factors, generally classified respectively as nuclear and cytoplasmic competence. Nuclear competence characterizes the quality of oocyte chromatin and spindle (Mattson and Albertini, 1990; Albertini, 1992
), which is essentially governed by the level of the metaphase-promoting factor (MPF) activity (Hashimoto and Kishimoto, 1988
; Murray, 1989
; Verde et al., 1990
; 1992). MPF governseither directly or indirectlyall processes linked to metaphase entry in eukaryotic cells, such as nuclear envelope breakdown (Ookata et al., 1992
), histone phosphorylation (Collas, 1999
) and microtubule polymerization (Charrasse et al., 2000
). Mitogen-activated protein (MAP) kinase (a serine/threonine kinase) is another principal regulator of oocyte maturation, and its action in regulating cell cycle events may be uncoupled from MPF in mammalian oocytes (Sun et al., 1999
). The results of recent studies have shown that MAP kinase is more important than MPF in controlling chromatin and microtubule behaviour in both mouse (Verlhac et al., 1994
) and porcine oocytes (Sun et al., 2001
; 2002). Activation of MAP kinases occurs after germinal vesicle breakdown (GVBD) and keeps microtubules and chromatin from entering into an interphase configuration (Sun et al., 1999
). In human oocytes, extrusion of the first polar body and formation of the MII oocyte occurs at 32 h after ovarian stimulation, this being 68 h before ovulation (Bomsel-Helmreich et al., 1987
). The presence of a functional spindle ensures the fidelity of chromosome segregation, and is thus an essential feature of healthy MII oocytes (Eichenlaub-Ritter et al., 1986
; Van Blerkom and Henry, 1992
; Volarcik et al., 1998
; Van Blerkom and Davis, 2001
). Ageing of MII oocytes, thermal changes and insufficient oxygen supply throughout the entire culture period (Hu et al., 2001
; Wang et al., 2001
) may cause concomitantly a deterioration of the spindle and displacement of chromosomes from the spindle equator (Eichenlaub-Ritter et al., 1986
).
Cytoplasmic maturation can be considered as the sum of the processes by which the mammalian oocyte changes from a developmentally incompetent cell to one with the capacity to support fertilization and early embryonic development. One of these changes concerns the redistribution of cell organelles named cortical granules (CGs) (Ducibella et al., 1990; 1993; Yoshida et al., 1993
). CGs are small (0.20.6 µm diameter) secretory granules which were first identified using electron microscopy and originate from the Golgi body (Gulyas, 1976
; Manna et al., 2001
). During oocyte growth, the CGs increase in number and migrate toward the cortex, assuming a position 0.40.6 µm below the plasma membrane (Ducibella et al., 1988
; 1994). In large germinal vesicle (GV) mouse oocytes, most of the CGs are uniformly localized in the cortex, while a population of CGs remains in the interior. Meiotic maturation is accompanied by an additional movement of CGs residing in the interior to the periphery of the oocyte, as well as the formation of a CG-free domain (CGFD) overlying the MII spindle (Ducibella et al., 1988
; 1990; Connors et al., 1998
). The cortical accumulationbut not formation of the CGFDis observed during meiotic maturation in others mammals, such as cat (Byers et al., 1992
), pig (Sun et al., 2001
), cattle (Izadyar et al., 1998
) and human (Ghetler et al., 1998
). CGs contain mucopolysaccharides and several enzymes such as proteases, peroxidase and acid phosphatase (Flechon, 1970
; Moller and Wassarman, 1989
; Hoodbhoy and Talbot, 1994
). Electron microscopic studies have shown that the CGs positioned beneath the plasma membrane undergo exocytosis in response to an inositol triphosphate (IP3)-induced calcium rise generated by fertilization or parthenogenetic activation (Schultz and Kopf, 1995
) and extrude their contents into the perivitellin space (Chamow and Hedrick, 1986
). This release modifies the extracellular coat of the oocyte (Connors et al., 1998
), thus inducing an extracellular block to polyspermy.
Another criterion of cytoplasmic maturation is the ability to decondense the sperm chromatin following sperm penetration (Navara et al., 1995) and to transform it into the male pronucleus. Decondensation of the spermatozoon is considered to occur independently from the resumption of meiosis, under the influence of a cytoplasmic factor called sperm decondensation factor (SDF), the synthesis of which is induced during normal oocyte maturation (Moor and Gandolfi, 1987
). In contrast, changes in sperm chromatin beyond the initial decondensation stage depend on cytoplasmic conditions which also permit female pronucleus formation; that is, the decrease in MPF activity triggered by sperm entry (Adenot et al., 1991
; Gook et al., 1998
; Kovacic and Vlaisavljevic, 2000
; Bao et al., 2002
). Moreover, in-vitro insemination of metaphase I oocytes leads to condensation of male chromatin into premature chromosome condensation (PCC) (Clarke and Masui, 1986
; 1987).
