Experimental evidence that changes in oocyte growth influence meiotic chromosome segregation

C.A. Hodges1, A. Ilagan1, D. Jennings1, R. Keri2, J. Nilson2 and P.A. Hunt1,3

1 Department of Genetics and 2 Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and interpretation
 Discussion
 Acknowledgements
 References
 
BACKGROUND: It is well known that the fidelity of meiotic chromosome segregation is greatly reduced with increasing maternal age in humans. More recently, direct studies of human oocytes have demonstrated a striking age-related increase in oocytes exhibiting gross disturbances in chromosome alignment on the meiotic spindle. This abnormality, termed congression failure, has been postulated to be causally related to human non-disjunction and to result from subtle alterations in folliculogenesis that develop with advancing reproductive age. METHODS: Immunofluorescence staining, conventional cytogenetic analysis and spectral karyotyping of oocytes from mouse models were used to investigate the hypothesis that changes in the regulation of folliculogenesis induce meiotic defects. RESULTS: Mutations that affect oocyte growth were found to increase the frequency of congression failure at first meiotic metaphase. Importantly, increased congression failure was correlated with meiotic non-disjunction, suggesting a cause-and-effect relationship. CONCLUSIONS: Our findings support the hypothesis that congression failure results from disturbances in the complex interplay of signals regulating folliculogenesis and that these changes subtly alter the late stages of oocyte growth, increasing the risk of a non-disjunction error. These findings have important implications for human aneuploidy, since they suggest that it may be possible to develop prophylactic treatments for reducing the risk of age-related aneuploidy.

Key words: age-related aneuploidy/folliculogenesis/meiosis/non-disjunction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and interpretation
 Discussion
 Acknowledgements
 References
 
In the human, a significant number of conceptions are lost as a result of errors in chromosome segregation during the meiotic divisions. The reason for the high incidence of meiotic errors in our species remains unclear; however, the incidence of errors is strongly correlated with maternal age (Hassold and Chiu, 1985Go). Data from studies of the human maternal age effect over the past three decades have suggested that an age-related increase in meiotic errors is a characteristic of women of all racial groups (Hassold and Chiu, 1985Go) and that most meiotic errors have their genesis at meiosis I (MI) (Hassold and Hunt, 2001Go). There is variability among human chromosomes, both with respect to the error frequency and the extent to which age influences the rate of error (Hassold and Chiu, 1985Go). Nevertheless, for those chromosomes that have been analysed in detail, it is clear that both the number and the placement of the sites of recombination along the length of the chromosome arms influence the fidelity of homologue segregation at MI (Hassold and Hunt, 2001Go). The recognition that certain exchange configurations are `susceptible' to meiotic segregation errors led to the postulation that both prenatal meiotic events (i.e. the initiation of recombination) and postnatal events (i.e. the effect of age) contribute to the high incidence meiotic errors in our species (Lamb et al., 1996Go).

The complexity of a meiotic process in which chromosome segregation is influenced both by prenatal events and age has significantly complicated the study of human meiotic non-disjunction. Studies of human trisomy have provided considerable information on the impact of age but not the underlying molecular mechanism of error. Further, since the effect appears to be unique to the human female, studies in lower eukaryotes have provided little insight. However, recent direct studies of human oocytes by us (Volarcik et al., 1998Go) and by others (Battaglia et al., 1996Go) have provided evidence for a meiotic phenotype that may be related to non-disjunction. Specifically, in both laboratories, immunofluorescence studies of oocytes from different-aged donors revealed an age-related increase in oocytes with gross aberrations in spindle morphology and chromosome alignment; as the chromosomes appeared unable to move to the equator of the MI spindle, we termed this condition `congression failure' (Figure 1a,bGo). Because the oocyte acquires the competency to reinitiate and complete the meiotic divisions during the late stages of growth in the adult ovary, we interpreted this age-related disturbance in chromosome alignment as evidence that the maternal age effect on chromosome segregation results from a decline in the growth process of the human oocyte (Volarcik et al., 1998Go).



