Hum. Reprod. Advance Access originally published online on September 2, 2005
Human Reproduction 2006 21(1):240-247; doi:10.1093/humrep/dei283
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Meiotic abnormalities in in vitro-matured marmoset monkey (Callithrix jacchus) oocytes: development of a non-human primate model to investigate causal factors
1 Human IVF Center, Department of Biology, Medical University, Sofia, 1431- Bulgaria. 2 Department of Reproductive Biology, German Primate Centre, Kellnerweg 4, Goettingen D-37077, Germany 3 Current address: Department of Gynaecological Endocronology and Reproductive Medicine, University Women’s Clinic, University of Bonn, Bonn D-53105, Germany
4 To whom correspondence should be addressed. E-mail: pnayudu{at}gwdg.de
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Abstract |
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BACKGROUND: Meiotic abnormalities are thought to be a major causal factor of low embryo development rates, for embryos developed from in vitro-matured oocytes. A new non-human primate model, in the common marmoset, is being developed to facilitate investigation of the mechanisms involved. METHODS: Oocytes were dissected from antral follicles from three size classes. They were allowed to mature in vitro for only 24 h, in order to focus the investigation on the rapidly maturing oocytes. Chromosome spreads were visualized with Giemsa staining, and spindles /chromosomes with fluorescently labelled anti-
-tubulin antibody combined with a DNA fluorochrome. RESULTS: 40% of the oocytes had reached metaphase II (MII) after 24 h. Of the MII oocytes selected for karyotyping, readable chromosomal spreads were obtained from 64%. Overall, 63% of these presented a normal haploid chromosome number of 23,X, with all abnormal karyotypes occurring in the oocytes from small follicles. For another group of MII oocytes, where meiotic spindles were visualized, only half of the MII oocytes displayed well-formed spindles and apparently correct chromosomal alignment. CONCLUSIONS: This work provides the first information on the normal and aneuploid MII meiotic chromosome sets for the marmoset oocyte, and demonstrates a high rate of chromosomal and spindle abnormality among rapidly maturing oocytes from small antral follicles.
Key words: aneuploidy/in vitro maturation/marmoset/meiosis/oocyte
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Introduction |
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A substantial proportion of human embryos are lost before their implantation in the uterus and therefore too early to be detected as pregnancies even by elevated HCG levels. It has been calculated that
47% of in vivo-fertilized human embryos are lost in the first 2 weeks (Leridon, 1973
; Racowsky, 2002). It is most likely that suboptimal oocyte quality is a major source of this loss. Further, although birth rates for assisted reproduction techniques have now reached approximately the levels expected for in vivo (25–35% per cycle) (Racowsky, 2002; Macklon et al., 2002); this rate is achieved only through the practice of multiple embryo transfer (usually two or three).
Numerous studies of in vitro-produced embryos have shown that aberrations in the first meiotic division resulting in aneuploidy are the likely major causal factor (Wells et al., 1999; Wolstenholme and Angell, 2000; Hodges et al., 2002; Hunt and Hassold, 2002) in embryo loss in vitro. But, since the data on the natural oocyte meiotic error rates for the human fertile population are limited (Gras et al., 1992; Battaglia et al., 1996; Volarcik et al., 1998; Munné et al., 2000; Sandalinas et al., 2001), the contribution of the oocyte to embryonic aneuploidy in vivo remains unclear. From in vitro data, the published rates of chromosomal abnormality vary greatly with a range between 8 and 60% of oocytes of IVF-treated women with strong evidence for age-related increases (Plachot et al., 1987; Angell et al., 1991; Pellestor, 1991; Edirisinghe et al., 1997; Pellestor et al., 2002; Zhivkova, 2003; Verlinsky and Kuliev, 2004).
