1 Research Laboratories of Schering AG, Berlin and 2 Fakultät für Biologie, Gentechnologie/Biotechnologie, University Bielefeld, Bielefeld, Germany
3 To whom correspondence should be addressed at: University of Bielefeld, Universitätsstrasse 25, 33501 Bielefeld, Germany. e-mail: Suna.Cukurcam{at}Uni-Bielefeld.de
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Abstract |
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Key words: maturation/meiosis/mouse/non-disjunction/oocyte
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Introduction |
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The mechanisms responsible for the resumption of meiosis in vivo are governed by the LH surge (Adashi, 1994; Downs, 2001
). However, maturation-competent, fully grown oocytes from a number of species including humans are capable of undergoing spontaneously meiotic maturation to metaphase II in vitro after liberation from their intrafollicular environment and release from the meiosis-arresting activities of the follicle cells and components of the follicular fluid (Pincus and Enzmann, 1935
; Edwards, 1965
; Trounson et al., 1994
; 1998; 2001; Barnes et al., 1995
; Cha and Chian 1998
; Cobo et al., 1999
; Chian et al., 2000
). Maturation in the absence of a follicular environment may be sub-optimal and produce oocytes with limited developmental potential (Trounson et al., 2001
).
Intermediates of the cholesterol biosynthetic pathway, C-29 sterols, were identified in a recent study (Byskov et al., 1997). These compounds may act as a physiological signal downstream from the LH surge, reinitiating the meiotic cycle in oocytes contained within their normal follicular endowment. Follicular fluid-meiosis-activating sterol (FF-MAS) is a lipophilic sterol (4,4-dimethyl-5
-cholest-8,14,24-trien-3
-ol) which is present in follicular fluid and ovary tissue. Dependent upon oocyte transmitted signals, FF-MAS seems to be produced by the somatic compartment of the follicle, and is capable of initiating meiosis in isolated naked, or cumulus cell-enclosed oocytes from several species, when cultured in the presence of meiotic inhibitors (Byskov et al., 1995
; 2002). Accordingly, synthetically produced FF-MAS dose-dependently stimulated germinal vesicle breakdown (GVBD) in meiotically arrested rodent oocytes in the presence of hypoxanthine (HX), isobutyl-methyl-xanthine (IBMX) or dbcAMP (Grøndahl et al., 1998
; 2000; Hegele-Hartung et al., 1999
; 2001; Downs et al., 2001
). Micromolar concentrations of FF-MAS have been detected in follicular fluid of non-rodent species (Baltsen et al., 2001
). A concentration of 10 µmol/l or >20 µmol/l synthetically produced FF-MAS has been most effective in overcoming meiotic inhibition in rodent oocytes (Hegele-Hartung et al., 2001
), and in support of human oocyte survival and maturation (Andersen et al., 1999
; Grøndahl et al., 2000
; Cavilla et al., 2001
) respectively. Hence, this dose level was chosen for use in the present study as it may be relevant for use in assisted reproduction.
Meiotic resumption induced in rodent oocytes by FF-MAS to overcome meiotic arrest by inhibitors is relatively delayed and extended compared with spontaneous maturation (Hegele-Hartung et al., 1999). In contrast with FF-MAS induced maturation in rodents, spontaneous maturation in isolated human oocytes appeared rather accelerated by FF-MAS. Concomitantly, FF-MAS enhanced human oocyte survival by yet unknown mechanisms under these conditions (Grøndahl et al., 2000
; Cavilla et al., 2001
). It has been speculated previously that the quality and survival of mammalian oocytes could possibly be supported by an FF-MAS-related delay in cytoplasmic ageing processes at metaphase II (Hegele-Hartung et al., 1999
). The signalling events governing resumption of maturation of mammalian oocytes within the Graafian follicle downstream from the hormonal stimulus are still largely unknown and under investigation. Accordingly, most previous studies have focused on the molecular events in FF-MAS-induced early steps of resumption of maturation (Færge et al., 2001
; Byskov et al., 2002
), while the basis of other potentially beneficial (or adverse) effects of FF-MAS on oocyte quality and developmental potential have not been analysed in detail. Therefore, the decision was made to investigate in the present study the distal influences of FF-MAS on oocyte maturation, which may either improve or compromise developmental competence and health of the embryo, using isolated denuded oocytes of the mouse as a model. Although it appears that FF-MAS supports human oocyte survival, it was essential to show that there is no adverse influence of the sterol on chromosome segregation before considering using the sterol in clinical practice and in assisted reproduction in humans. Therefore, FF-MAS-induced meiotic progression as well as nuclear maturation was assessed in denuded mouse oocytes induced to resume maturation in vitro, and fidelity of chromosome segregation was analysed after exposure to FF-MAS in the presence and absence of HX.
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Materials and methods |
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Animals and treatments
Inbred female CBA/Ca mice (Harlan-Winkelmann, Borchen, Germany) were maintained under a 12 h light:dark cycle (07:00-19:00) and fed ad libitum. Oocytes were obtained from the ovaries of hormonally untreated 6- to 16-week-old CBA/Ca mice at diestrous. Isolation of oocytes was performed as previously described (Eichenlaub-Ritter and Boll, 1989; Soewarto et al., 1995
).
Oocytes from large antral follicles were cultured in 1 ml -MEM medium supplemented as described above in Nuclon 4-well dishes (Eichenlaub-Ritter and Betzendahl, 1995
). Spontaneous maturation and progression to metaphase II [polar body (PB) formation] was analysed after 16 h of culture at 37°C in an atmosphere of 5% CO2 in air in the absence or presence of 10 µmol/l FF-MAS or 10 µmol/l lanosterol. As a negative control for predivision (here defined as precocious separation of chromatids prior to anaphase II; Angell et al., 1993
; Wolstenholme and Angell, 2000
; see below) mouse oocytes were cultured in M16 medium. Maturation was considered normal when at least 65% of the cells in the control group (
-MEM without HX and FF-MAS) extruded the first PB during 16 h of culture.
