1 Follicle Biology Unit, Centre for Reproductive Medicine, University Hospital and Medical School, Dutch-speaking Brussels Free University, Brussels, Belgium and 2 Institute of Gentechnology/Microbiology, Faculty of Biology, University of Bielefeld, Bielefeld, Germany
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Abstract |
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Key words: ageing/oocyte/oxygen/pre-antral follicle culture/spindle
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Introduction |
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Mouse models have been established to study factors essential for oocyte development during in-vitro folliculogenesis (reviewed by Hartshorne, 1997; Van den Hurk et al., 1997). By designing the appropriate conditions for follicle growth and differentiation, a large percentage of oocytes can be matured to metaphase II (MII) after reinitiation of meiosis by human chorionic gonadotrophin (HCG) and recombinant epidermal growth factor (rEGF) (Smitz et al., 1998). These oocytes are capable of normal embryonic development following in-vitro fertilization (Cortvrindt et al., 1996
, 1998
; Smitz et al., 1996
). Using this culture system, 60% of grown oocytes fertilize, and 60% of the fertilized oocytes develop in vitro to blastocysts. This final yield is lower than from in-vivo-grown follicles from age-matched animals (100% fertilized, 60% blastocyst formation) so that culture conditions should be further improved, although live offspring were obtained after in-vitro culture of follicles from prepubertal mice.
The formation of a functional spindle, completion of first meiotic division and high fidelity of chromosome segregation are essential features of a healthy oocyte (Eichenlaub-Ritter et al., 1986, 1988a
,Eichenlaub-Ritter et al., b
, 1995
; Battaglia et al., 1996
; Eichenlaub-Ritter, 1998
; Van Blerkom and Henry, 1998
; Volarcik et al., 1998
). Spindle morphology and chromosome alignment can be used as one indicator for the oocyte's capacity to form a chromosomally balanced embryo. Therefore, anti-tubulin immunofluorescence has now been employed to study spindle formation and chromosome behaviour in oocytes grown and matured in pre-antral follicle culture under different conditions.
Maturation to MII appears to be slower in in-vitro follicle culture as compared with in-vivo maturation (Cortvrindt et al., 1996). Prolongation of culture past 16 h post HCG addition may further increase the yield of oocytes in MII. However, ageing of oocytes at MII may cause concomitantly a deterioration of the spindle and displacement of chromosomes from the spindle equator (Eichenlaub-Ritter et al., 1986
, 1988b
). To determine the optimal time for harvest of oocytes with normal spindles after reinitiation of meiosis by HCG/EGF for in-vitro fertilization, spindles and chromosome alignment were compared between mouse oocytes retrieved from culture 16 or 20 h post HCG and rEGF addition.
Under conditions set by a previously developed pre-antral follicle culture (Cortvrindt et al., 1996), follicles deteriorated rapidly, and few were able to emit a polar body (PB) and mature to MII when oxygen tension was reduced to 5% throughout the 12-day culture period (Smitz et al., 1996
). Denuded oocytes isolated in HEPES-buffered, ungassed media in an atmosphere of ~20% O2 in air from antral follicles will mature to MII when placed into pre-equilibrated bicarbonate-buffered medium and cultured with 5% O2 and 5% CO2 to MII. Since oocytes cease to be directly coupled to their follicle cells by gap junctions during the final stages after initiation of resumption of maturation, the oxygen tension was lowered to 5% from the time of HCG/rEGF addition in one experimental group. It was expected that the oocytes might be protected against reactive oxygen species (ROS) generation once they resumed maturation under such conditions. The rate of meiosis reinitiation and progression to MII was then compared with the rate of maturation in the 20% oxygen group.
To assess the influence of sexual maturity of the animal and the presence or absence of follicle cells during maturation in vitro on spindle morphology and chromosome behaviour, spindles of oocytes from follicle culture were qualitatively and timely compared with those grown and matured in vivo or with those in denuded, in-vitro-matured oocytes obtained from antral follicles of prepubertal as well as sexually mature, adult female mice.