The aim of the present study, which involved the close collaboration of clinicians and cell biologists, was to evaluate oocyte and/or sperm defects in cases of very low rates of fertilization or complete fertilization failure. For that purpose, an a posteriori multiparametric analysis was undertaken of human oocytes which failed to produce pronucleated zygotes after IVF. Patients presented with tubal disease, endometriosis, polycystic ovary syndrome (PCOS) or unexplained infertility. The decision was made to analyse both female and male chromatin, together with the microtubular array, as well as CG distribution. When used in conjunction with the patient clinical data, these observationswhich may be gathered using regular microscopic equipmentare aimed towards understanding the physiological causes of fertilization failure, and represent valuable tools for the future orientation of therapeutics in the treatment of infertility.
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Materials and methods |
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Oocyte collection
At 36 h after stimulation, oocytes were retrieved using transvaginal ultrasound-guided aspiration. Cumulus-enclosed oocytes were collected in IVFTM-20 medium (Vitrolife; Mölndalsvägen, Gothenburg, Sweden) and left intact for conventional IVF. Cumulusoocyte complexes were inseminated with
2x105 motile sperm in 1 ml Scandinavian IVF medium at 37°C in 5% CO2 in an humidified atmosphere. Fertilization was assessed at 1720 h after insemination (day 1) by the presence of two pronuclei and polar bodies. On day 2, normal embryos were transplanted to the uterine cavity or, if necessary, frozen for later transplantation. Oocytes remaining at the MII stage at 48 h after insemination were removed and used for further multiparametric analysis. Patients from ICSI programmes who had not been inseminated (e.g. azoospermia due to complete absence of spermatozoa) donated control oocytes at the MII stage. The MII-stage controls were analysed on the same day as puncture.
Fluorescence staining of meiotic spindles and chromosomes
Oocytes were fixed in 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at 37°C for 30 min; this fixative is known to preserve cellular structures and antigenic sites (Grootenhuis et al., 1996). Oocytes were then rinsed in PBS, permeabilized by the addition of 1% Triton X-100 in PBS for 20 min, and incubated in PBS + 2% bovine serum albumin (BSA). Incubation with the primary antibody, a mouse monoclonal antibody (IgG) raised against
-tubulin (Clone DM1A; Amersham Pharmacia, France) diluted 1:400 in PBS + 2% BSA, was performed overnight at 4°C. Oocytes were rinsed several times in PBS, and incubated for 1 h at room temperature with the secondary antibody, a fluoroscein isothiocyanate (FITC)-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, USA) diluted 1:200 in PBS + 2% BSA. Excess antibody was washed out in PBS. The oocytes were labelled using 2 µg/ml Hoechst 33342 (Riedel de Haen, Germany) for 20 min, transferred into PBS, and then mounted under a coverslip in citifluor (Citifluor Products, Canterbury, UK).
Stained oocytes were examined using an inverted microscope (Carl Zeiss, Oberkochen, Germany) that was equipped for epifluorescence and fitted with appropriate filter combinations. Images were recorded through a charge-coupled device (CCD) camera (Type KAF 1400, 12-bit range; Photometrics, Tucson, AZ, USA) cooled to 10°C, and coupled to the IPLAB spectrum imaging software (Vysis, France). Acquired images were processed using Adobe Photoshop 6.0 (Adobe Systems, Mountain View, CA, USA).
Analysis of CG distribution
Cortical granules were labelled with lens culinaris agglutinin (LCA); this lectin was reported previously to bind specifically to the CGs content and exudate (Ducibella et al., 1994). Oocytes were first fixed as above in 2% PFA in PBS for 30 min at 37°C, and washed in PBS containing 2% BSA. The ZP was then removed mechanically with a small needle and narrow-mouth glass pipettes. Previous attempts to use pronase (0.25%) or
-chymotrypsin (0.01%) before fixation failed, because: (i) a large number of oocytes, in which the ZP was not easily detached, were lysed due to over-exposure to enzymes; and (ii)
-chymotrypsin and pronase caused holes to develop in the plasma membrane, thereby rendering the results of the CG labelling uninterpretable. Labelling of CGs was subsequently performed according to a previously published method (Ghetler et al., 1998
). Briefly, oocytes were incubated in 5 µg/ml biotin-conjugated LCA (Sigma-Aldrich Chimie, France) for 30 min, and stained with 2 µg/ml rhodamine redstreptavidin (Jackson ImmunoResearch) for 30 min. Finally, oocytes were labelled with 1 µg/ml Sytox green (Molecular Probes, Eugene, OR, USA), which stains double-stranded DNA for 15 min, transferred into PBS, mounted under a coverslip in citifluor, and then observed on an inverted microscope (Nikon France, Paris, France) equipped with Bio-Rad LaserSharp MRC-1024 confocal laser scanning software (Elexience, Paris, France). The objective lens was a Nikon Fluor oil-immersion 100x (NA 1.3), and the pinhole was set at between 1.5 and 2.8 µm. The 488 nm and 568 nm wavelengths of the laser were used for excitation of fluorescein (or Sytox green) and rhodamine respectively. Optical sections were imaged at 5 µm intervals.
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Results |
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Quality of the metaphasic spindle
The status of the metaphasic plate (assembly of chromosomes at the spindle equator) represents a more advanced characterization of oocyte quality. Some 53% of all MII oocytes exhibited a normal spindle (Figure 2A,c,d), though this proportion was higher in cortical (70% on average) than in centripetal MII (46% on average) (Table III).