Although it is likely that a major proportion of these chromosomal defects have their origin in the oocyte, it is difficult to isolate the major causal factors using clinical material since many different factors may influence rates of aneuploidy. For example, subnormal ovary function, follicular origin, and the hormonal stimulation treatment all may negatively influence oocyte quality. Further complication in the interpretation of results is that the oocytes available are usually only those which have failed to fertilize or failed to cleave 48 h after fertilization, and that the culture conditions themselves may influence chromosomal and spindle normality (Hodges et al., 2002; Eichenlaub-Ritter et al., 2004; Trussler et al., 2004). However, that the time in culture does not significantly contribute to the aneuploidy rate has been suggested by the similarity of results obtained by the few studies of aneuploidy in fresh uninseminated oocytes (Gras et al., 1992; Sandalinas et al., 2002).
Finally, the growing practice of in vitro-maturing (IVM) human oocytes harvested from smaller antral follicles as an alternative approach for obtaining oocytes for fertilization (Wynn et al., 1998; Mikkelsen et al., 1999; Wright et al., 1999; Combelles et al., 2005) makes it increasingly urgent to determine both the rate and causal factors of meiotic abnormalities. In order to facilitate this aim, the establishment of in vitro systems, using a species as close to human as possible, where the animal variables as well as the in vitro maturation conditions can be controlled and manipulated would be desirable. Only through this approach will it be possible to acquire fundamental information on the causal factors necessary to develop strategies to reduce the incidence of meiotic errors.
IVM is already being carried out on the oocytes of two species of non-human primates, the rhesus (Schramm et al., 2002, 2003) and the marmoset (Gilchrist et al., 1997; Isachenko et al., 2000 and unpublished data; Faerge et al., 2001; Nayudu et al., 2003). A limited number of rhesus oocyte metaphases have been investigated (Schramm et al., 2002) and similarities between human and rhesus female meiotic disturbances have been noted, but no detailed studies have been published to date. Marmosets have the practical advantage of a larger number of maturable oocytes per ovary from small and medium-sized antral follicles without the necessity of ovarian stimulation. Therefore it has been chosen as the model species for the present study. The focus of the present investigation has been to provide initial information about marmoset oocyte chromosomes, and to evaluate the rate of aneuploidy and spindle abnormalities occurring in in vitro-matured oocytes from different antral follicle classes. This study is expected to provide a basis for further investigations into the mechanisms responsible for oocyte chromosomal abnormalities in human and non-human primates, and provide necessary information for treatments designed to protect the quality of oocytes during their growth in the ovary and during in vitro maturation.
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Animals
All procedures were carried out according to German Animal Experimentation Law (Animal Experiment Permission # 509.42502/08-01-03). Animals were housed according to standard German Primate Centre practice for this species (Gilchrist et al., 1997
; Isachenko et al., 2002). Ovarian cycles were monitored by plasma progesterone measured by enzyme immunoassay according to a technique developed by Heistermann et al. (1993). Plasma was obtained twice a week by femoral vein puncture. Cycles were either natural or were controlled by a luteolytic dose of Estrumate (Mallinckrodt Vet GmbH, Burgwedel, Germany), which is a prostaglandin F2 analogue (Summers et al., 1985). Ovaries from five young adult marmosets (aged 25–36 months) were used in this study. In all cases Estrumate had been administered 7 days before the ovarectomies, which were carried out under injectable anaesthesia on the morning of day 7 of the follicular phase to increase the probability that advanced follicles were present. The animals were thereafter killed by an overdose of anaesthetic, and the organs distributed to other experimenters.
Oocyte collection
The ovaries were transferred into modified Leibovitz L-15 medium (Gibco–Invitrogen, Karlsruhe, Germany) supplemented with 5% heat-inactivated fetal calf serum (Seromed, Biochrom, Berlin, Germany), 1.0 µmol/l Glutamax (Gibco–Invitrogen), and penicillin–streptomycin (Sigma–Aldrich, Taufkirchen, Germany). Using previously published visual criteria (Gilchrist et al., 1995), non-degenerate antral follicles with a minimum diameter of 700 µm diameter were mechanically dissected and sorted by size. Follicles were measured using an ocular scale at a standard magnification and classified according to diameter into three groups: small (700–1000 mm), middle (1000–1400 mm) and large antral follicles (>1400 mm) as previously published (Gilchrist et al., 1997). The cumulus–oocyte complexes (COC), obtained by puncture of the antral follicles in the same medium as above with the exception of an increased percentage of fetal calf serum (FCS) (10% Seromed–Biochrom), were sorted according to whether the cumulus cells were completely intact and tightly attached to the oocyte or not according to previously published criteria (Gilchrist et al., 1995).