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Materials and methods |
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IVM oocytes were obtained from 24-day-old (prepubertal) or adult (3-month-old) hybrid females primed with 5 IU pregnant mare's serum (PMS; Intergonan, Intervet, Tönishofen, Germany). At 48 h after PMS administration, oocytes were released from large antral follicles and mechanically denuded prior to in-vitro maturation for 15 h in M16 medium (Sigma, Deisenhofen, Germany) in 5% CO2 in air (Figure 1). This group was included as a reference group since the oocytes matured without follicle cells. In our experience oocytes of this strain reach MII at about 12 h past isolation, and do not exhibit any sign of ageing, such as detachment of the spindle from the cortex or deterioration of spindles when processed for fluorescence at 15 h of culture.
The in-vivo controls (MII oocytes, Figure 1) consisted of ovulated oocytes isolated from the ampulla either of 26-day-old or sexually mature, young adult (3-month-old; adult) hybrid mice after priming with 5 IU PMS and administration of 5 IU HCG (Predalon®; Organon, Oberschleißheim, Germany) 48 h later and oocyte retrieval 14 h later. A time of 14 h corresponds to the optimal time when oocytes of this strain are suitable for in-vitro fertilization. The analysis on IVM and MII oocytes was carried out on oocytes obtained from at least five independent experiments performed independently in two laboratories.
Conditions of pre-antral follicle culture and media
Pre-antral (100130 µm) follicles obtained from 12- to 14-day-old hybrid mice were isolated in L15 medium + 10% fetal calf serum (FCS; Gibco, Eggenstein, Germany) + penicillin/streptomycin and cultured individually in 20 µl droplets (1 follicle per droplet) of -minimal essential medium (
-MEM with glutamax; Gibco) with 5% FCS, and with 5 µg/ml insulin, 5 µg/ml transferrin and 5 ng/ml selenium (ITS; Sigma). In addition, 100 mIU/ml recombinant FSH (rFSH; kindly donated by Ares Serono, Geneva, Switzerland), and 10 mIU/ml of recombinant LH (LH; Ares Serono) was present in culture medium, according to previously established conditions (Cortvrindt et al., 1996
, 1998
). On each second day, 10 µl of culture medium from each drop was removed and replaced by freshly prepared pre-equilibrated medium (Cortvrindt et al., 1996
). On day 12 of culture, half of the volume of the culture droplet was removed and replaced by the same culture medium in which HCG/EGF was added. This caused mucification of the follicle-enclosed cumulus and progression of meiosis in its oocyte. The COC were collected at the preset time point (16 or 20 h), and the cumulus cells mechanically removed by pipetting the COC gently in and out through a pre-pulled glass pipette.
Indirect anti-tubulin immunofluorescence (spindle and chromosome staining)
The preparation of oocytes for indirect immunofluorescence was carried out (using the same conditions and protocols) independently by two laboratories and different investigators. Results were matching, and there was no significant inter-laboratory or inter-operator variation. IVM and in-vivo-matured MII oocytes from young and adult females were processed repeatedly, and in parallel. For practical reasons, oocytes from follicle culture were processed for indirect immunofluorescence separately from IVM or in-vivo-matured MII oocytes using identical protocols for extraction and fixation. The different groups (low and high oxygen tension) were processed in parallel, and those matured for 16 h and 20 h on the same day. There was no significant inter-experiment variation.