View larger version (48K):
[in this window]
[in a new window]
 
Figure 1. Confocal images of human and mouse oocytes. Examples of meiosis II (MII) arrested human oocytes exhibiting congression failure (a, b); of the successive stages of the first meiotic division in mouse oocytes (c–e); and of the types of chromosome anomalies in mouse oocytes scored in this study (f–h). The oocytes in all panels have been stained with an antibody to ß-tubulin to visualize the meiotic spindle (green) and propidium iodide to visualize the chromosomes (red). (a) An MII arrested human oocyte showing both the first polar body (bottom left) and the second meiotic spindle (top right). Note that although a normal MII spindle is present, the chromosomes have failed to assemble at the spindle equator (congression failure). (b) Higher magnification view of the MII spindle in a human oocyte exhibiting congression failure. (c) A mouse oocyte at early prometaphase showing condensed chromosomes surrounded by a mass of microtubules. (d) A slightly later stage of prometaphase, showing evidence of pole formation and of the early movement of the chromosomes to the spindle equator. (e) An oocyte exhibiting the tight metaphase I configuration that is achieved 8–10 h after the resumption of the division. (f) An oocyte exhibiting a loose `metaphase I' (actually late prometaphase) configuration with one bivalent that would be scored as an outlier. (g) An oocyte from an XYPOS female exhibiting an early MI bipolar spindle but little evidence of chromosome congression (e.g. compare with d); this configuration would be scored as congression failure. (h) An MII arrested oocyte exhibiting congression failure on the second meiotic spindle. The image in (a) is at 40% magnification relative to (bh).

 
This hypothesis suggests that even subtle changes (e.g. changes in cycle length, slightly elevated FSH levels etc.) that influence the final stages of oocyte growth may have a profound effect on the meiotic process. It also makes an important assumption, namely that there is a causal relationship between the observed chromosome alignment defects and meiotic non-disjunction. Although, intuitively, failure to properly align the chromosomes on the spindle seems likely to increase the frequency of segregation errors at anaphase, this presupposes that anaphase occurs normally in such cells. Based on our understanding of cell cycle control mechanisms this seems unlikely, i.e. studies of mitotic cells and male meiotic cells suggest that a checkpoint mechanism that monitors both spindle assembly and chromosome attachment and alignment operates at the metaphase/anaphase transition (Burke 2000Go; Shah and Cleveland 2000Go). Hence, cells with gross aberrations in chromosome alignment would be expected to delay or arrest at metaphase. Our previous studies of oocytes from two mouse mutants, however, suggest that, although the spindle assembly portion of the checkpoint mechanism is functional, the chromosome monitoring aspect is ineffective or absent during mammalian female meiosis (LeMaire-Adkins et al., 1997Go; Woods et al., 1999Go). Thus, the fate of human oocytes exhibiting congression failure during the meiotic divisions remains an intriguing question.

Because age-related non-disjunction appears to be unique to the human, designing experimental approaches to study the mechanism of human meiotic defects has proved difficult. However, the hypothesis that subtle changes in the complex interplay of signals that regulate folliculogenesis may impact the meiotic process is testable in an experimental mammal. Accordingly, we initiated meiotic studies of mouse oocytes in several situations in which folliculogenesis is disrupted. In this report, we summarize data from these studies that suggest that: (i) congression failure at MI is associated with a primary defect in either the intraovarian or extraovarian environment, but that it is not merely a symptom of oocyte immaturity; (ii) congression failure at MI is not associated with metaphase arrest or a delay in anaphase onset; and (iii) congression failure is correlated with an increase in meiotic non-disjunction. Taken together, these data provide support for the hypothesis that congression failure is a consequence of disturbances in either the paracrine or endocrine regulation of follicle growth, and that it predisposes to errors in chromosome segregation at the first meiotic division. In addition, these studies provide further evidence that the control of mammalian female meiosis differs markedly from mitotic cell division and from meiotic cell division in some animals, lacking a stringent chromosome-mediated checkpoint mechanism to delay the onset of anaphase in the event of disturbances in chromosome behaviour.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and interpretation
 Discussion
 Acknowledgements
 References
 