Oocyte IVM
The COC were thereafter transferred into 50 µl drops of maturation medium. The medium consisted of modified minimal essential medium (MEM) (Gibco–Invitrogen,) under light paraffin oil (Sigma–Aldrich). This method has been published in brief (Isachenko et al., 2000), and the full details and optimization are currently submitted for publication elsewhere (E.Isachenko et al., unpublished data). Therefore the modification of this procedure is described here only in brief. The base medium was supplemented with 20% heat-inactivated FCS, 10 IU recombinant hFSH (Gonal-F; Serono Pharma, Geneva, Switzerland) (10 IU/ml hCG (Pregnesin Serono), and 1 µg/ml 
-estradiol (Sigma–Aldrich). Additionally 1.0 mmol/l Glutamax (Gibco-Invitrogen) 0.5 mmol/L, sodium pyruvate and 10 mmol/l sodium lactate, 50 µg/ml L-ascorbic acid (all Sigma–Aldrich) were present. The oocytes were matured in groups of 5–10 for 24 h in a gas atmosphere of 5% CO2 in air at 38.4°C. The expanded cumulus cells were removed after the maturation with hyaluronidase (Sigma–Aldrich) and mechanical pipetting. Thereafter the denuded oocytes were scored for the presence of a polar body, and photographed conventionally using a Zeiss Axiovert microscope under differential interference contrast optics. Immediately thereafter the oocytes were proportionally (based on follicle size) allocated to one of two groups and processed for chromosome karyotyping, or fluorescent imaging of the spindle and chromatin.
Chromosomal spread preparation
A modified Tarkowski (1966) technique, the purpose of which was to minimize the risk of individual chromosome loss while promoting optimal chromosome spreading, was used for preparing oocyte chromosomal spreads. The zonae pellucidae were removed by acid Tyrode solution, pH 2.0 adjusted by HCl. The oocytes were incubated for 15 ± 5 min at room temperature in hypotonic solution (1% sodium citrate) in dH2O with 2% bovine serum albumin (BSA) (Sigma) to prevent adhesion to the dish, placed in minimal volume onto slides cleaned immediately before with acetone, dried (while being observed under a dissecting microscope) and fixed in a small drop of methanol/acetic acid (3:1). Fixation was controlled under phase-contrast microscope. Three to five drops of fixative were added and dried until the cytoplasm could no longer be detected (Delimitreva, 2002; Zhivkova, 2003).
Cytogenetic analysis
After 2 days at room temperature or overnight on a warm plate at 60°C, the slides were dehydrated and rehydrated in alcohol series (70%, 80%, absolute ethanol and back), stained with a standard Giemsa solution (Merck, Darmstadt, Germany). Chromosomal spreads were visualized by conventional light microscopy (Zeiss Axiophot) under oil without coverslip. The image was recorded digitally using an Open Lab Image Analysis system (Improvision, Coventry, UK). Each chromosome preparation was examined and the chromosomes counted by two independent observers. Marmoset somatic chromosome nomenclature proposed by Sherlock et al. (1996) was employed for tentative chromosome identification and karyotyping.