Processing of oocytes for anti-tubulin immunofluorescence was carried out as described previously (Eichenlaub-Ritter and Boll, 1989; Eichenlaub-Ritter and Betzendahl, 1995
). All procedures were carried out on a heated stage. In short, the zona pellucida was digested with 7 mg/ml pronase (Boehringer, Mannheim, Germany) in M2 medium with 4 mg/ml bovine serum albumin (BSA; Sigma). Care was taken to maintain the temperature during the procedure. Zona-free oocytes were extracted for 4060 min at 36°C (50 mmol/l KCl, 5 mmol/l EGTA, 0.5 mmol/l MgCl2, 25 mmol/l HEPES, 20 µmol/l phenyl-methyl sulphonyl fluoride (PMSF), 25% glycerol, 2% Triton X-100; pH 6.75) (Eichenlaub-Ritter et al., 1988a
,b
, 1995
; Eichenlaub-Ritter and Betzendahl, 1995
; Soewarto et al., 1995
), placed onto a poly-L-lysine coated slide and fixed for 612 min in cold methanol (20°C). This fixation protocol avoided mechanical flattening of the round oocytes for attachment to the slide, for example by centrifugation prior to or during fixation. Since oocyte cytoplasm was extracted before fixation, the spindle usually became attached with its long axis to the slide, and measurements of pole-to-pole distance did not require three-dimensional reconstruction of optical sections with this fixation protocol, as is necessary with most aldehyde-based fixation procedures. After rehydration in phosphate-buffered saline (PBS; Oxoid, Basingstoke, Hampshire, UK), slides were incubated with the first antibody (monoclonal mouse anti-alpha-tubulin antibody (Sigma; diluted 1:400 in PBS), followed by the second antibody [fluorescein isothiocyanate (FITC)-labelled rabbit anti-mouse antibody; Sigma], and the chromosomes were stained with 10 µg/ml 4',6-diamidino-2-phenylindole (DAPI; Sigma). Finally, slides were immersed in PBS with 20% glycerol containing 2 mg/ml diaminobicyclooctane (DABCO; Sigma) as antifade. The slides were viewed with a Zeiss Axiophot microscope equipped with filter sets for FITC and DAPI fluorescence. Images were either recorded on Kodak T-MAX 400 negative film or Alpha-400ASA slide film. Images were later scanned and processed using Adobe PhotoShop or PowerPoint software.
For confocal microscope analysis (Fluoview, Olympus, Hamburg, Germany) chromosomes were stained with propidium iodine instead of DAPI.
Analysis of spindle size and chromosome alignment
The centre of the two outermost microtubule organizing centres (MTOC) at each spindle pole were used to determine spindle width at poles. Figure 2 indicates how measurements were carried out using a micrometer on the fluorescent microscope, or on spindle images produced by the confocal laser microscope. Taking the middle of the imaginary line presented by the two polar plates as end points, the length of the spindle was determined. Spindle width was measured at the spindle equator and the relative quotient of spindle width at equator versus width at poles was determined as an additional parameter for spindle shape. Measurements performed independently by two investigators gave reproducible results.
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Statistical analysis
Oocyte maturation data were compared by contingency analysis followed by 2 testing. A Student's t-test was applied for comparison of means from oocyte diameters and quantitative assessment of spindle shape.
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Results |
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The maturation rate between oocytes from experimental groups (20% or 5% O2) in pre-antral follicle culture was significantly different (P < 0.001) after 16 h or 20 h of harvest. Comparable with earlier observations (Cortvrindt et al., 1996, 1998
), nearly half of all oocytes matured continuously in normal oxygen (20%) emitted a PB. There was non-significant increase in the rate of oocytes undergoing cytokinesis upon final culture for 20 h instead of 16 h (Table I
). Some 34.2% of oocytes had still an intact germinal vesicle (GV) at 16 h (Table I
). In addition, some oocytes of the 20% oxygen group appeared to be capable of resuming maturation and undergoing GVBD later, since the percentage of oocytes with GV decreased from 34.2% to 27.5% upon prolonged culture (Table I
). Finally, 49.5% oocytes emitted a PB.