In a second set of experiments, FF-MAS-induced maturation was analysed in oocytes cultured in -MEM + 3 mmol/l HX. Since FF-MAS-induced GVBD occurs with a delay of about 711 h after oocyte isolation and the start of culture (Hegele-Hartung et al., 1999
; Downs et al., 2001
), oocytes in the HX groups were cultured for 22 h in total to determine the rate of PB formation and maturation to metaphase II. The
-MEM+HX group served as a negative control. Oocytes matured for 22 h in
-MEM+HX with 10 µmol/l lanosterol (an inactive precursor of FF-MAS; Færge et al., 2001
) were used as a second control to demonstrate the specificity of FF-MAS activities. Only those oocytes from experiments with less than 20% spontaneous maturation in the HX-control group were considered blocked by HX and therefore included in the analysis.
Analysis of meiotic maturation
Meiotic progression was analysed in in-vitro-matured oocytes by determining the number of meiotically blocked or incompetent oocytes with an intact germinal vesicle (GV), those resuming maturation and undergoing GVBD, those extruding a first PB, or those activated and forming pronuclei after 16 h of spontaneous maturation without HX or 22 h of FF-MAS-stimulated maturation in the presence of HX respectively.
Analysis of nuclear maturation and chromosomal constitution
Spreading, hypotonic treatment, fixation and C-Banding of oocytes was carried out as described previously (Eichenlaub-Ritter and Boll, 1989; Soewarto et al., 1995
) using a modified Tarkowski method (Tarkowski, 1966
). Chromosomes were analysed by phase-contrast with an Axiophot microscope (Zeiss, Oberkochem, Germany) equipped with a sensitive CCD camera (Sensi Cam; PCO CCD Imaging, Kelheim, Germany).
Numbers of oocytes with nucleus (GV), meiosis I chromosomes (bivalents) or metaphase II chromosomes (dyads) without or with chromatids (monads) were determined. Oocytes with 20 metaphase II chromosomes or with an equivalent number of chromatids, for instance, 15 dyads plus five pairs of monads of equal size (Figure 1D) were scored as euploid. Oocytes with >20 metaphase II chromosomes or the equivalent number of chromatids were regarded as hyperploid (Figure 1B). All oocytes containing 16 dyads up to 19 dyads plus a single monad or an equivalent number of monads were scored as hypoploid. Oocytes containing about 40 metaphase II dyads were designated as diploids (diploid metaphase II stages; Figure 1C). The percentage of diploids was determined in relation to all oocytes with metaphase II chromosomes (see text), or in relation to those metaphase II oocytes in which ploidy could unambiguously be determined (Table I), although the diploid group is relatively over-represented under these conditions.
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Analysis of spindle formation and chromosome behaviour
Measurement of tubulin-immunofluorescence of oocytes was carried out as described previously (Eichenlaub-Ritter and Betzendahl, 1995; Hegele-Hartung et al., 1999
). Briefly, the zona pellucida was removed by rapid pronase digestion and gentle pipetting. Oocytes were then extracted in a microtubule-stabilizing buffer at 37°C containing non-ionic detergent and glycerol. Extracted oocytes were attached to poly-L-lysine-coated slides and fixed in 20°C cold methanol. After washing, samples were reacted with a monoclonal anti-
-tubulin antibody (Sigma), and a fluoroscein isothiocyanate (FITC)-conjugated second antibody (Eichenlaub-Ritter and Betzendahl, 1995
; Soewarto et al., 1995
). Chromosomes were stained with 4,6-diamidino-2-phenylindole (DAPI; 10 µg/ml in phosphate-buffered saline). Spindle formation was analysed using an Axiophot microscope equipped with filters for FITC and DAPI fluorescence. The number of oocytes with normal barrel-shaped bipolar spindle or with aberrant or no spindle was recorded. Number of oocytes with all chromosomes aligned at the equator of the metaphase II spindle or those with displacement of one or several chromosomes was analysed quantitatively. Additionally, the number of cytoplasmic asters in each oocyte was determined. Cytoplasmic asters were defined as all microtubular asters distinct from the spindle, including those asters located in the vicinity of the main spindle body. Images of spindles were recorded using a sensitive CCD camera and processed with Adobe Photoshop or PowerPoint software.
Statistical analysis
Statistical analysis was carried out using either a 2- test with Yates correction or a t-test.
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Results |
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In contrast with spontaneously matured oocytes, over 80% of all oocytes cultured for 22 h in the presence of -MEM+HX remained blocked in GV stage. A similarly high number of meiotically arrested oocytes was found in the group cultured in
-MEM+ HX+10 µmol/l lanosterol. In both groups, all oocytes which escaped the HX-block matured to meiosis II (Table I). This was significantly different for the
-MEM+HX+FF-MAS group. In the presence of 10 µmol/l FF-MAS, almost 70% of oocytes (28.5% + 41.4%; Table I) were induced to resume maturation, and over 40% of all oocytes emitted a PB (lower left panel in Table I), corresponding to almost 60% of the oocytes initially induced to resume maturation by FF-MAS. Since about 90% of all oocytes extruded a PB in spontaneous maturation (left upper panel in Table I), FF-MAS-stimulated maturation was significantly less effective in supporting cytokinesis as compared with spontaneous maturation.
Nuclear maturation of mouse oocytes
Nuclear maturation was determined after spreading of oocytes at the end of culture, at 16 h or 22 h respectively (Table I, middle panel). Over 90% of oocytes maturing spontaneously in vitro in the -MEM control, in the
-MEM+lanosterol group, the group cultured in M16 medium, and the oocytes cultured in
-MEM+FF-MAS possessed metaphase II chromosomes. Less than 6% of oocytes of these three experimental groups of spontaneously maturing oocytes had bivalent chromosomes, and remained arrested at meiosis I.