Fluorescent visualization of spindle and metaphase plate
Tubulin and chromatin were visualized using a modification of a method previously published for marmoset oocytes (Gilchrist et al., 1995). Oocytes were fixed in prewarmed (37°C) 2% paraformaldehyde (Merck, Darmstadt, Germany) in Dulbecco’s phosphate buffered saline (DPBS; Sigma) with 0.04% Triton X-100 (Serva, Heidelberg, Germany) for 1 h. Afterwards they were washed twice in DPBS (Sigma) plus 0.3% BSA (Sigma) for 10 min. Thereafter they were stored overnight at 4°C in DPBS + 0.3% BSA + 0.02% sodium azide. For spindle visualization the fixed oocytes were incubated in mouse monoclonal anti--tubulin (1/2500; ICN, Meckenheim, Germany) in DPBS with 0.3% BSA and 1 µl/ml Tween-20 (Merck) for 90 min at 37°C. The staining was performed with fluorescein isothiocyanate (FITC) conjugated to goat anti-mouse F(ab)2 (1/100; ICN) together with 10 µg/ml Hoechst 33258 (Hoechst Aventis, Paris, France) in DPBS + 0.3% BSA for 45 min at 37°C in the dark. The oocytes were subsequently washed three times in DPBS + 0.3% BSA + 1 µl/ml Tween 20 and thereafter impregnated with Mowiol 4–88 mounting medium (Hoechst) in four concentration steps, 5, 10 30 and 50% in DPBS + 0.3% BSA and 0.02% sodium azide for 10 min each. The oocyte was then placed in a drop of 100% Mowiol on a clean glass slide and covered with a cover glass. These were stored in the dark until they were photographed conventionally under oil using the x100 objective with an Axiovert microscope under epifluorescence illumination with FITC excitation at 490 and Hoechst at 365 nm.
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Results |
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A total of 166 oocytes with attached cumulus cells from different follicle size classes were selected for IVM. After 24 h in vitro maturation 117 oocytes were prepared for cytogenetic analysis: four oocytes from large, eight from middle-sized and 105 from small follicles. Forty-nine additional oocytes were selected proportionally from each follicle size group from four animals for fluorescent spindle and chromosome staining.
The oocytes used for chromosome spreading were classified according to their meiotic progression after the 24 h maturation period. The results are shown in Table I. Of the total of 117 oocytes, 47 (40%) had reached metaphase II (MII). It can also be observed that the proportion of oocytes which matured varied markedly among the individual animals. Chromosomal number was determined in 30 out the 47 MII oocytes (64%). The remaining MII plates could not be accurately analysed because of chromosomal clustering and multiple overlapping or incomplete condensation of chromosomes. Fourteen oocytes (12%) were arrested at metaphase I (MI) and two (2%) at anaphase I (AI). Thirty-four oocytes (29%) were at different points of dictyate–MI transition, defined as germinal vesicle breakdown (GVBD). An intact nucleus with single nucleolus (defined as germinal vesicle stage, GV) was observed in 12 oocytes (10%), and eight (7%) had a necrotic appearance with absence of nuclear material and are not included in Table I.
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From the 30 MII oocytes for which karyotypes could be obtained, only 19 (63%) had a normal haploid chromosome number 23,X. Figure 1 illustrates a normal MII haploid chromosomal set (Figure 1A) together with a tentative chromosome identification (Figure 1B) according to the Sherlock system. Numerical chromosomal abnormalities were observed in the remaining 11 oocytes (37%). Two types of aneuploidy were recorded. Single chromatids were recorded in three oocytes, and whole missing or extra chromosomes were observed in seven oocytes: five cases with hyperhaploidy and two with hypohaploidy, including three spreads with extreme aneuploidy (12, 31 and 33 chromosomes). Furthermore, one oocyte had a complete diploid chromosomal set: 46,XX. Of the MII oocytes from small follicles, 15/26 (58%) exhibited normal karyotypes. Eleven (42%) were chromosomally abnormal, which represents all of the aneuploid MII oocytes in the sample. All four readable MII derived from the 12 large and middle follicles were normal, but larger numbers are required to confirm this relationship. Distribution of mature and chromosomally normal oocytes according to the follicle size is shown in Table II.