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When patients were grouped according to their age, it was noted that oocytes from older patients (aged >40 years) showed a higher proportion of aberrant MII, such as chromosome misalignment or completely disrupted spindles (69% for 40- to 45-year-old women versus 35% for 26- to 33-year-old) (Table III). Remarkably, this increase was seen to be particularly drastic for oocytes that were mature at the time of retrieval (centripetal), but was much lower in the case of oocytes that were not mature at the time of retrieval (cortical) (Table III).
In contrast, control oocytes presented a cortical MII plate with a symmetric, barrel-shaped meiotic spindle which was radially oriented, and there were no detectable cytoplasmic asters (Figure 2A,a). In addition, these control oocytes with a normal spindle generally did not show any morphological abnormalities: the ZP was of normal thickness, the perivitellin space was limited to the site of normal PB1 extrusion, and the cytoplasm was without vacuoles or dense granules.
Cortical reaction
Migration and redistribution of CGs in the cortex is a common attribute of cytoplasmic maturation. The CGs may undergo exocytosis after activation of the oocyte at fertilization, and this is termed the cortical reaction.
A total of 33 oocytes was analysed for the distribution of CGs, organization of the meiotic spindle, and alignment of chromosomes on the metaphasic plate. A good correlation between nuclear maturation of the oocyte and CG localization was found, with three cases being identified:
1. CGs distributed as large aggregates in the entire cytoplasm, with very few at the level of the plasma membrane (Figure 2B,a). This distribution was similar to that seen in human GV oocytes (data not shown). In that case, abnormalities were observed in most cases (88%) in the organization of the meiotic spindle and metaphasic plate (Figure 2B,a).
2. CGs mainly located at the periphery of the oocyte. Below this ring of CGs, small CG aggregates still remained in the cytoplasm (Figure 2B,b). The spindle and metaphasic plate in these oocytes were well organized, but located cortically. Their position with respect to the PB could not be analysed as it was detached from the oocyte when the ZP was removed (Figure 2B,b). In this case, it could be suggested that the delay of CG translocation and fusion with the plasma membrane reflects an uncompleted nuclear maturation at the time of puncture. It is believed that fertilization failure and infertility in those cases might be attributable to uncoupling of the nuclear and cytoplasmic maturation processes.
3. All CGs fused with the plasma membrane, so that it was uniformly labelled (Figure 2B). In this case, the spindle and chromosomes were well organized and in the centripetal position (Figure 2B,a). Thus, cytoplasmic maturationwhen estimated by the migration of CGsseems to be associated with the achievement of nuclear maturation at the time of puncture.
Combination of cellular and clinical analyses
From a clinical point of view, the analysis of oocytes not fertilized after IVF may be of diagnostic value. IVF cycles without recognized male factor for infertility, but without cleavage after IVF, may reveal a sperm defectespecially when the majority of oocytes have well-organized chromosomes and spindle and are not penetrated by sperm. On the other hand, the presence of sperm chromatin in some uncleaved oocytes excluded the possibility of sperm penetration incapacity; in this case, an oocyte problem could be suspected.
Classified as fertilizable were those oocytes which had: (i) chromosomes that were well aligned on the metaphasic plate and in the middle of a barrel-shaped spindle; (ii) the chromosomes and meiotic spindle in a centripetal position; and (iii) CGs that were exclusively cortical. Classified as unfertilizable were those oocytes which had one of the following defects: (i) chromosomes totally dispersed on a well-organized spindle; (ii) chromosomes outside the metaphasic plate; (iii) aberrations in the spindle; and (iv) still condensed chromatin with interphasic microtubules. All of these meiotic defects were generally accompanied by poor migration of the CGs.
In addition to the quality of the oocytes as assessed above, data relating to the age of the patients, the pathology as indicated by clinicians, the number of IVF attempts, and the protocols of ovarian stimulation were given. The present study focused especially on endometriosis, PCOS and idiopathic infertility. When the majority of a patients oocytes were fertilizable, but not fertilized at the first attempt, then a new attempt was recommended. In such cases, a good pregnancy rate was obtained with this new attempt, irrespective of the pathology encountered (Table IV), the pregnancy rate being similar after either IVF or ICSI (38 versus 32%). By contrast, when the majority of the oocytes was judged to be non-fertilizable at a first attempt, then no pregnancy was registered at a second attempt (Table IV). These findings clearly demonstrate the reliability of the a posteriori multiparametric analysis utilized in the present study.