In contrast, there was a significant block in nuclear maturation in the oocytes cultured in the presence of HX and HX+lanosterol compared with the groups maturing spontaneously without HX (P < 0.001) (Table I, middle panel). Only a few oocytes escaped the HX block and matured to metaphase II (<17%). There was no oocyte in these two groups, which possessed bivalent meiosis I chromosomes. In contrast to the other HX groups, most of the oocytes cultured in the presence of HX+FF-MAS developed to meiosis I or II during 22 h of culture (69.5% in total; Table I, middle panel, last row). Only slightly more than 20% of the oocytes in this group possessed bivalents, whereas around 50% had metaphase II chromosomes. From the 351 oocytes (112 plus 239; Table I) which were induced to resume maturation by FF-MAS in the presence of HX, over two-thirds (239/351 = 68.1%) reached metaphase II (activated not included). However, from the oocytes with metaphase II chromosomes, 56 (corresponding to 23.4% of all metaphase II oocytes; MII, middle panel of Table I; Figure 1C) contained a diploid set of metaphase II chromosomes. Therefore, nuclear maturation to metaphase II appears to be asynchronous to aspects of cytoplasmic maturation like cytokinesis in FF-MAS stimulated oocytes. Accordingly, all oocytes with a diploid set of metaphase II chromosomes except one was from the GVBD group, which suggested that anaphase I occurred in the absence of cytokinesis. In conclusion, FF-MAS overcame the meiotic arrest by HX, and induced nuclear maturation to metaphase II in the majority of oocytes. Events associated with cytoplasmic, cortical maturation required for PB formation were also induced, but possibly delayed and asynchronous relative to nuclear maturation in a portion of the oocytes stimulated to resume maturation by FF-MAS in the presence of HX.
Errors in chromosome segregation in mouse oocytes maturing with and without FF-MAS
In order to assess the influences of FF-MAS on fidelity in chromosome segregation, the number of euploid oocytes (Figure 1A), hypoploids and hyperploids with >20 metaphase II chromosomes/dyads was determined (Figure 1B), as well as diploids with about 40 metaphase II chromosomes (Figure 1C). Separately, the number of oocytes possessing precociously separated chromatids/monads (Figure1D) was determined, irrespective of ploidy (Table I, right panel, Predivision). The number of euploid oocytes in the control was only about 60%, which appeared mainly due to a high number of hypoploid oocytes with <20 dyads. The percentage of euploid oocytes was significantly higher, and that of hypoploid oocytes lower, in the group of oocytes cultured in M16 medium (>80% and >15% respectively; P < 0.001). The oocytes cultured in -MEM appeared to be more fragile during handling and therefore possibly also more susceptible to loss of chromosomes during spreading compared with those cultured in M16 medium. Accordingly, hypoploidy was significantly higher in all oocytes of the
-MEM groups compared with the M16 group. Oocytes matured in the presence of HX+FF-MAS had only comparatively low rates of hypoploidy (<10%), so that oocytes of this group may be less susceptible to spreading-related chromosome loss.
However, many of the oocytes from the HX+FF-MAS group had two sets of metaphase II chromosomes (diploid metaphase II). Diploidy is shown as relative rate per metaphase II spreads with analysable numbers of chromosomes (Diploids in right panel of Table I). When calculated relative to all oocytes with metaphase II chromosomes (MII in middle panel of Table I), diploidy was 1.7, 1.7, 0, 3.3 and 23.4% for oocytes matured in -MEM, in M16 medium, in
-MEM+lanosterol, in
-MEM+FF-MAS, and in
-MEM+HX+FF-MAS respectively. Regardless of whether diploidy was analysed with respect to all metaphase II oocytes or only those with clearly recognized chromosome numbers, diploidy was highest in the HX+FF-MAS group. Since there was no significant difference in diploidy rate between spontaneously matured oocytes cultured with or without FF-MAS in the absence of HX, chromosome segregation without cytokinesis did not appear to be a priori related to the presence of FF-MAS. Instead, the delayed resumption of maturation by FF-MAS in HX-containing media may be responsible for an uncoupling of cytokinesis from nuclear maturation in the HX+FF-MAS group.
Importantly, the rate of hyperploidya conclusive marker for errors in chromosome segregation at meiosis Idid not differ significantly between the groups of oocytes matured in either the presence or absence of FF-MAS. Neither was any significant adverse influence of FF-MAS on the fidelity of chromosome segregation found when statistical analysis excluded the diploids and hypoploids in each group. In conclusion, chromosomal analysis of in-vitro-matured mouse oocytes suggests that fidelity of chromosome segregation at anaphase I is normal in the presence of FF-MAS.
Precocious chromatid separation in relation to culture media and FF-MAS
Unexpectedly, it was found that oocytes matured in -MEM medium frequently possessed pairs of chromatids/monads at meiosis II (Predivision, right panel of Table I). This was probably mainly due to sub-optimal culture conditions inducing a balanced precocious separation of monads at metaphase II (Figure 1D, arrows). Centromeres of pairs of monads were frequently still oriented towards each other but appeared in the process of separation. Predivision (precocious loss of chromosome cohesion) was calculated by determining the numbers of oocytes containing chromatids separated for more than their lengths (Table I, right column). More than one-third of all spontaneously matured oocytes cultured in
-MEM contained chromatids/monads beside of metaphase II chromosomes (39.5%). In contrast, only 2.5% of all oocytes in the M16 group exhibited predivision (P < 0.001). The presence of FF-MAS in spontaneous maturation without HX reduced the overall percentage of oocytes with chromatids significantly in the
-MEM group relative to the
-MEM control to 27.5% (P < 0.001). Maturation induced by FF-MAS in
-MEM+HX resulted also in only 15% of oocytes with chromatids. This was significantly lower compared with the
-MEM control, but still significantly higher compared with the M16 group (P < 0.001). The percentage of oocytes with precociously separated chromatids was also high in the oocytes matured spontaneously in
-MEM+lanosterol. Therefore, FF-MAS appears to have a specific intrinsic property to protect chromosomes from loss of cohesion, and presegregation before anaphase II and, possibly, random segregation at completion of second meiosis.
Influences of FF-MAS on spindle formation and chromosome behaviour
Indirect immunofluorescence showed that the majority of oocytes in all groups possessed normal bipolar, barrel-shaped (Figure 2A) or trapezoid-like spindles with broad spindle poles (Figure 2B) (Table II). The highest number of oocytes which reached metaphase II, emitted a PB and had normal spindles, was found in the group matured in the presence of HX and FF-MAS. Accordingly, the relative percentage of aberrant metaphase II spindles with asymmetric shape (Figure 2C and D) was higher in both groups of spontaneously matured mouse oocytes as compared with the group cultured in the presence of HX and FF-MAS, though differences did not reach statistical significance (Table II). The majority of metaphase II oocytes maturing with or without FF-MAS had well-aligned chromosomes (Figure 2A' and B'). Again, the highest percentage of oocytes with well-aligned chromosomes was found in the group cultured in the presence of FF-MAS+HX. Only a few oocytes had totally dispersed chromosomes at metaphase II (Figure 2C' and D').