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In 16 of the MII oocytes with a readable main chromosome set, the first polar body (PB) also was located on the slide, but only six of these PB showed countable chromosomal spreads (example of a normal countable PB MII spread is in Figure 2.). Five of these cases had 23 chromosomes in both sets (23 + 23) and one displayed correct total chromosome number, but with hypohaploid main set and hyperhaploid PB set (21 + 25). The remaining 10 PB were fragmented, with apoptotic chromatin, and therefore could not be evaluated for chromosome number.
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An additional type of chromosomal abnormality involving incomplete condensation (referred to as ‘long’ chromosomes) was also observed. This defect was primarily observed in the chromosomes of one animal (marmoset 3). Sixty one per cent of oocytes (17/28) derived from this individual, defined as mature according to the presence of polar bodies, had ‘long’ chromosomes (Figure 3). This abnormality occurred both in the oocyte and PB for each affected oocyte. In the oocytes of two additional animals, individual examples of ‘long’ chromosomes were observed in one oocyte each, but in both instances only limited number of chromosomes were involved, with the remaining having normal metaphase appearance. A further 12 countable chromosome spreads were obtained from 14 MI oocytes (one from a middle-sized and 11 from small follicles). All had normal total chromosome number, although separated bivalents were observed in two oocytes from one animal (Figure 4).
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Fluorescent visualization of the spindle and chromosomes was also carried out on 49 oocytes from four of the marmosets (oocytes from one animal were not included because of the small total number of oocytes recovered). Approximately one-third of the oocytes were selected proportionally from the follicle size classes for this treatment: one from a large follicle, five from middle size, and 43 from small follicles including a random selection of GVBD and MII oocytes. GV oocytes were excluded because of their lack of spindle. The distribution of oocytes according to their meiotic progression and spindle normality from each animal and in total is presented in Table III. Nineteen from 49 (39%) of the oocytes were defined as mature based on the presence of a MII spindle and a polar body. Nearly half of these (10) were found to have a well-formed spindle without free tubulin fibres and well-aligned chromosomes (Figure 5). The tendency for fast destruction of polar bodies noted with the chromosome spreads was confirmed by spindle observations (Figure 5A and B). Fourteen oocytes were classified as MI based on the lack of a polar body. Half of these (7/14) displayed normal spindle organization (Figure 6). Although low numbers limit the interpretation at this stage, this suggests that for MI oocytes, in spite of the high normoploidy rate, spindle abnormality at this stage is likely to lead to aneuploidy in the MII stage. For those animals where ‘long’ chromosomes could be observed in the spreads, a similar phenomenon was also observable within the spindle, thereby verifying the validity of this finding (Figure 5B).
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These results, differentiated according to follicle size class, are presented in Table IV. The one oocyte from a large follicle was arrested at MI with a disorganized spindle. The five oocytes from middle-sized follicles had normal spindles; four (80%) were MII and one was MI. Of the 43 oocytes from small follicles 15/43 (35%) were MII and only five of these (33%) were classified as having a normal spindle. Twelve of the 43 (28%) were MI. Six of these (50%) were classified as having a normal spindle. Five were in transition stages (anaphase I and telophase I: example shown in Figure 7) and four had remained at GVBD stage and three were degenerative.
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Discussion |
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This study is part of a larger one aimed at establishing the common marmoset as a primate model for human in the investigation of oocyte maturation in vitro (Gilchrist et al., 1995
; 1997; Isachenko et al., 2000; E.Isachenko et al., unpublished data). The rationale for the use of a primate species as a model for the human is based on their evolutionary closeness to our species. The marmoset is advantageous over the more commonly used macaque in that the limitations on material are less stringent, and sufficient numbers of maturable oocytes per individual can be obtained without hormonal stimulation. Further, fully matured and incompletely matured oocytes as well as cleaving embryos can be made available for unrestricted experimentation in contrast to the clinical situation.
Since a fundamental aim of the present investigation was to analyse the innate variation in the ploidy of the oocytes in isolation from the effects of the in vitro conditions, only a single in vitro maturation condition, that shown in our laboratory to produce the best maturation rate among those so far tested (Isachenko et al., 2000; E.Isachenko et al. unpublished data), was employed. Further in an attempt to reduce the variables at the animal level, no exogenous gonadotrophin treatment was used, and the age range of the animals was restricted to between 2 and 3 years (they are considered adult at 2 years).