Culture of follicles in the presence of only 5% oxygen for the last 16 h or 20 h resulted in a significant reduction in the numbers of oocytes emitting a PB (less than one-fifth of the high-oxygen group; P < 0.001; Table I). There was a significant difference (P < 0.05) in the number of oocytes arrested in GV stage between the 5% and 20% oxygen group at 20 h post HCG/rEGF addition, but not at 16 h post HCG/rEGF addition (34.2 versus 37.4%). This indicates that the arrest in GV stage was irreversible in the low-oxygen group, while some oocytes cultured under normal oxygen conditions were able to resume maturation at a later time after addition of hormone. Over 40% of all oocytes in the low-oxygen group, as compared with only 20% of the high-oxygen group, were arrested in GVBD (Table I
).
In the in-vivo controls, all the oocytes ovulated in 26-day or adult mice possessed a PB (Table I). In-vitro maturation of isolated, denuded oocytes of both age groups of mice was around 90% (with culture in M16 medium at 5% CO2 in air) (Table I
).
Meiotic progression and spindle formation in oocytes from pre-antral follicle culture
All stages of spindle formation were observed in oocytes retrieved from pre-antral follicle culture 16 h or 20 h after stimulation by HCG and rEGF (Figure 3), suggesting that maturation is not highly synchronous in such populations of follicles, or that maturation became frequently arrested prior to MII. Some oocytes still possessed a GV, and did not form a spindle. Prophase I stages were characterized by the presence of condensed chromosomes after GVBD and formation of many asters of microtubules, predominantly in the vicinity of the chromosomes in the oocytes from follicle culture (arrowhead in Figure 3A
). Prometaphase I spindles were present in some oocytes. Such spindles had the characteristic `barrel shape' due to the presence of multiple MTOC at the flat spindles' poles. In these typical prometaphase I oocytes, bivalent chromosomes were still located away from the spindle equator and therefore might have been in the process of congression or were meiotically blocked at the time of fixation (Figure 3F
). Metaphase I was designated as the stage where a bipolar spindle was present and all chromosomes were aligned at the equator (Figure 3A
). Such stages could be observed in both the low- and the high-oxygen groups. Some anaphase I and telophase I stages were also detected at 16 h or 20 h after HCG/rEGF addition. Chromosomes had characteristically separated to the two spindle poles in late anaphase I or in telophase I (Figure 3B
, C, G) in spindles with a more or less constricted interpolar region. Many oocytes from pre-antral follicle culture had MII spindles with an anastral shape, flat poles, and well-aligned chromosomes (Figure 3D
), as is characteristic for normal, ovulated oocytes (see Figure 3A
), while others were anastral and barrel-shaped but their chromosomes appeared scattered around the spindle equator (Figure 3H
, H').
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All IVM oocytes ovulated in 26-day-old mice and retrieved 14 h after stimulation by HCG had MII spindles. In adult mice, one anaphase I and one telophase I stage were detected among 111 ovulated oocytes. Therefore, in-vivo maturation of oocytes appears to be generally completed at 14 h after stimulation by gonadotrophin.
Among 123 IVM oocytes obtained from 26-day-old mice and matured outside of a follicle, only 9% were still in prometaphase I/metaphase I stage, while the rest (91%) had MII spindles 15 h after isolation and culture in M16 medium. Similarly, 90% of oocytes obtained from adult mice matured spontaneously in vitro after isolation from follicles possessed a MII spindle. Moreover, 5% had a GV and the rest of this group were in meiosis I. Therefore, maturation in vivo appeared to be highly synchronous and completed in most oocytes of 26-day-old mice matured in vitro for 15 h when oocytes were obtained from PMS-primed cycles.