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Discussion |
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In contrast to maturation in vivo, as well as spontaneous maturation of mouse oocytes where GVBD takes place within the first 2 h of meiotic resumption (Verlhac et al., 1994; Lu et al., 2000
), GVBD occurs with a delay of several hours in FF-MAS-stimulated maturation in the mouse oocyte (Hegele-Hartung et al., 1999
; Downs et al., 2001
). Thus, oocytes were analysed exclusively after 22 h of culture for meiotic progression to metaphase II in this group. Unlike spontaneous maturation (Hampl and Eppig, 1995
), resumption of maturation in the presence of HX by FF-MAS requires protein synthesis (Grøndahl et al., 2000
), and appears to involve activation of mitogen-activated protein (MAP) kinases, possibly through G protein receptors (Færge et al., 2001
; Grøndahl et al., 2003
). cAMP levels appear to remain fairly high in FF-MAS-stimulated maturation (Grøndahl et al., 2003
). A sustained high activity of cAMP-dependent kinase may interfere with the translation of c-mos mRNA (Lazar et al., 2002
) and may be causal to the asynchrony in aspects of cytoplasmic maturation required for cytokinesis and nuclear maturation events, in this way causing high rates of diploid metaphase II oocytes. Diploidy was not related to the presence of FF-MAS per se, as rates were low in the group of oocytes maturing spontaneously with FF-MAS without HX. Two sets of metaphase II chromosomes in an oocyte may assemble on a common spindle (Soewarto et al., 1995
), or on two spindles respectively (Balakier et al., 2002
), as also clearly seen with tubulin immunofluorescence in the present study in one case. When diploid metaphase II oocytes become fertilized and emit one or two PBs, they result in a two or three pronuclear, triploid, dygynic 1-cell embryo, which may develop to the blastocyst stage but later is destined to die (Pergament et al., 2000
). A doseresponse study was not performed to define threshold concentrations of FF-MAS for inducing meiotic resumption or resulting in uncoupling of anaphase I from cytokinesis. There is a possibility that only high concentrations of FF-MAS delay cytokinesis dramatically, so that this results in asynchrony with anaphase I progression. However, the present authors are not aware of any previous study supporting such notion. There was no indication that the relative rate of oocytes with PB formation decreased with increasing FF-MAS concentrations, according to a meiosis arresting effect. Rather, it is assumed that it is mainly the initial delay in resumption of meiosis triggered by FF-MAS in oocytes cultured in HX-containing media (Hegele-Hartung et al., 1999
) that is causal to the generation of diploid oocytes.
Two sets of metaphase II chromosomes have also been observed in mouse oocytes spontaneously maturing in vitro in the presence of cytochalasinsdrugs which disturb organization of the actin cytoskeleton (Kubiak et al., 1991; Soewarto et al., 1995
). Spindle migration and cytokinesis requires actin and actin-regulating proteins (Soewarto et al., 1995
; Terada et al., 2000
; Leader et al., 2002
). Cortical granule transport also involves microfilaments (Connors et al., 1998
). Migration of cortical granules to the oolemma was delayed in mouse oocytes matured in FF-MAS- and HX-containing media (Hegele-Hartung et al., 1999
). Therefore, FF-MAS may be relatively ineffective in inducing cytoplasmic maturation events involving dynamic alterations in the actin cytoskeleton. Failure of the spindle and chromosomes to migrate and attach to the cell cortex may therefore have prevented the complex interactions between the spindle and the cortical cytoskeleton that normally take place (Maro et al., 1986
; Leader et al., 2002
) to synchronize chromosome segregation with cytokinesis. Taken together, the present observations suggest that FF-MAS might be a physiologically important factor in support of nuclear maturation, but that additional signalling events are probably required to act concomitantly in vivo to induce release from meiotic arrest and effectively induce full cytoplasmic as well as nuclear maturation events. Based on the present observations it appears that FF-MAS could contribute to support nuclear maturation of immature human oocytes, which would be especially important under conditions compatible with normal cytoplasmic maturation and cytokinesis (Grøndahl et al., 2000
).
Activation of maturation-promoting factor (MPF) is required for resumption of meiosis at oogenesis in mammals (Choi et al., 1991; Heikinheimo and Gibbons, 1998
). FF-MAS seems to activate MPF by bypassing the reduction in cAMP-levels (Grøndahl et al., 2003
) and probably also the reduction in activity of c-AMP-dependent kinase, which is believed to initiate meiotic resumption in vivo, in response to the LH surge (Dekel, 1988
; Downs, 2002
). Recent reports suggest that inactivation of a stimulatory G protein (Gs) (Mehlmann et al., 2002
) and an activation of AMP-activated protein kinase (AMPK) are critical for resumption of meiosis in the mammalian oocyte (Downs et al., 2002
). In addition to AMPK (Downs et al., 2002
), phosphodiesterase (PDE) (Conti et al., 2002
) presumably also acts upstream of activation of the cdc25b phosphatase, which dephosphorylates critical residues on cyclin-dependent kinase in activation of MPF (Lincoln et al., 2002
). In contrast to FF-MAS-stimulated maturation, the kinetics of GVBD induced by AMPK activators were not delayed in denuded and cumulus-enclosed oocytes maintained in meiotic arrest by inhibitors (Downs et al., 2002
). The present observations therefore suggest that FF-MAS is especially effective in supporting the nuclear maturation events, but under physiological conditions in the ovary and in the presence of meiotic inhibitors in vitro may require additional other critical and essential factors, which are indispensable to induce all aspects of meiotic resumption and acquisition of full meiotic and developmental competence at oogenesis.