The first study of meiotic chromosomes of the marmoset (in spermatocytes and MI oocytes) was provided by Chandley (1989). We have presented and evaluated the first normal and abnormal metaphase II oocyte chromosomal sets for the marmoset. Aneuploidy among MII oocytes was found only with those originating from small antral follicles and this was consistent with a rate of spindle abnormality, which roughly paralleled the aneuploidy rate. Other abnormalities such as incomplete chromosome condensation and premature chromatid separation have been noted, and were also confined to oocytes from small follicles. It is supposed that these variations are related to disturbed co-ordination between follicular development, nuclear maturation and cytoplasmic competence (Combelles et al., 2002). Additionally, since the incompletely matured and degenerated oocytes also originated predominantly from the small antral follicles, this is presumably a function of the developmental stage as well as the atresia status of this class of follicles. In contrast to MII oocytes, the rate of numerical chromosomal defects for oocytes that had only reached the MI stage was low. But the associated high rate of spindle abnormality suggests that the following progression would result in MII aneuploidy (if the cell cycle control is compromised) or meiotic delay or arrest. Our results are in accordance with the studies that define the imperfect action of the cell cycle control in the first meiotic division as a main cause for unusually frequent aneuploidy of mammalian oocytes (Hassold, 1998; Steuerwald et al., 2001; Hunt and Hassold, 2002).
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The diploid karyotype of common marmoset monkey Callithrix jacchus was first reported by Benirschke et al. (1962)
. The diploid chromosome number is 46: 23 chromosomal pairs (22 pairs of autosomes and two sex chromosomes) for C.J. and its closest sister species, in contrast to other known marmoset Callithrix species, which have 22 chromosomal pairs (Dutrillaux and Couturier, 1881; Seuanez et al., 1988; Nagamachi et al., 1992, 1996). The basic chromosomal nomenclature for Callithrix jacchus was proposed by Perrotez (1974). Several alternative nomenclatures based on chromosome banding using marker band combinations for human chromosomes have been suggested subsequently. In the present study we have used the system based on chromosomal size and the original nomenclature (Benirshke et al., 1962) proposed by Sherlock et al. (1996). The current report is the first for the haploid chromosome set of the mature marmoset oocyte. Unequivocal identification of the chromosomes has not yet been achieved, and is an important next step for further progress. Studies are underway comparing different FISH strategies for their effectiveness with marmoset chromosomes.
We have applied a variant of the Tarkowski method for spreading oocyte MII plates with one-step fixation because our experience with human oocytes and embryos (Delimitreva et al., 2001, 2005; Delimitreva, 2002; Zhivkova, 2003) indicates that this method minimizes the risk of chromosomal loss. In the present study hypohaploidy was recorded in <20% of the aneuploid MII oocytes. In contrast hyperhaploidy was the most common occurrence, supporting our previous findings that this method does not lead to overspreading of metaphase plates and chromosomal loss. We were able to support this assumption by the fact that all analysed MI oocytes had correct total chromosome number, and, in the cases where the chromosomes of the polar body were also countable, the total number was always correct. The oocyte chromosomes were karyotyped after conventional Giemsa staining without banding of the chromosomes. This classic approach was used because our experience with human oocytes and the fact that extreme condensation of oocyte chromosomes precludes interpretable banding results (Pellestor et al., 1993; Delhanty, 2001). The percentage of successfully analysed oocytes obtained by us in this study (64%) is close to the results for human oocytes from a range of authors and our own previously published studies (Plachot et al., 1988; Almeida and Bolton, 1993; Kamiguchi et al.,1993; Zhivkova, 2003).