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Discussion |
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In contrast, in the present study an incidence of 3545.5% abnormalities was found in organization of the spindle and metaphasic plate in patients aged between 26 and 33 years, while an incidence of 69% was found in those women aged 4045 years. This difference suggests a strong dependence on patient agea result which is in line with earlier reports which showed more frequent defects in chromosome alignment and spindle formation in MII oocytes from aged, non-stimulated women (Battaglia et al., 1996; Battaglia and Miller, 1997
; Volarcik et al., 1998
), together with a higher incidence of errors in chromosome segregation in the first meiotic division (Volarcik et al., 1998
). The 69% incidence of abnormalities found in the present study among non-fertilized MII oocytes from 40- to 45-year old women was slightly less than the value of 79% observed among MII oocytes from women of the same age but without ovarian stimulation (Battaglia et al., 1996
). This led to the consideration that more oocytes from 40- to 45-year-old women, after being fertilized, present with defects in their meiotic spindle and chromosome organization. Indeed, 91% of these oocytes (n = 22) did not achieve pregnancy after implantation, most likely as a consequence of the bad organization of the spindle and chromosomes before fertilization. In fact, it appears that meiotic aberrations (i.e. chromosome misalignment and spindle disruption) do not prevent meiotic progression from MI to MII, but rather meiosis seems to proceed by default (Liu and Keefe, 2002
). Although ageing-associated infertility has also been found in mice, age-related abnormalities in meiotic spindle organization and chromosome alignment have not been recognized (Eichenlaub-Ritter et al., 1988
; Van Blerkom and Davis, 2001
). In addition, the aneuploidy rate does not increase significantly with mouse age (Zuccotti et al., 1998
). Much higher rates of meiotic aberration were found in old, senescence-accelerated mouse (SAM) oocytes (Liu and Keefe, 2002
). SAM mice exhibit mitochondrial dysfunction and oxidative damage early during ageing (Mori et al., 1998
). Oxidative stress has been shown to induce disturbances in chromosomal distribution in the MII spindle of mouse oocytes (Tarin et al., 1998
), whereas mitochondrial dysfunction might compromise the activity of microtubule motor proteins as a consequence of a reduced energy supply (Liu and Keefe, 2002
). In this respect, the severe disturbances seen in old SAM oocytes may mimic better the human situation.
Besides defects in the extent of nuclear maturation and/or organization of the meiotic chromosomes, defects in the oocyte cytoplasm might also account for fertilization failures. One outstanding feature of correct cytoplasmic maturation is migration of the CGs. In fact, a strong correlation between CG translocation and quality of the metaphasic plate was observed, as no CG translocation was seen in oocytes with abnormalities in the meiotic spindle and metaphasic plate organization, whereas CGs were translocated in all normal centripetal MII-stage oocytes. Microtubules have been suggested to be involved in correct CG translocation (Kim et al., 1996; Abbott et al., 2001
), and a role for microfilaments has also been proposed in mouse (Tahara et al., 1996
; Connors et al., 1998
), hamster (DiMaggio et al., 1997
) and porcine (Kim et al., 1996
) oocytes. An alternate hypothesis is that unfertilized MII-stage oocytes are deficient in a Ca2+-dependent signalling component necessary for CG translocation and subsequent extrusion (Abbott et al., 2001
). In mouse oocytes, artificial CG extrusion was induced by a Ca2+ ionophore (Tatone et al., 1999
), while inhibition of calcium-dependent protein kinase II (CAMK II) negatively affected CG exocytosis and MPF inactivation (Tatone et al., 2002
), and blocked transit from MII to anaphase II (Johnson et al., 1998
). The simultaneous effect of the CAMK II on CG migration and MPF activity suggests that disorganization of the spindle, blocking of the cortical reaction and premature condensation of the sperm chromatin that was seen in many of the oocytes analysed might all be related to anomalies in CAMK II activity. The confirmation of a physiological role for CAMK II during human fertilization requires further investigation, however.
Finally, the absence of sperm in 85.5% of analysed oocytes after IVF failure is very surprising, and hardening of the ZP during insemination is the most suspected cause (De Felici et al., 1985; Bedford and Kim, 1993
). The released CG content is thought to be responsible for ZP hardening. In humans, a reduction in the number of CGs has been reported in pre-ovulatory oocytes (Rousseau et al., 1977
), fertilized oocytes (Lopata et al., 1980
) and IVF fertilization-failed oocytes (Ducibella et al., 1995
). On the other hand, the possibility that the ZP may undergo changes that are not dependent on CG exocytosis has been previously reported (De Felici and Siracusa, 1982
; Dolci et al., 1991
). For example, one group (Talevi et al., 1997
) reported that fertilization-failed human oocytes showed alterations in carbohydrate distribution of the ZP that were not related to cortical reaction. In this case, hardening is probably a consequence of intrinsic modifications of the zona components which might block sperm penetration.
Although the zona and oolemma barriers may be bypassed with ICSI (Palermo et al., 1992), the results of the present study show that the difference between pregnancy rates obtained with a new attempt of IVF or ICSI in the case of non-male sterility and good quality oocytes, is not significant (38 versus 32%). In turn, ICSI is often associated with reduced blastocyst formation (Dumoulin et al., 2000
; Griffiths et al., 2000
), sperm DNA fragmentation (Sakkas et al., 1999
) and an increase in the rate of aneuploidy (Bernardini et al., 1997
). The technique itself may have negative effects on embryonic development (Griffiths et al., 2000
; Dumoulin et al., 2001
). These arguments do not support the use of the ICSI in all treatments of assisted reproductive technologies.