FF-MAS and fidelity of chromosome segregation
The results of previous studies have suggested that FF-MAS contributes to oocyte health and developmental competence. Thus, FF-MAS accelerated the in-vitro maturation of human oocytes and increased fertilization rate in the mouse (Hegele-Hartung et al., 1998; Grøndahl et al., 2000
; Cavilla et al., 2001
). The present study provides novel insights into potentially important aspects of FF-MAS properties, which may mediate these activities. Unexpectedly, it was found that
-MEM is sub-optimal for mouse oocyte culture. In denuded oocytes of this and other strains of mice (unpublished observations), a high proportion of chromosomes was detected which had separated their chromatids precociously when matured in
-MEM to meiosis II. It is assumed that maturation in
-MEM fails to provide essential components for normal oocyte maturation, causing the chromatid cohesion at the centromeres of sister chromatids to weaken. Frequently, chromatids were seen in different areas of the slide and totally separated from each other, suggesting that they separated prior to spreading of the oocytes. Separation of chromatids prior to anaphase I would induce chiasma resolution and a high risk for random segregation and aneuploidy in oocytes. However, high rates of hyperploid oocytes were not detected, and so it can be assumed that the chromatids segregated when the oocytes had already progressed to meiosis II during culture in
-MEM.
This first report analysing chromosomal constitution in oocytes matured in the presence of FF-MAS provides evidence that FF-MAS is compatible with high fidelity and normal chromosome segregation at meiosis I in oocytes of the mouse. Few oocytes of the FF-MAS groups possessed more than 20 metaphase II chromosomes and therefore had suffered from non-disjunction. The rate was within the normal range observed in untreated, in-vitro-matured mouse oocytes in the present and previous studies (Eichenlaub-Ritter and Betzendahl, 1995; Eichenlaub-Ritter, 1998
). In essence, in spite of sub-optimal culture conditions in
-MEM, and the micromolar concentrations of FF-MAS in the medium, the fidelity of chromosome segregation at meiosis I was high and not disturbed by FF-MAS or its inactive precursor, lanosterol. Although additional studies with human oocytes are required, the results of the present investigation suggest that FF-MAS has no adverse effect on segregation of chromosomes at first meiosis of mammalian oogenesis at concentrations relevant for in-vitro maturation (Grøndahl et al., 2000
).
By contrast, it was found that the presence of FF-MAS in the culture medium significantly reduced the frequency of precocious chromatid segregation. This effect was specific to FF-MAS and not mediated by lanosterol. Both oocytes matured spontaneously in presence of FF-MAS, and those stimulated by FF-MAS to overcome HX-induced meiotic arrest, had significantly less often chromatids compared with the positive -MEM control. Therefore, the protective effect does not appear to be related to the mechanisms responsible for meiotic resumption, but rather relies on a molecule-specific influence of FF-MAS on oocytes, which may be related to its chemical properties. One group (Boer et al., 2001
) investigated the relationship between the molecular electrostatic potential and activity of FF-MAS and some FF-MAS-related active and inactive sterols. These authors proposed that the locally high and dense negative electrostatic potential as compared with that of inactive molecules may be responsible for the specific meiosis-inducing activity of FF-MAS and other sterol derivatives with similar properties. Future studies on this structurefunction relationship may show whether electrostatic characteristics are also of significance in the protective effects of MAS on chromosome cohesion.
It is clear that the maturation of denuded oocytes, as examined in the current study, differs significantly from that in cumulus-enclosed oocytes or in oocytes maturing within a follicle in vivo. However, the analysis of factors supporting the maturation of naked oocytes is also of clinical significance, for example when immature human oocytes from ICSI patients are considered for in-vitro maturation and IVF. Several recently published studies have reported that the quality of these oocytes can be improved by optimizing culture media (e.g. Chian and Tan, 2002). In a recent workshop report (ESHRE Campus 2003, Frascati, April 10-12, 2003), Helen Picton (University of Leeds, UK) provided tentative evidence that the majority of immature, naked, in-vitro-matured human oocytes may be chromosomally abnormal. It is still not known to what extent deficiencies in paracrine and autocrine signalling prior to resumption of maturation or sub-optimal in-vitro maturation conditions contribute to the low quality of such oocytes. To improve human oocyte maturation under these conditions, it is important to identify follicle cell-derived factors and culture conditions, which may compensate for the removal of cumulus during oocyte retrieval, and may thus help to improve oocyte quality. The present observations suggest that the presence of FF-MAS in the maturation medium might have a specific, previously unnoticed, protective effect.
Protective influence of FF-MAS on chromosome cohesion
Cohesion between the centromeres of sister chromatids up to the initiation of anaphase II is essential for correct chromosome disjunction in meiosis (Nasmyth, 2001). The results of the present study suggest, for the first time, that the presence of FF-MAS during maturation helps to protect oocytes from a precocious loss of cohesion between centromeres of chromatids. This may have contributed to the increased fertilization rate and high developmental potential of oocytes matured in FF-MAS seen in previous studies (Hegele-Hartung et al., 1998
). FF-MAS could therefore be used in assisted reproduction in in-vitro maturation to obtain chromosomally normal oocytes and embryos, which are capable of supporting preimplantation development and implantation. Meanwhile, a whole series of experiments has been carried out aimed at identifying the factors in induction of predivision, and potential mechanisms causing this aberration related to culture in
-MEM but not in the less complex M16 medium. It appears that the presence and concentration of carbohydrates are critical, and that a change in the concentration of carbohydrates will also reduce the predivision rate in mouse oocytes cultured in
-MEM without FF-MAS (unpublished observations). These extensive data have not been included in the present report as this topic was not the subject of this investigation, which focused specifically on the activity and safety of FF-MAS in oocyte in-vitro maturation. Rather, these other data will be presented in a later publication describing the precocious chromosome segregation under defined culture conditions of naked and cumulus-enclosed mouse oocytes which were more susceptible to predivision than those of the CBA strain examined herein. Preliminary observations with oocytes from this more-susceptible strain imply that there is no precocious chromatid segregation in cumulus-enclosed mouse oocytes cultured in
-MEM (S.Cukurcam et al., unpublished results). This suggests that granulosa cells provide all factors to protect oocytes from predivision in vivo and in vitro when oocytes are within a follicle or in a cumuluscell complex during culture. Therefore, FF-MAS may act synergistically to other granulosa cell-derived factors in vivo and during in-vitro maturation to support normal maturation and chromosome segregation. As ageing may influence granulosa cell function and junctional communication, it is possible that compromised folliculogenesis inducing an altered, sub-optimal microenvironment as well as insufficient expression of FF-MAS in response to oocyte signals (Byskov et al., 1997
), might contribute to the high risk of chromosomes separating precociously. This, in consequence, may result in an age-related loss of control of chromosome segregation. Several studies conducted in large cohorts of unfertilized or donated human oocytes have supported the concept that loss of chromatid cohesion is causal to chromosomal imbalance in oocytes (Pellestor et al., 2002
; Sandalinas et al., 2002
). However, further studies in human oocytes are required to demonstrate whether FF-MAS might help in obtaining high-quality oocytes for ART, especially in cases where ageing may compromise folliculogenesis.