The rate of readable chromosomal spreads from four of the animals was 80% (24/30), thereby validating the method for use with the marmoset. However, one of the five monkeys had ‘long’ (not fully condensed) chromosomes and as a result, only one-third of the MII plates were readable from this animal. That the same long chromosome structure was seen with the oocytes processed for spindle evaluation indicated that this was not a methodological artefact. Additionally, since the aneuploidy rate for this monkey was comparable to other individuals (with five from six countable chromosomal spreads being normal) and that the alignment of the ‘long’ chromosomes on the metaphase plate was correct, this defect was thought to be unrelated to disturbances of chromosomal distribution. Although the functional significance remains unclarified, the presence of ‘long’ chromosomes both in oocytes and corresponding PB from this one individual could be a sign of asynchrony between nuclear and cytoplasmic meiotic progression (Combelles et al., 2002), and appeared to be largely an animal-specific defect. Also interesting was the presence of single ‘long’ chromosomes in otherwise apparently normal MII plates in single oocytes from two other animals.
In the present study, maturation rate of oocytes (for 24 h) from large and middle-sized follicles was higher than for oocytes from small follicles, but the total maturation rate was lower than that reported by us in a previous study (Isachenko et al., 2000 and unpublished data) for maturation for over 30 h under the same conditions. Oocytes from larger follicles completed their maturation within the 24 h while a proportion of the oocytes from smaller follicles required a longer time. The shorter time was employed to avoid spindle ageing in the faster-maturing oocytes, and it is of theoretical and practical interest, that all MII karyotypes and spindles from middle-sized and large antral follicles were normal. In contrast the MII oocytes from small antral follicles exhibited a relatively high frequency of abnormal karyotypes and spindles. This means that rapidly maturing oocytes from small antral follicles are potentially the major source of aneuploidy, and as such present a considerably higher risk as a source of oocytes for embryo production than oocytes from larger follicles. The fact that, in contrast, the tubulin structure of the oocytes still in transition stages appeared normal supports the suggestion that the more slowly maturing oocytes from small follicles may represent a healthier subpopulation.
Aneuploidy may be due to several different possible cell cycle dysfunctions which can be distinguished by the pattern of the abnormality: (i) mitotic non-disjunction in some primordial cells (gonadal mosaicism) leads to abnormal number of bivalents at MI; (ii) premature disjunction or non-disjunction of bivalents during MI results in extra or missing chromosomes in MII oocytes; (iii) premature separation of chromatids during MI results in extra or missing single chromatids at MII (Angell, 1997; Pellestor et al., 2002). In our study of marmoset IVM, all MI spreads had normal total number of univalents, so we could exclude gonadal mosaicism as a reason for oocyte aneuploidy. In total, the overall frequency of whole chromosome aneuploidy in analysed marmoset oocytes exceeded aneuploidy due to single chromatids, which means the most prevalent cause of aneuploidy was premature disjunction/non-disjunction of bivalents during MI. It will be important therefore to compare these results with those obtained from older marmosets to determine if the single chromatid-based aneuploidy increases and to evaluate the causal factors and possible preventive strategies for both types of aneuploidy.
In conclusion, the present investigations have provided the first information on the chromosomes of the in vitro-matured marmoset MII oocyte. The results have shown that the maturation rate of oocytes from larger follicles is higher (after 24 h) than those from smaller follicles. Of particular importance was the observation that for those oocytes that had reached MII after 24 h, aneuploidy and spindle defects occurred only in oocytes originating from smaller antral follicles (700–1000 µm diameter) and that the most frequent cause was premature disjunction/non-disjunction of bivalents. This result indicates that a significant risk exists in using rapidly maturing oocytes from small antral follicles for embryo production after in vitro maturation. The results further indicate that the marmoset monkey may provide a suitable model for critical investigations, both in vivo and in vitro, of factors that may influence oocyte aneuploidy.
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The authors wish to thank Susane Rensing and Anette Schrod of the Department of Primate Husbandry for performing surgical procedures, and Jutta Hagedorn of the Comparative Endocrinology Laboratory for hormone measurements for cycle monitoring.
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References |
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Submitted on June 7, 2005; revised on July 29, 2005; ; accepted on August 4, 2005
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