Animals
In the mouse ovary, the first cohort of follicles initiates growth shortly after birth, and by 26–28 days of age most have reached the antral stage, and the oocytes they contain have acquired the competency to resume and complete the first meiotic division and arrest at metaphase II. Thus, to determine the impact of oocyte immaturity on meiotic chromosome behaviour, partially and fully meiotically competent oocytes from 18–20 and 26–28 day old females respectively were compared. For studies of oocytes from control and mutant females, animals were generated as follows: (i) males and females of the LT/Sv inbred strain were mated to generate LT/Sv females; (ii) C57BL/6 males carrying a single copy of an altered LH gene [LHßCTP transgenics (Risma et al., 1995Go)] were mated to C57BL/6 females to produce transgenic and control sibling females; and (iii) normal C57BL/6 females were mated to fertile, phenotypically male C57BL/6 hermaphrodites carrying the Mus domesticus poschivinus Y (YPOS) chromosome to produce XY sex-reversed females and XX control siblings (Eicher et al., 1982Go). PCR typing using tissue obtained by ear punching was used to distinguish XY versus XX and transgenic versus normal siblings (Mroz et al., 1999Go).

Oocyte collection, culture, and fixation
Oocytes at the germinal vesicle stage (GV oocytes) were liberated from antral follicles using 26 gauge needles, placed in 10 µl drops of Waymouth's medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum and 0.23 mmol/l sodium pyruvate overlaid with Squibb mineral oil, and incubated at 37°C in an atmosphere of 5% CO2 in air. After 2 h in culture, oocytes were scored for evidence of germinal vesicle breakdown (GVBD), indicating resumption of the first meiotic division. Only oocytes resuming meiosis within the first 2 h of culture were included in the study. To obtain oocytes at metaphase of the first meiotic division, oocytes were incubated for a total of 6–10 h. At the end of the culture period, oocytes were fixed, stained and analysed as described below. To obtain oocytes arrested at metaphase II, oocytes were maintained in culture for 16 h, and those exhibiting polar body extrusion were fixed for analysis.

Oocytes from all females were embedded in a fibrin clot (bovine fibrinogen type IV; Calbiochem, La Jolla, CA, USA; bovine thrombin; Sigma, St Louis, MO, USA) attached to a microscope slide as previously described (Hunt et al., 1995Go) and immediately fixed in 2% formaldehyde, 1% Triton X-100, 0.1 mmol/l PIPES, 5 mmol/l MgCl2 and 2.5 mmol/l EGTA, for 30 min at 37°C. Following fixation, oocytes were washed for 10 min in 0.1% normal goat serum (NGS; Gibco BRL)/phosphate-buffered saline (PBS), blocked for at least 1 h at 37°C in PBS wash solution containing 10% NGS, 0.02% sodium azide and 0.1% Triton X-100, and stored at 4°C.

Immunofluorescence staining
Oocytes were incubated with a 1:2000 dilution in PBS of a primary mouse monoclonal antibody to acetylated tubulin (Sigma), washed in 10% NGS/PBS, and detected with an fluoroscein isothiocyanate-conjugated goat anti-mouse IgG (Accurate Chemical, Westbury, NY, USA). Prior to analysis, oocytes were counter-stained with 100 ng/ml propidium iodide and a coverslip applied with 50% glycerol/4xstandard saline citrate (SSC) and 0.1 µg/ml p-phenylenediamine mounting medium and sealed with rubber cement. Oocytes were analysed on a Zeiss Axioplan epifluorescent microscope and three-dimensional images were collected on a BioRad MRC 600 confocal system. The meiotic stage of individual oocytes was classified on the basis of chromosome configuration and spindle morphology as described previously (LeMaire-Adkins et al., 1997Go).