Based on the results of the present study, it may be concluded that an analysis of three complementary componentsDNA, spindle and CGsrequires only the use of a fluorescence microscope, yet permits rapid assessment of oocyte quality. Further simplification of these protocols would render the analyses both rapid and amenable to the clinical situation. Moreover, all three factorswhen analysed on a complete set of oocytes from the same patientprovide information relating to potential causes of IVF failure. They should therefore be considered as part of an oocyte quality evaluation needed to select the clinically assisted fertilization method best suited to an individual patient.
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Acknowledgements |
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References |
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---|
Adenot, P.G., Szollosi, M.S., Geze, M. Renard, J.P. and Debey, P. (1991) Dynamics of paternal chromatin changes in live one-cell mouse embryo after natural fertilization. Mol. Reprod. Dev., 28, 2334.[ISI][Medline]
Albertini, D.F. (1992) Cytoplasmic microtubular dynamics and chromatin organization during mammalian oogenesis and oocyte maturation. Mutat. Res., 296, 5768.[ISI][Medline]
Bao, S., Obata, Y., Ono, Y., Futatsumata, N., Niimura, S. and Kono, T. (2002) Nuclear competence for maturation and pronuclear formation in mouse oocytes. Hum. Reprod., 17, 13111316.
Battaglia, D.E. and Miller, M. (1997) The aging oocyte. Endocrinologist, 7, 15.[ISI]
Battaglia, D.E., Goodwin, P., Klein, N.A. and Soules, M.R. (1996) Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum. Reprod., 11, 22172222.[Abstract]
Bedford, J.M. and Kim, H.H. (1993) Sperm/egg binding patterns and oocyte cytology in retrospective analysis of fertilization failure in vitro. Hum. Reprod., 8, 453463.[Abstract]
Belsey, M.A., Eliasson, R., Gallegos, A.J. Moghissi, K.S., Paulson, C.A. and editors (1980) World Health Organization: Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. Press Concern, MNR Prasad, Singapore.
Bernardini, L., Martini, E., Geraedts, J.P. Hopman, A.H., Lanteri, S., Conte, N. and Capitanio, G.L. (1997) Comparison of gonosomal aneuploidy in spermatozoa of normal fertile men and those with severe male factor detected by in-situ hybridization. Mol. Hum. Reprod., 3, 431438.[Abstract]
Bomsel-Helmreich, O., Huyen, L.V., Durand-Gasselin, I., Salat-Baroux, J. and Antoine, J.M. (1987) Timing of nuclear maturation and cumulus dissociation in human oocytes stimulated with clomiphene citrate, human menopausal gonadotropin, and human chorionic gonadotropin. Fertil. Steril., 48, 586595.[ISI][Medline]
Byers, A.P., Barone, M.A., Donoghue, A.M. and Wildt, D.E. (1992) Mature domestic cat oocyte does not express a cortical granule-free domain. Biol. Reprod., 47, 709715.[Abstract]
Caligara, C., Navarro, J., Vargas, G., Simon, C., Pellicer, A. and Remohi, J. (2001) The effect of repeated controlled ovarian stimulation in donors. Hum. Reprod., 16, 23202323.
Chamow, S.M. and Hedrick, J.L. (1986) Subunit structure of a cortical granule lectin involved in the block to polyspermy in Xenopus laevis eggs. FEBS Lett., 206, 353357.[CrossRef][ISI][Medline]
Charrasse, S., Lorca, T., Doree, M. and Larroque, C. (2000) The Xenopus XMAP215 and its human homologue TOG proteins interact with cyclin B1 to target p34cdc2 to microtubules during mitosis. Exp. Cell Res., 254, 249256.[CrossRef][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, 10391046.[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, 831840.[Abstract]
Collas, P. (1999) Sequential PKC- and Cdc2-mediated phosphorylation events elicit zebrafish nuclear envelope disassembly. J. Cell Sci., 112 (Pt 6), 977987.