Characteristic effects of FF-MAS on the microtubular cytoskeleton
In agreement with previous studies (Hegele-Hartung et al., 1999), it can be shown that spindles have normal morphology, that chromosomes are well aligned in FF-MAS-matured oocytes, comparable with the control, and that oocytes maturing in HX+FF-MAS- containing media possess abundant cytoplasmic asters. Such asters were only infrequently observed in oocytes maturing spontaneously in the presence of FF-MAS. Therefore, the assembly of cytoplasmic microtubular asters appears characteristic for the delayed FF-MAS-stimulated maturation rather than the presence of FF-MAS per se. Spindle structure and microtubule configurations are intricately related to the activity of kinases in the MOS-gene family/MAP kinase signalling pathway in mammalian oocytes (e.g. Verlhac et al., 1994
; 1996; 2000; Oh et al., 1998
; Lefebvere et al., 2002
). It can be speculated that delayed maturation, the specific pattern of MAP kinase activation (Færge et al., 2001
), or a sustained protein kinase (PK) A activity are causal to the distinct cytoskeletal characteristics in FF-MAS-stimulated oocyte maturation. Analysis of MPF and MAP kinase activity profiles in these oocytes is currently in progress. Importantly, the present observations provide a quantitative analysis showing that FF-MAS supports congression to and alignment of chromosomes at the spindle equator at meiosis II and, thus, presumably contributes to the control of chromosome segregation at anaphase II. The significance of aster formation for developmental potential warrants further investigation.
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Anderiesz, C., Ferraretti, A., Magli, C., Fiorentino, A., Fortini, D., Gianaroli, L., Jones, G.M. and Trounson, A.O. (2000) Effect of recombinant human gonadotrophins on human, bovine and murine oocyte meiosis, fertilization and embryonic development in vitro. Hum. Reprod., 15, 11401148.
Andersen, C.Y., Baltsen, M. and Byskov, A.G. (1999) Gonadotrophin-induced resumption of oocyte meiosis and meiosis-activating sterols. Curr. Top. Dev. Biol., 41, 163185.[ISI][Medline]
Angell, R.R., Xian, J. and Keith, J. (1993) Chromosome anomalies in human oocytes in relation to age. Hum. Reprod., 8, 10471054.[Abstract]
Balakier, H., Bouman, D., Sojecki, A., Librach, C. and Squire, J.A. (2002) Morphological and cytogenetic analysis of human giant oocytes and giant embryos. Hum. Reprod., 17, 239423401.
Baltsen, M., Bogh, I.B. and Byskov, A.G. (2001) Content of meiosis activating sterols in equine follicular fluids: correlation to follicular size and dominance. Theriogenology, 56, 133145.[CrossRef][ISI][Medline]
Barnes, F.L., Crombie, A., Gardner, D.K., Kausche, A., Lacham-Kaplan, O., Suikkari, A.M., Tiglias, J., Wood, C. and Trounson, A.O. (1995) Blastocyst development and birth after in vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching. Hum. Reprod., 10, 32433247.[Abstract]
Boer, D.R., Kooijman, H., van der Louw, J., Groen, M., Kelder, J. and Kroon, J. (2001) Relation between the molecular electrostatic potential and activity of some FF-MAS related sterol compounds. Bioorg. Med. Chem., 9, 26532659.[CrossRef][ISI][Medline]
Burns, K.H. and Matzuk, M.M. (2002) Minireview: genetic models for the study of gonadotropin actions. Endocrinology, 143, 28232835.
Byskov, A.G., Andersen, C.Y., Nordholm, L., Thøgersen, H., Xia, G., Wassmann, O., Andersen, J.V., Guddal, E. and Roed, T. (1995) Chemical-structure of sterols that activate oocyte meiosis. Nature, 374, 559562.[CrossRef][ISI][Medline]
Byskov, A.G., Yding Andersen, C., Hossaini, A. and Guoliang, X. (1997) Cumulus cells of oocyte-cumulus complexes secrete a meiosis-activating substance when stimulated with FSH. Mol. Reprod. Dev., 46, 296305.[CrossRef][ISI][Medline]
Byskov, A.G., Andersen, C.Y. and Leonardsen, L. (2002) Role of meiosis activating sterols, MAS, in induced oocyte maturation. Mol. Cell. Endocrinol., 187, 189196.[CrossRef][ISI][Medline]
Cavilla, J.L., Kennedy, C.R., Baltsen, M., Klentzeris, L.D., Byskov, A.G. and Hartshorne, G.M. (2001) The effects of meiosis-activating sterol on in vitro maturation and fertilization of oocytes from stimulated and unstimulated ovaries. Hum. Reprod., 16, 547555.
Cha, K.Y. and Chian, R.C. (1998) Maturation in vitro of immature human oocytes for clinical use. Hum. Reprod. Update, 4, 103120.
Chian, R.C. and Tan, S.L. (2002) Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles. Reprod. Biomed. Online, 5, 2532.
Chian, R.C., Buckett, W.M., Tulandi, T. and Tan, S.L. (2000) Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum. Reprod., 15, 165170.