Metaphase II chromosome preparations and spectral karyotyping
Chromosome preparations of MII arrested oocytes were made using a modification of a published technique (Tarkowski, 1966Go). Briefly, oocytes were hypotonized in 1% citrate, moved to a small drop of acidified water on a microscope slide, and fixed in situ with several drops of 3 parts methanol:1 part acetic acid. For conventional cytogenetic analysis, slides were denatured briefly (~40 s at 72°C) in 70% formamide in SSC to accentuate the centromeric region and stained with 400 mg/ml 4',6-diamidino-2-phenylindole (DAPI). For spectral karyotyping, the probe was denatured and pre-annealed according to the manufacturer's specifications (Applied Spectral Imaging, Inc., Migdal Ha'Emek, Israel). The slides were soaked in 2xSSC, dehydrated through an ethanol series, allowed to air dry, and denatured at 72°C for 45 s in 70% formamide in SSC. Immediately after being denatured, slides were dehydrated in a cold ethanol series, dried, and hybridized for 48 h at 37°C with the pre-annealed probe (SkyPaint probe mixture for mouse chromosomes; SkyPaint Kit M-10). Following hybridization, the slides were washed, counterstained, mounted according to the manufacturer's specifications, and stored in the dark until analysed.


    Results and interpretation
 Top
 Abstract
 Introduction
 Materials and methods
 Results and interpretation
 Discussion
 Acknowledgements
 References
 
In previous studies of human oocytes, we observed a striking age-related increase in the number of oocytes that exhibited gross aberrations in chromosome alignment on the MII spindle (Volarcik et al., 1998Go) (Figure 1a,bGo). We postulated that such `congression failure' reflected compromised oocyte growth in the reproductively aged human ovary, and that it is but one manifestation of the abnormal meiotic phenotype that develops with advancing reproductive age in the human female (Volarcik et al., 1998Go).

Is congression failure simply a reflection of oocyte immaturity?
To determine if defects in meiotic chromosome alignment are a symptom of oocyte immaturity, we first analysed mouse oocytes from early antral follicles. Analysis of the first wave of growing oocytes in the immature mouse ovary has revealed that the oocyte acquires the competence to undergo the meiotic divisions during the late stages of follicle growth: Oocytes isolated from pre-antral follicles from 12 day old females are incapable of undergoing any nuclear maturation. However, when follicles reach the early antral stage several days later (e.g. 15–20 day old females), the oocytes have acquired partial meiotic competence; they will reinitiate meiosis when placed in culture but will arrest at metaphase I. In contrast, oocytes from slightly more mature follicles (e.g. from 22 day old females) exhibit full meiotic competence, resuming and completing the first meiotic division and arresting at metaphase of MII when released from the follicle and placed in culture (Eppig, 1994Go).

The majority of MI arrested oocytes isolated from the antral follicles of sexually immature (26–28 day old) C57BL/6 females will spontaneously resume meiosis (as evidenced by nuclear envelope breakdown) within the first 2 h of culture. At this stage, immunostaining with an antibody to ß-tubulin reveals highly condensed chromosomes and a disorganized mass of microtubules (Figure 1cGo). Within several hours, the microtubules become organized into a bipolar spindle with the broad poles characteristic of MI in the female. The congression of the chromosomes to the spindle equator is contemporaneous, and the chromosomes are loosely aligned at the spindle equator by the time the poles become evident (Figure 1dGo). Representative data for oocytes analysed after 8 h in culture are shown in Table IaGo. In our laboratory, these results are highly reproducible; the vast majority of oocytes from C57BL/6 females exhibit a normal MI configuration with the chromosomes aligned at the spindle equator (Figure 1eGo); however, a proportion (6–7%) have one or two poorly aligned bivalents (e.g. Figure 1fGo), and in 1–2% the MI spindle is fully formed but the majority of the chromosomes have not aligned at the equator (congression failure; identical in appearance to Figure 1gGo). Similarly, the majority of oocytes that extrude the first polar body and enter metaphase arrest after extended time in culture exhibit a normal MII metaphase configuration, with the chromosomes tightly aligned at the spindle equator.