Spindle morphology in oocytes from follicle culture and in-vitro- and in-vivo-matured oocytes
Spindle morphology appeared normal in most oocytes retrieved from follicles after continuous culture in an atmosphere with 20% O2. Oocytes in metaphase I possessed typical anastral, barrel-shaped spindles. Condensed chromosomes were congressed at the spindle equator at the end of the culture. Small, cytoplasmic asters could often be discerned (red arrowheads in Figure 3), as in ovulated MII oocytes (arrowhead in Figure 4B
) oocytes possessing late anaphase I or telophase I spindles, there was no evidence for lagging of chromosomes in the equatorial, central part of the spindle in the group of oocytes matured under normal oxygen conditions (Figure 3B
, C). At MII, most oocytes had square, anastral spindles and mostly well-aligned chromosomes (Figure 3D
, D ').
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Metaphase II presents the end point of development before fertilization in oocytes matured either in vivo or in vitro. Therefore, spindle structure was compared at this stage between the different experimental groups. Most MII spindles of oocytes from pre-antral follicle culture had the characteristic flat spindle poles (Figure 3D), comparable to what is also found in ovulated oocytes obtained from adult, sexually mature mice after hormonal stimulation with PMS and HCG (Figure 4A
). In pre-antral follicle culture, the shape of the spindle was sometimes not as regular and symmetric as found generally in in-vivo matured oocytes. Often, astral fibres appeared to extend from the outer rim of the polar plane into the cytoplasm, giving the spindle a cushion-like appearance (Figures 3D and 4D
). The equator appeared smaller than the polar plane in some such spindles (Figure 4D
). Other spindles in oocytes from pre-antral follicle culture had a characteristic trapezoid-like shape with a smaller pole as compared to the spindle equator (Figure 4E
). Most spindles of pre-antral follicle culture appeared square in shape (Figure 4F
), and were possibly slightly less compact in the IVG oocytes as compared with those in oocytes ovulated in vivo in adult mice. In-vitro-matured, denuded oocytes of 26-day-old or adult mice appeared longer and more slender when compared to spindles in the oocytes of pre-antral follicle culture (Figure 4C
).
To determine whether there were consistent differences in spindle shape between IVG, IVM and ovulated MII oocytes, spindle length (X), width at the equator (Y') and width at both poles (Y) were measured, taking the outermost centre of microtubular asters at poles as reference points (see Figure 2). The dimensions of spindles from in-vivo-matured and ovulated oocytes differed between adult and 26-day-old mice (Figure 4A
, B). Oocytes from adult mice had very compact, barrel-shaped spindles, the width of spindles at the poles and the equator being similar. In contrast, oocytes from 26-day-old mice were on average slightly and significantly longer, and had narrower spindle equators and poles (Table III
, Figure 4B
). The overall width of spindles at the equator was in a similar range in spindles of oocytes ovulated in adult and 26-day-old mice. In consequence, spindles from 26-day-old mice had a rather slender shape with pointed poles, as compared with the rectangular shape of spindles of oocytes from adult mice (compare also Figure 4A and B
).
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Metaphase II spindles in denuded oocytes matured to MII in vitro were also examined. Initially, they appeared similar in shape to those from oocytes obtained from 26-day-old mice after in-vivo ovulation. IVM oocytes of 26-day-old or adult mice had also a rather slender shape and relatively pointed poles (Figure 4C). Quantitative analysis confirmed that both oocytes from adult and 26-day-old mice matured in vitro were significantly longer and had exceptionally small poles (6 and 6.4 µm respectively) when compared with those of oocytes of the adult females matured in vivo (11.1 µm) and those of IVM oocytes from follicle culture (10.912.5 µm; Table III
). As a result, the ratio of equator width to pole width was about 2, but it was much lower (1.221.38) in the spindles of oocytes matured in vivo in adult mice or such from pre-antral follicle culture. The average ratio was 1.58 in oocyte spindles of prepubertal mice matured and ovulated in vivo (Table III
).