Choi, T., Aoki, F., Mori, M., Yamashita, M., Nagahama, Y. and Kohmoto, K. (1991) Activation of p34cdc2 protein kinase activity in meiotic and mitotic cell cycles in mouse oocytes and embryos. Development, 113, 789795.[Abstract]
Cobo, A.C., Requena, A., Neuspiller, F., Aragon, S.M., Mercader, A., Navarro, J., Simon, C., Remohi, J. and Pellicer, A. (1999) Maturation in vitro of human oocytes from unstimulated cycles: selection of the optimal day for ovum retrieval based on follicular size. Hum. Reprod., 14, 18641868.
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]
Conti, M., Andersen, C.B., Richard, F., Mehats, C., Chun, S.Y., Horner, K., Jin, C. and Tsafriri, A. (2002) Role of cyclic nucleotide signaling in oocyte maturation. Mol. Cell. Endocrinol., 187, 153159.[CrossRef][ISI][Medline]
Dekel, N. (1988) Regulation of oocyte maturation. The role of cAMP. Ann. N. Y. Acad. Sci., 541, 211216.[ISI][Medline]
Downs, S.M. (2001) A gap-junction-mediated signal, rather than an external paracrine factor, predominates during meiotic induction in isolated mouse oocytes. Zygote, 9, 7182.[CrossRef][ISI][Medline]
Downs, S.M. (2002) The biochemistry of oocyte maturation. Ernst Schering Res. Found. Workshop, 41, 8199.[Medline]
Downs, S.M., Ruan, B. and Schroepfer, G.J., Jr (2001) Meiosis-activating sterol and the maturation of isolated mouse oocytes. Biol. Reprod., 64, 8089.
Downs, S.M., Hudson, E.R. and Hardie, D.G. (2002) A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes. Dev. Biol., 245, 200212.[CrossRef][ISI][Medline]
Edwards, R.G. (1965) Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature, 208, 349351.[ISI][Medline]
Eichenlaub-Ritter, U. (1998) Genetics of oocyte ageing. Maturitas, 30, 143169.[CrossRef][ISI][Medline]
Eichenlaub-Ritter, U. and Boll, I. (1989) Nocodazole sensitivity, age-related aneuploidy, and alterations in the cell cycle during maturation of mouse oocytes. Cytogenet. Cell Genet., 52, 170176.[ISI][Medline]
Eichenlaub-Ritter, U. and Betzendahl, I. (1995) Chloral hydrate induced spindle aberrations, metaphase I arrest and aneuploidy in mouse oocytes. Mutagenesis, 10, 477486.[Abstract]
Eppig, J.J., Wigglesworth, K. and Pendola, F.L. (2002) The mammalian oocyte orchestrates the rate of ovarian follicular development. Proc. Natl Acad. Sci. USA, 99, 28902894.
Færge, I., Terry, B., Kalous, J., Wahl, P., Lessl, M., Ottesen, J.L., Hyttel, P. and Grøndahl, C. (2001) Resumption of meiosis induced by meiosis-activating sterol has a different signal transduction pathway than spontaneous resumption of meiosis in denuded mouse oocytes cultured in vitro. Biol. Reprod., 65, 17511758.
Grøndahl, C. (2002) FF-MAS and its role in mammalian oocyte maturation. Ernst Schering Res. Found. Workshop, 41, 177193.[Medline]
Grøndahl, C., Ottesen, J.L., Lessl, M., Faarup, P., Murray, A., Gronvald, F.C., Hegele-Hartung, C. and Ahnfelt-Ronne, I. (1998) Meiosis-activating sterol promotes resumption of meiosis in mouse oocytes cultured in vitro in contrast to related oxysterols. Biol. Reprod., 58, 12971302.[Abstract]
Grøndahl, C., Hansen, T.H., Marky-Nielsen, K., Ottesen, J.L. and Hyttel, P. (2000) Human oocyte maturation in vitro is stimulated by meiosis-activating sterol. Hum. Reprod., 15, 310.
Grøndahl, C., Breinholt, J., Wahl, P., Murray, A., Hansen, T.H., Færge, I., Stidsen, C.E., Raun, K. and Hegele-Hartung, C. (2003) Physiology of meiosis-activating sterol: endogenous formation and mode of action. Hum. Reprod., 18, 122129.
Hampl, A. and Eppig, J.J. (1995) Translational regulation of the gradual increase in histone H1 kinase activity in maturing mouse oocytes. Mol. Reprod. Dev., 40, 915.[ISI][Medline]
Hegele-Hartung, C., Lessl, M. and Ottesen, J.T. (1998) Oocyte maturation can be induced by a synthetic meiosis-activating sterol (MAS) leading to an improvement of IFV rate in mice. Hum. Reprod., 11, 193.
Hegele-Hartung, C., Kuhnke, J., Lessl, M., Grondahl, C., Ottesen, J., Beier, H.M., Eisner, S. and Eichenlaub-Ritter, U. (1999) Nuclear and cytoplasmic maturation of mouse oocytes after treatment with synthetic meiosis-activating sterol in vitro. Biol. Reprod., 61, 13621372.
Hegele-Hartung, C., Grützner, M., Lessl, M., Grondahl, C., Ottesen, J. and Brännström, M. (2001) Activation of meiotic maturation in rat oocytes after treatment with follicular fluid meiosis-activating sterol in vitro and ex vivo. Biol. Reprod., 64, 418424.
Heikinheimo, O. and Gibbons, W.E. (1998) The molecular mechanisms of oocyte maturation and early embryonic development are unveiling new insights into reproductive medicine. Mol. Hum. Reprod., 4, 745756.[Abstract]
Hillier, S.G. (2001) Gonadotropic control of ovarian follicular growth and development. Mol. Cell. Endocrinol., 179, 3946.[CrossRef][ISI][Medline]
Joyce, I.M., Pendola, F.L., Wigglesworth, K. and Eppig, J.J. (1999) Oocyte regulation of kit ligand expression in mouse ovarian follicles. Dev. Biol., 214, 342353.[CrossRef][ISI][Medline]
Kubiak, J., Paldi, A., Weber, M. and Maro, B. (1991) Genetically identical parthenogenetic mouse embryos produced by inhibition of the first meiotic cleavage with cytochalasin D. Development, 111, 763769.[Abstract]
Lazar, S., Galiani, D. and Dekel, N. (2002) cAMP-Dependent PKA negatively regulates polyadenylation of c-mos mRNA in rat oocytes. Mol. Endocrinol., 16, 331341.