View this table:
[in this window]
[in a new window]
 
Table I. Chromosome alignment at metaphase in oocytes from control and mutant females
 
The synchrony of follicle development in the immature mouse ovary provided a means of determining if the ability to orchestrate chromosome alignment on the MI spindle is linked to the acquisition of meiotic competence. To assess the effect of meiotic immaturity on chromosome alignment, we compared MI spindle formation and chromosome alignment in oocytes with only partial meiotic competence from 18–20 day old females produced on the C57BL/6 inbred strain background (Table IbGo). At this age, some oocytes have acquired the competence to resume meiosis but cannot initiate anaphase, and thus arrest at metaphase of the first division. Overall, comparatively few oocytes from 18–20 day old females resumed meiosis when placed in culture (e.g. <20% exhibited nuclear envelope breakdown after 2 h in culture as compared to >70% of oocytes from 26–28 day old females). Among those that did and were analysed after 8 h in culture, nearly 20% exhibited a metaphase configuration with one or two unaligned bivalents, or outliers. This value was significantly increased over that observed for controls (7%), i.e. fully competent oocytes from 26–28 day old females [{chi}2 = 4.14 (df =1), P < 0.05]. However, this apparently reflected delayed chromosome alignment; when cultured for extended time (e.g. 16–18 h), none of the oocytes initiated anaphase, but the frequency of MI cells with misaligned bivalents declined to control levels (Table IbGo).

Thus, the analysis of partially competent oocytes suggests that congression failure is not simply a symptom of meiotic immaturity. However, to determine if the inability to organize chromosomes on the MI spindle is symptomatic of defects in the acquisition of competence, we analysed oocytes from females produced on the LT/Sv inbred stain background. Oocytes from these females exhibit a high frequency of MI arrest, whether matured in vitro or in vivo, and have a propensity for parthenogenetic activation that results in a high incidence of ovarian teratomas (Eppig et al., 1996Go). Oocytes from LT/Sv females exhibit abnormal centrosomal characteristics, and minor abnormalities in pole structure are evident among those that arrest at MI (Albertini and Eppig, 1995Go). Although the underlying molecular defect remains unknown, recent studies suggest that it is oocyte intrinsic (Eppig et al., 2000Go), and results in a delay in the acquisition of the competence to trigger anaphase onset (Hirao and Eppig, 1999Go). To determine if the centrosomal and/or cell cycle control defects in these oocytes influence meiotic chromosome behaviour, we analysed chromosome alignment at metaphase I (Table IcGo). Similar to partially competent oocytes and controls, we detected no cells exhibiting congression failure. However, the incidence of metaphase cells with outliers was high, both at the 8 h timepoint (which includes both oocytes capable of completing MI and those that will become arrested) and at the 16–18 h timepoint (which includes only MI arrested oocytes). While differences in strain background preclude formal comparisons of the LT/Sv and control data (Table Ia–cGo), it seems reasonable to conclude that this abnormality is increased in oocytes from LT/Sv females.

In general, the data from studies of partially competent oocytes from normal C57BL/6 females and oocytes with defects in the acquisition of meiotic competence from LT/Sv females are strikingly similar: although the frequency of cells with minor disturbances in chromosome alignment was slightly increased in both, gross disturbances in the congression of the chromosomes to the spindle equator was not a feature of either. Hence, these results provide experimental evidence that congression failure is not simply a reflection of oocyte immaturity.

Is congression failure a reflection of disturbances in the regulation of folliculogenesis?
To determine if congression failure is a symptom of compromised oocyte growth, we initiated meiotic studies of two different mouse mutants with defects in the process of folliculogenesis. We chose for these studies a mutant in which the primary defect is extraovarian (the LHßCTP transgenic mouse) and one in which the primary defect is intraovarian (the XYPOS sex-reversed female). Due to the complexity of the interplay between signals from the hypothalamus, pituitary and ovary, a primary defect at one level ultimately influences regulation at all levels. However, to minimize the influence of secondary effects, all studies were conducted on oocytes derived from the first wave of follicles that initiate growth in the immature ovary.