Connors, S.A., Kanatsu-Shinohara, M., Schultz, R.M. and Kopf, G.S. (1998) Involvement of the cytoskeleton in the movement of cortical granules during oocyte maturation, and cortical granule anchoring in mouse eggs. Dev. Biol., 200, 103115.[CrossRef][ISI][Medline]
De Felici, M. and Siracusa, G. (1982) Survival of isolated, fully grown mouse ovarian oocytes is strictly dependent on external Ca2+. Dev. Biol., 92, 539543.[ISI][Medline]
De Felici, M., Boitani, C. and Cossu, G. (1985) Synthesis of glycoconjugates in mouse primordial germ cells. Dev. Biol., 109, 375380.[ISI][Medline]
DiMaggio, A.J., Jr, Lonergan, T.A. and Stewart-Savage, J. (1997) Cortical granule exocytosis in hamster eggs requires microfilaments. Mol. Reprod. Dev., 47, 334340.[CrossRef][ISI][Medline]
Dolci, S., Bertolani, M.V., Canipari, R. and De Felici, M. (1991) Involvement of carbohydrates in the hardening of the zona pellucida of mouse oocytes. Cell. Biol. Int. Rep., 15, 571579.[ISI][Medline]
Dor, J., Seidman, D.S., Ben-Shlomo, I., Levran, D., Ben-Rafael, Z. and Mashiach, S. (1996) Cumulative pregnancy rate following in-vitro fertilization: the significance of age and infertility aetiology. Hum. Reprod., 11, 425428.[Abstract]
Ducibella, T., Anderson, E., Albertini, D.F., Aalberg, J. and Rangarajan, S. (1988) Quantitative studies of changes in cortical granule number and distribution in the mouse oocyte during meiotic maturation. Dev. Biol., 130, 184197.[ISI][Medline]
Ducibella, T., Duffy, P., Reindollar, R. and Su, B. (1990) Changes in the distribution of mouse oocyte cortical granules and ability to undergo the cortical reaction during gonadotropin-stimulated meiotic maturation and aging in vivo. Biol. Reprod., 43, 870876.[Abstract]
Ducibella, T., Kurasawa, S., Duffy, P., Kopf, G.S. and Schultz, R.M. (1993) Regulation of the polyspermy block in the mouse egg: maturation-dependent differences in cortical granule exocytosis and zona pellucida modifications induced by inositol 1,4,5-trisphosphate and an activator of protein kinase C. Biol. Reprod., 48, 12511257.[Abstract]
Ducibella, T., Duffy, P. and Buetow, J. (1994) Quantification and localization of cortical granules during oogenesis in the mouse. Biol. Reprod., 50, 467473.[Abstract]
Ducibella, T., Dubey, A., Gross, V., Emmi, A., Penzias, A.S., Layman, L. and Reindollar, R. (1995) A zona biochemical change and spontaneous cortical granule loss in eggs that fail to fertilize in in vitro fertilization. Fertil. Steril., 64, 11541161.[ISI][Medline]
Dumoulin, J.C., Coonen, E., Bras, M., van Wissen, L.C., Ignoul-Vanvuchelen, R., Bergers-Jansen, J.M., Derhaag, J.G., Geraedts, J.P. and Evers, J.L. (2000) Comparison of in-vitro development of embryos originating from either conventional in-vitro fertilization or intracytoplasmic sperm injection. Hum. Reprod., 15, 402409.
Dumoulin, J.M., Coonen, E., Bras, M., Bergers-Janssen, J.M., Ignoul-Vanvuchelen, R.C., van Wissen, L.C., Geraedts, J.P. and Evers, J.L. (2001) Embryo development and chromosomal anomalies after ICSI: effect of the injection procedure. Hum. Reprod., 16, 306312.
Eichenlaub-Ritter, U., Chandley, A.C. and Gosden, R.G. (1986) Alterations to the microtubular cytoskeleton and increased disorder of chromosome alignment in spontaneously ovulated mouse oocytes aged in vivo: an immunofluorescence study. Chromosoma, 94, 337345.[ISI][Medline]
Eichenlaub-Ritter, U., Chandley, A.C. and Gosden, R.G. (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, 220226.[ISI][Medline]
Fishel, S., Aslam, I., Lisi, F., Rinaldi, L., Timson, J., Jacobson, M., Gobetz, L., Green, S., Campbell, A. and Lisi, R. (2000) Should ICSI be the treatment of choice for all cases of in-vitro conception? Hum. Reprod., 15, 12781283.
Flechon, J.E. (1970) Nature glycoproteique des granules corticaux de loeuf de lapine. J. Microsc., 9, 221242.[ISI]
Ghetler, Y., Raz, T., Ben Nun, I. and Shalgi, R. (1998) Cortical granules reaction after intracytoplasmic sperm injection. Mol. Hum. Reprod., 4, 289294.[Abstract]
Gook, D., Osborn, S.M., Bourne, H., Edgar, D.H. and Speirs, A.L. (1998) Fluorescent study of chromatin and tubulin in apparently unfertilized human oocytes following ICSI. Mol. Hum. Reprod., 4, 11301135.[Abstract]
Griffiths, T.A., Murdoch, A.P. and Herbert, M. (2000) Embryonic development in vitro is compromised by the ICSI procedure. Hum. Reprod., 15, 15921596.
Grootenhuis, A.J., Philipsen, H.L., de Breet-Grijsbach, J.T. and van Duin, M. (1996) Immunocytochemical localization of ZP3 in primordial follicles of rabbit, marmoset, rhesus monkey and human ovaries using antibodies against human ZP3, J. Reprod. Fertil. Suppl., 50, 4354.[Medline]
Gulyas, B.J. (1976) Ultrastructural observations on rabbit, hamster and mouse eggs following electrical stimulation in vitro. Am. J. Anat., 147, 203218.[ISI][Medline]
Hashimoto, N. and Kishimoto, T. (1988) Regulation of meiotic metaphase by a cytoplasmic maturation-promoting factor during mouse oocyte maturation. Dev. Biol., 126, 242252.[ISI][Medline]
Hoodbhoy, T. and Talbot, P. (1994) Mammalian cortical granules: contents, fate, and function. Mol. Reprod. Dev., 39, 439448.[ISI][Medline]
Horne, G., Atkinson, A., Brison, D.R., Radford, J., Yin, J.A., Edi-Osagie, E.C., Pease, E.H. and Lieberman, B.A. (2001) Achieving pregnancy against the odds: successful implantation of frozen-thawed embryos generated by ICSI using spermatozoa banked prior to chemo/radiotherapy for Hodgkins disease and acute leukaemia. Hum. Reprod., 16, 107109.