Chromosome alignment at MII in oocytes matured within or outside of a follicle
The behaviour of chromosomes in those oocytes reaching MII after maturation in follicle culture under different conditions was also compared. In fact, most oocytes of the ambient oxygen group had well-aligned chromosomes (Figures 3D, D
and 4D, D '; Table II
). Of the 108 oocytes ovulated in vivo in 26-day-old mice, and the 111 oocytes from adult females, all (100%) had all chromosomes aligned (Figure 4B
, B '). Similarly, all oocytes matured in vitro outside of a follicle from the 26-day-old mice had well-aligned chromosomes. In only 1.8% of those oocytes obtained from adult mice was a slight scattering of chromosomes at the equator detected (Figure 4C
, C '); this was not scored as chromosome displacement according to the criteria used.
Similar to the situation in in-vivo-matured or denuded IVM oocytes, in only 13% of all the MII oocytes from pre-antral follicles cultured continuously in an atmosphere of 5% CO2 in air (20% O2 group) were individual chromosomes located outside of the equator (Table II, right column). However, this rate increased to 27% upon culture to 20 h post HCG/rEGF addition (Table II
). The percentage of MII oocytes from follicle culture with displaced, unordered chromosomes was significantly higher in the low-oxygen as compared to the normal-oxygen group, with 34.8% unaligned chromosomes at 16 h and 50% at 20 h retrieval of oocytes (Table II
; Figure 4F
, F '). When the rate of oocytes at MII with well-aligned chromosomes from all oocytes obtained in pre-antral follicle culture was calculated, about 40% of all oocytes from the normal-oxygen group belonged to this high-quality group. In contrast, only 11.7 and 11.3% of oocytes with this high quality were collected in the low-oxygen group after final maturation for 16 or 20 h respectively (Table II
).
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Discussion |
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The present study provides evidence that oocytes from pre-antral follicle culture form functional, normal-appearing meiosis I spindles and progress to MII with well-aligned chromosomes. Spindle shape at MII in oocytes from in-vitro-grown follicles closely resembled that of oocytes of adult mice matured and ovulated in vivo after hormonal stimulation by PMS and HCG. Consistently, spindles in oocytes from pre-antral follicle culture acquired an anastral, barrel shape with flat poles, as is characteristic of mammalian oocytes. The pole-to-pole distance of spindles of cultured oocytes was also in a range close to that observed in IVM oocytes. Some consistent differences between the in-vivo and pre-antral follicle group concerned astral fibres which frequently extended from the rim of the spindle poles into the cytoplasm, giving some spindles in oocytes from follicle culture a cushion-like appearance. The impression of a rather square and not so much rectangular shape of the spindle in the oocytes from pre-antral follicle culture was related to the significant differences in spindle length and more compacted spindle equator in IVM oocytes as compared to follicle culture, although these differences were rather subtle. However, average diameters at the equator or pole did not differ greatly between oocytes from in-vivo maturation in adult females and those from pre-antral follicle culture. Possibly the inability of obtaining oocytes of a similar final volume as in vivo from in-vitro-cultured early pre-antral follicle stages might explain the subtle differences in spindle morphology and the failure of some oocytes to acquire full meiotic and developmental competence (Eppig et al., 1992; Gilchrist et al., 1995
).
The observations made in the present study show that oocytes initially grown inside a follicle in vivo in prepubertal, 26-day-old mice but resuming maturation in vitro without follicle cells, had a tendency to form spindles with a less compacted shape and more pointed poles as that seen in cultures of pre-antral follicles from 14-day-old mice or those of in-vivo matured and ovulated oocytes in adult females. The IVM group of oocytes consistently formed rather long, slender spindles with significantly smaller diameters at the spindle pole compared to the former groups. This observation suggests that it is not only cytoplasmic maturity that is obtained during follicle growth, but also signalling and paracrine interactions between the oocyte and its somatic compartment and milieu at and prior to the resumption of maturation which influence spindle formation in oocytes. Molecules and signals obtained by granulosa cells, as well as factors produced inside of the oocyte in response to such signalling events, might act directly on cellular organization.