Leader, B., Lim, H., Carabatsos, M.J., Harrington, A., Ecsedy, J., Pellman, D., Maas, R. and Leder, P. (2002) Formin-2, polyploidy, hypofertility and positioning of the meiotic spindle in mouse oocytes. Nature Cell Biol., 4, 921928.[CrossRef][ISI][Medline]
Lefebvre, C., Terret, M.E., Djiane, A., Rassinier, P., Maro, B. and Verlhac, M.H. (2002) Meiotic spindle stability depends on MAPK-interacting and spindle-stabilizing protein (MISS), a new MAPK substrate. J. Cell Biol., 157, 603613.
Leonardsen, L., Stromstedt, M., Jacobsen, D., Kristensen, K.S., Baltsen, M., Andersen, C.Y. and Byskov, A.G. (2000) Effect of inhibition of sterol delta 14-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro. J. Reprod. Fertil., 118, 171179.
Lincoln, A.J., Wickramasinghe, D., Stein, P., Schultz, R.M., Palko, M.E., De Miguel, M.P., Tessarollo, L. and Donovan, P.J. (2002) Cdc25b phosphatase is required for resumption of meiosis during oocyte maturation. Nature Genet., 30, 446449.[ISI][Medline]
Lu, Z., Xia, G., Byskov, A.G. and Andersen, C.Y. (2000) Effects of amphotericin B and ketoconazole on mouse oocyte maturation: implications on the role of meiosis-activating sterol. Mol. Cell. Endocrinol., 164, 191196.[CrossRef][ISI][Medline]
Maro, B., Johnson, M.H., Webb, M. and Flach, G. (1986) Mechanism of polar body formation in the mouse oocyte: an interaction between the chromosomes, the cytoskeleton and the plasma membrane. J. Embryol. Exp. Morphol., 92, 1132.[ISI][Medline]
Mehlmann, L.M., Jones, T.L. and Jaffe, L.A. (2002) Meiotic arrest in the mouse follicle maintained by a Gs protein in the oocyte. Science, 297, 13431345.
Nasmyth, K. (2001) Disseminating the genome: joining, resolving, and separating sister chromatids during mitosis and meiosis. Annu. Rev. Genet., 35, 673745.[CrossRef][ISI][Medline]
Oh, B., Hampl, A., Eppig, J.J., Solter, D. and Knowles, B.B. (1998) SPIN, a substrate in the MAP kinase pathway in mouse oocytes. Mol. Reprod. Dev., 50, 240249.[CrossRef][ISI][Medline]
Pellestor, F., Andreo, B., Arnal, F., Humeau, C. and Demaille, J. (2002) Mechanisms of non-disjunction in human female meiosis: the co-existence of two modes of malsegregation evidenced by the karyotyping of 1397 in-vitro unfertilized oocytes. Hum. Reprod., 17, 21342145.
Pergament, E., Confino, E., Zhang, J.X., Roscetti, L., Xien Chen, P. and Wellman, D. (2000) Recurrent triploidy of maternal origin. Prenat. Diagn., 20, 561563.[CrossRef][ISI][Medline]
Pincus, G. and Enzmann, E.V. (1935) The comparative behavior of mammalian eggs in vivo and in vitro. J. Exp. Med., 62, 655675.
Sandalinas, M., Marquez, C. and Munne, S. (2002) Spectral karyotyping of fresh, non-inseminated oocytes. Mol. Hum. Reprod., 8, 580585.
Soewarto, D., Schmiady, H. and Eichenlaub-Ritter, U. (1995) Consequences of non-extrusion of the first polar body and control of the sequential segregation of homologues and chromatids in mammalian oocytes. Hum. Reprod., 10, 23502360.[Abstract]
Tarkowski, A.K. (1966) An air-drying method for chromosome preparations from mouse eggs. Cytogenetics, 5, 394400.[ISI]
Terada, Y., Simerly, C. and Schatten, G. (2000) Microfilament stabilization by jasplakinolide arrests oocyte maturation, cortical granule exocytosis, sperm incorporation cone resorption, and cell-cycle progression, but not DNA replication, during fertilization in mice. Mol. Reprod. Dev., 56, 8998.[CrossRef][ISI][Medline]
Trounson, A.O., Wood, C. and Kausche, A. (1994) In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil. Steril., 62, 353362.[ISI][Medline]
Trounson, A., Anderiesz, C., Jones, G.M., Kausche, A., Lolatgis, N. and Wood, C. (1998) Oocyte maturation. Hum. Reprod., 13, 5262.[Medline]
Trounson, A., Anderiesz, C. and Jones, G. (2001) Maturation of human oocytes in vitro and their developmental competence. Reproduction, 121, 5175.
Tsafriri, A., Cao, X., Vaknin, K.M. and Popliker, M. (2002) Is meiosis activating sterol (MAS) an obligatory mediator of meiotic resumption in mammals. Mol. Cell. Endocrinol., 187, 197204.[CrossRef][ISI][Medline]
Varani, S., Elvin, J.A., Yan, C., DeMayo, J., DeMayo, F.J., Horton, H.F., Byrne, M.C. and Matzuk, M.M. (2002) Knockout of pentraxin 3, a downstream target of growth differentiation factor-9, causes female subfertility. Mol. Endocrinol., 16, 11541167.
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.
Verlhac, M.H., Kubiak, J.Z., Weber, M., Geraud, G., Colledge, W.H., Evans, M.J., and Maro, B. (1996) Mos is required for MAP kinase activation and is involved in microtubule organization during meiotic maturation in the mouse. Development, 122, 815822.
Verlhac, M.H., Lefebvre, C., Guillaud, P., Rassinier, P. and Maro, B. (2000) Asymmetric division in mouse oocytes: with or without Mos. Curr. Biol., 10, 13031306.[CrossRef][ISI][Medline]
Wolstenholme, J. and Angell, R.R. (2000) Maternal age and trisomy a unifying mechanism of formation. Chromosoma, 109, 435438.[ISI][Medline]
Submitted on February 27, 2003; accepted on May 30, 2003.