Hu, Y., Betzendahl, I., Cortvrindt, R., Smitz, J. and Eichenlaub-Ritter, U. (2001) Effects of low O2 and ageing on spindles and chromosomes in mouse oocytes from pre-antral follicle culture. Hum. Reprod., 16, 737748.
Izadyar, F., Hage, W.J., Colenbrander, B. and Bevers, M.M. (1998) The promotory effect of growth hormone on the developmental competence of in vitro matured bovine oocytes is due to improved cytoplasmic maturation. Mol. Reprod. Dev., 49, 444453.[CrossRef][ISI][Medline]
Johnson, J., Bierle, B.M., Gallicano, G.I. and Capco, D.G. (1998) Calcium/calmodulin-dependent protein kinase II and calmodulin: regulators of the meiotic spindle in mouse eggs. Dev. Biol., 204, 464477.[CrossRef][ISI][Medline]
Kim, N.H., Funahashi, H., Abeydeera, L.R., Moon, S.J., Prather, R.S. and Day, B.N. (1996) Effects of oviductal fluid on sperm penetration and cortical granule exocytosis during fertilization of pig oocytes in vitro. J. Reprod. Fertil., 107, 7986.[Abstract]
Kovacic, B. and Vlaisavljevic, V. (2000) Configuration of maternal and paternal chromatin and pertaining microtubules in human oocytes failing to fertilize after intracytoplasmic sperm injection. Mol. Reprod. Dev., 55, 197204.[CrossRef][ISI][Medline]
Liu, L. and Keefe, D.L. (2002) Ageing-associated aberration in meiosis of oocytes from senescence-accelerated mice. Hum. Reprod., 17, 26782685.
Lopata, A., Sathananthan, A.H., McBain, J.C., Johnston, W.I. and Speirs, A.L. (1980) The ultrastructure of the preovulatory human egg fertilized in vitro. Fertil. Steril., 33, 1220.[ISI][Medline]
Manna, C., Rienzi, L., Greco, E., Sbracia, M., Rahman, A., Poverini, R., Siracusa, G. and De Felici, M. (2001) Zona pellucida solubility and cortical granule complements in human oocytes following assisted reproductive techniques. Zygote, 9, 201210.[CrossRef][ISI][Medline]
Mattson, B.A. and Albertini, D.F. (1990) Oogenesis: chromatin and microtubule dynamics during meiotic prophase. Mol. Reprod. Dev., 25, 374383.[ISI][Medline]
Moller, C.C. and Wassarman, P.M. (1989) Characterization of a proteinase that cleaves zona pellucida glycoprotein ZP2 following activation of mouse eggs. Dev. Biol., 132, 103112.[ISI][Medline]
Moor, R.M. and Gandolfi, F. (1987) Molecular and cellular changes associated with maturation and early development of sheep eggs. J. Reprod. Fertil. Suppl., 34, 5569.[Medline]
Mori, A., Utsumi, K., Liu, J. and Hosokawa, M. (1998) Oxidative damage in the senescence-accelerated mouse. Ann. N. Y. Acad. Sci., 854, 239250.
Murray, A.W. (1989) Cyclin synthesis and degradation and the embryonic cell cycle. J. Cell Sci. Suppl., 12, 6576.[Medline]
Navara, C.S., Simerly, C., Zoran, S. and Schatten, G. (1995) The sperm centrosome during fertilization in mammals: implications for fertility and reproduction. Reprod. Fertil. Dev., 7, 747754.[ISI][Medline]
Ola, B., Afnan, M., Sharif, K., Papaioannou, S. Papaioannou, S., Hammadieh, N. and Barratt, C.L. (2001) Should ICSI be the treatment of choice for all cases of in-vitro conception? Considerations of fertilization and embryo development, cost effectiveness and safety. Hum. Reprod., 16, 24852490.
Ookata, K., Hisanaga, S., Okano, T., Tachibana, K. and Kishimoto, T. (1992) Relocation and distinct subcellular localization of p34cdc2-cyclin B complex at meiosis reinitiation in starfish oocytes. EMBO J., 11, 17631772.[Abstract]
Palermo, G., Joris, H., Devroey, P. and Van Steirteghem, A.C. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, 1718.[ISI][Medline]
Palermo, G.D., Takeuchi, T. and Rosenwaks, Z. (2002) Technical approaches to correction of oocyte aneuploidy. Hum. Reprod., 17, 21652173.
Rousseau, P., Meda, P., Lecart, C., Haumont, S. and Ferin, J. (1977) Cortical granule release in human follicular oocytes. Biol. Reprod., 16, 104111.[ISI][Medline]
Sakkas, D., Mariethoz, E., Manicardi, G., Bizzaro, D., Bianchi, P.G. and Bianchi, U. (1999) Origin of DNA damage in ejaculated human spermatozoa. Rev. Reprod., 4, 3137.