Chromosomes in vertebrate oocytes are able to recruit MTOC (Eichenlaub-Ritter et al., 1988a; Messinger and Albertini, 1991
), but organization of the oocyte spindle is dependent on the expression of distinct classes of microtubule-motor proteins (Heald et al., 1996
; Walczak et al., 1998
; Mountain et al., 1999
). Differences in expression patterns were reported between human oocytes resuming maturation in vitro, and in-vivo-matured ones (Anderiez et al., 1998
). Differences may also exist in the expression, and hence in the availability, of spindle-associated or motor proteins between oocytes actively initiated to progress into M-phase of first meiosis by their contact and interactions with follicle and theca cells and those matured in vitro in this study; such differences might be responsible for the altered spindle shape.
In agreement with the concept of a crucial role of cellcell interactions and a balanced hormonal environment, the present observations therefore suggest that in-vitro maturation conditions with oocytes inside of a follicle produce oocytes in which spindle formation is more comparable to in-vivo controls than to oocytes obtained from IVM (devoid of normal somatic interactions).
There appears to be a correlation between aberrant spindles and unaligned chromosomes at MII, and errors in chromosome segregation and induction of aneuploidy due to post-ovulatory ageing or in response to chemical exposures of oocytes (Eichenlaub-Ritter et al., 1986; Yin et al., 1998b
). Unordered chromosomes in a MII-arrested mammalian oocyte may present the result of predivision of homologous chromosomes at metaphase I or failures of kinetochores to attach properly to spindle fibres and congress at the equator at MII (Angell, 1997
; LeMaire-Adkins et al., 1997
; Yin et al., 1998a
,b
). Prolonged arrest at MII increases the risk for untimely segregation of chromatids and errors in chromosome segregation at anaphase II (Eichenlaub-Ritter, 1998
; Mailhes et al., 1998
) and, in consequence, trisomy in the embryo (Eichenlaub-Ritter, 2000
).
Prolongation of oocyte culture by only 4 h did not visibly affect spindle structure in a consistent fashion in this study, unlike that found in spontaneously ovulated oocytes post-ovulatory aged in the ampullae in vivo for extended times (Eichenlaub-Ritter et al., 1986). However, the percentage of MII oocytes with displaced, unordered chromosomes was increased in both the normal- and low-oxygen groups as in in-vivo-aged oocytes. There was no consistent alteration in spindle shape associated with such chromosome displacement. Only subtle disturbances in spindle structure could have been responsible for the loss of attachment of kinetochores to spindle fibres and displacement of chromosomes from the equator during ageing in culture. Mammalian oocytes possess a cell cycle checkpoint causing meiotic arrest in the absence of a spindle (Eichenlaub-Ritter and Boll, 1989
; Brunet et al., 1999
). However, oocytes may fail to sense chromosome displacement (LeMaire-Adkins et al., 1997
; Yin et al., 1998b
) such that defects in chromosome congression on the first or second meiotic spindle may have profound effects on the fidelity of chromosome segregation. To prevent such risks, it appears therefore advisable to harvest the oocytes from pre-antral follicle culture already at 16 h after the addition of HCG and rEGF (or even earlier), although this may reduce the total yield of MII oocytes. Hormonal homeostasis modulates the kinetics of in-vivo maturation in mouse oocytes (Polanski, 1986
). Using follicle culture, it is now feasible to determine precisely the influence of LH and FSH on maturation kinetics, spindle formation and chromosome alignment in oocytes.