Schultz, R.M. and Kopf, G.S. (1995) Molecular basis of mammalian egg activation. Curr. Top. Dev. Biol., 30, 2162.[ISI][Medline]
Sun, Q.Y., Breitbart, H. and Schatten, H. (1999) Role of the MAPK cascade in mammalian germ cells. Reprod. Fertil. Dev., 11, 443450.[ISI][Medline]
Sun, Q.Y., Lai, L., Park, K.W., Bonk, A., Cabot, R., Park, K.W., Day, B.N., Prather, R.S. and Schatten, H. (2001) Dynamic events are differently mediated by microfilaments, microtubules, and mitogen-activated protein kinase during porcine oocyte maturation and fertilization in vitro. Biol. Reprod., 64, 879889.
Sun, Q.Y., Wu, G.M., Lai, L., Bonk, A., Cabot, R., Park, K.W., Day, B.N., Prather, R.S. and Schatten, H. (2002) Regulation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro. Biol. Reprod., 66, 580588.
Tahara, M., Tasaka, K., Masumoto, N., Mammoto, A., Ikebuchi, Y. and Miyake, A. (1996) Dynamics of cortical granule exocytosis at fertilization in living mouse eggs. Am. J. Physiol., 270, C1354C1361.
Talevi, R., Gualtieri, R., Tartaglione, G. and Fortunato, A. (1997) Heterogeneity of the zona pellucida carbohydrate distribution in human oocytes failing to fertilize in vitro. Hum. Reprod., 12, 27732780.[Abstract]
Tarin, J.J., Ten, J., Vendrell, F.J. and Cano, A. (1998) Dithiothreitol prevents age-associated decrease in oocyte/conceptus viability in vitro. Hum. Reprod., 13, 381386.[CrossRef][ISI][Medline]
Tatone, C., Iorio, R., Francione, A., Gioia, L. and Colonna, R. (1999) Biochemical and biological effects of KN-93, an inhibitor of calmodulin-dependent protein kinase II, on the initial events of mouse egg activation induced by ethanol. J. Reprod. Fertil., 115, 151157.[Abstract]
Tatone, C., Delle Monache, S., Iorio, R., Caserta, D., Di Cola, M. and Colonna, R. (2002) Possible role for Ca(2+) calmodulin-dependent protein kinase II as an effector of the fertilization Ca(2+) signal in mouse oocyte activation. Mol. Hum. Reprod., 8, 750757.
Van Blerkom, J. and Henry, G. (1992) Oocyte dysmorphism and aneuploidy in meiotically mature human oocytes after ovarian stimulation. Hum. Reprod., 7, 379390.[Abstract]
Van Blerkom, J. and Davis, P. (2001) Differential effects of repeated ovarian stimulation on cytoplasmic and spindle organization in metaphase II mouse oocytes matured in vivo and in vitro. Hum. Reprod., 16, 757764.
Van Wissen, B., Bomsel-Helmreich, O., Debey, P., Eisenberg, C., Vautier, D. and Pennehouat, G. (1991) Fertilization and ageing processes in non-divided human oocytes after GnRHa treatment: an analysis of individual oocytes. Hum. Reprod., 6, 879884.[Abstract]
Van Wissen, B., Eisenberg, C., Debey, P., Pennehouat, G., Auger, J. and Bomsel-Helmreich, O. (1992) In vitro DNA fluorescence after in vitro fertilization (IVF) failure. J. Assist. Reprod. Genet., 9, 564571.[ISI][Medline]
Verde, F., Labbe, J.C., Doree, M. and Karsenti, E. (1990) Regulation of microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus eggs. Nature, 343, 233238.[CrossRef][ISI][Medline]
Verde, F., Dogterom, M., Stelzer, E., Karsenti, E. and Leibler, S. (1992) Control of microtubule dynamics and length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts. J. Cell Biol., 118, 10971108.[Abstract]
Verlhac, M.H., Kubiak, J.Z., Clarke, H.J. and Maro, B. (1994) Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes. Development, 120, 10171025.
Vogel, R. and Spielmann, H. (1992) Genotoxic and embryotoxic effects of gonadotropin-hyperstimulated ovulation of murine oocytes, preimplantation embryos, and term fetuses. Reprod. Toxicol., 6, 329333.[CrossRef][ISI][Medline]
Volarcik, K., Sheean, L., Goldfarb, J., Woods, L., Abdul-Karim, F.W. 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]
Wang, W.H., Meng, L., Hackett, R.J., Odenbourg, R. and Keefe, D.L. (2001) Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy. Hum. Reprod., 16, 23742378.
Yoshida, M., Cran, D.G. and Pursel, V.G. (1993) Confocal and fluorescence microscopic study using lectins of the distribution of cortical granules during the maturation and fertilization of pig oocytes. Mol. Reprod. Dev., 36, 462468.[ISI][Medline]
Zuccotti, M., Boiani, M., Garagna, S. and Redi, C.A. (1998) Analysis of aneuploidy rate in antral and ovulated mouse oocytes during female aging. Mol. Reprod. Dev., 50, 305312.[CrossRef][ISI][Medline]
Submitted on January 8, 2003; accepted on March 13, 2003.