In order to test for beneficial or adverse influences of unphysiologically high oxygen tensions during IVM, maturation rate, spindle formation and chromosome behaviour were examined in oocytes placed into an atmosphere with a more physiological (i.e. reduced) oxygen concentration. Indeed, in the Fallopian tubewhere freshly ovulated oocytes remain shortly before actual fertilizationoxygen tension is low. However, the reduction in oxygen tension interfered severely with maturation of oocytes under our culture conditions, since low numbers of oocytes emitted a polar body. Earlier studies (Eppig and Wigglesworth, 1998) using different follicle culture techniques for oocyte growth and maturation in vitro suggested that an atmosphere of 5% CO2 in air (20% O2) may significantly reduce the rate of oocyte maturation and the developmental capacity of the obtained embryos. There may be a direct link between increases in ROS and H2O2 in oocytes and embryos, and developmental arrest (Nasr-Esfahani and Johnson, 1991
; Kwon et al., 1999
) and induction of apoptosis (Yang et al., 1998
). From our previous experience, denuded mouse oocytes are capable of developing to MII in an atmosphere of 5% CO2/5% O2/90% N2, so the meiotic arrest in pre-antral follicle culture with reduced oxygen tension during the last day prior to oocyte retrieval was unexpected.
There are two possibilities to explain the meiotic block in oocytes from our pre-antral follicle culture with low oxygen during the last 16 or 20 h. It may be primarily the follicle and theca cells which are dependent on sufficient oxygen supply for their metabolism during in-vivo and in-vitro folliculogenesis, especially at resumption of maturation (Boland et al., 1994). Since availability of oxygen depends entirely on diffusion in follicle culture, reduced oxygen tension could interfere with activity of follicle and theca cells, particularly when HCG and rEGF are added, and thus prevent initiation of resumption of maturation.
The very efficient depletion of oxygen by the large number of follicle cells (1530x103 cells) in the microdroplet may also have caused indirectly the meiotic arrest of oocytes such that oocytes had perhaps to resume maturation under near-anoxic conditions.
Oxygen supply and ATP appear to be important determinants of meiotic and developmental competence in in-vivo maturing oocytes. Antioxidants reversibly inhibited the spontaneous resumption of meiosis in mammalian oocytes (Takami et al., 1999), and chemicals which affected mitochondrial function and the intimate association and accumulation of mitochondria with the oocyte spindle caused meiotic arrest or delay, and predivision or non-disjunction of chromosomes (Yin et al., 1998a
). Under reduced oxygen concentrations, the activity of motor proteins and other regulatory factors in spindle formation (Huang et al., 1999
; Mountain et al., 1999
) and cell cycle-regulating proteins may be affected in oocytes, when mitochondrial functions are compromised and little ATP provided. Under such conditions, synchrony in cytoplasmic and nuclear maturation, spindle formation and fidelity of chromosome segregation could be severely affected.
Under an ambient oxygen tension as is used routinely (Cortvrindt et al., 1996), diffusion in pre-antral follicles appears sufficient to provide for this basic and essential oxygen supply, without inducing visible damage. Preliminary observations on blastocyst formation rates from oocytes derived from culture under high oxygen did not provide evidence for an adverse influence of a normal oxygen tension (20%) under the presently chosen culture conditions. Oocytes express antioxidant genes and proteins (El Mouatassim et al., 1999
), and antioxidants and proteins suppressing ROS are also present in follicular fluid and granulosa cells (Briggs et al., 1999
; Jozwik et al., 1999
) such that even high concentrations of ROS may be tolerated. Oestradiol which is produced normally in this culture system (Cortvrindt et al., 1996
) may have an additional protective activity to prevent damage by ROS (Lund et al., 1999
). The present study supports the notion that oxygen supply is a critical parameter during pre-antral follicle culture, particularly during resumption of maturation in oocytes when spindle assembly and chromosome alignments are occurring.
In summary, the observations made in this study stress the importance of appropriate interactions between oocytes, their somatic compartment and the culture medium composition, and the requirement for sufficient oxygen in oocyte maturation, spindle formation, and high fidelity of chromosome segregation. Growth and maturation of oocytes within normally differentiating somatic cells in a system such as described herein provides the opportunity to study the effects of culture environment on ultrastructural dynamics involved in chromosome segregation. Further studies are required to determine the significance of these effects on embryo quality and the ability to develop to term.
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Acknowledgements |
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Notes |
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References |
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Submitted on August 17, 2000; accepted on .