The LH ßCTP transgenic female
A genetically engineered modification of the LH ß-subunit results in LH hypersecretion in the LHßCTP mouse (Risma et al., 1995Go). Chronically elevated LH levels cause early onset of puberty and the rapid development of ovarian abnormalities, culminating in enlarged, cystic and haemorrhagic ovaries and infertility due to anovulation (Risma et al., 1995Go, 1997Go; Mann et al., 1999Go). Despite the extensive ovarian pathology, recent studies have demonstrated that oocytes from LHßCTP females are developmentally competent and can give rise to liveborn when transferred to pseudopregnant recipients (Mann et al., 1999Go). From the standpoint of oocyte growth, these females are particularly interesting; hypersecretion of LH not only results in altered FSH:LH ratios, but also in altered testosterone:estradiol ratios (Risma et al., 1995Go, 1997Go). Thus, oocyte growth occurs in a highly abnormal endocrine environment. To determine if this affects the meiotic process, we analysed chromosome alignment at metaphase I and among metaphase II arrested oocytes (Table IdGo). In contrast to the results from control oocytes, partially competent oocytes and oocytes from LT/Sv females, we observed a high level of congression failure at both MI (i.e. 16 out of 88 cells, or 18.2%) and MII metaphase (i.e. 22 out of 57 cells, or 38.6%) among oocytes from LHßCTP females (Table IdGo). The overall level of meiotic abnormalities was high; however, we noted considerable variation among siblings but an apparent correlation between the level of congression failure and the extent of progression of the ovarian pathology (e.g. congression failure was highest in oocytes from females exhibiting uterine hypertrophy and ovarian cysts). Because the LHßCTP females were not on an inbred background, the influence of genetic factors could not be ruled out. Hence, to eliminate genetic variability, the transgene was transferred to the C57BL/6 inbred strain by repeated backcrossing. The analysis of oocytes progressing through the first meiotic division revealed a similar level of congression failure (data not shown). Further, although variation within litters was reduced, variation among litters was correlated with the extent of ovarian pathology. Thus, the meiotic abnormalities observed among oocytes from LHßCTP females produced on both a heterogeneous and an inbred background provide strong evidence that a primary defect that influences the endocrine control of folliculogenesis can impact the meiotic process. In this instance, the endocrine abnormalities do not preclude normal development of the oocyte, as evidenced by the fact that liveborn young can be obtained by embryo transfer. However, the high frequency of congression failure raises the possibility that the genetic quality of the oocytes from these females may be compromised.

The XYPOS sex-reversed female
As an independent assessment of the meiotic impact of altered oocyte growth, we initiated meiotic studies of the XYPOS sex-reversed female. These females are the result of an inappropriate interaction between the testis-determining gene, Sry, and an autosomal gene or genes involved in sex determination that results when a Y chromosome of Mus musculus domesticus origin (e.g. YPOS or YDOM) is placed onto the inbred C57BL/6 inbred strain background (Eicher et al., 1982Go). Most oocytes in the sex-reversed ovary become atretic before the female reaches sexual maturity, and the resultant XY females are sterile. Previous studies have demonstrated, however, that the oocytes are capable of ovulation and fertilization but are incapable of development after fertilization (Merchant-Larios et al., 1994Go). The primary defect is believed to be oocyte-intrinsic (Amleh and Taketo, 1998Go; Amleh et al., 2000Go), and from the standpoint of oocyte growth, oocytes from these females are interesting because they display subtle defects in oocyte–somatic cell communication at the late stages of follicle growth (Vanderhyden et al., 1997Go). Since XO females produced on the same genetic background are fertile, inappropriate expression of a gene or genes from the Y chromosome must be responsible for the disturbances in oocyte–somatic cell communication in the developing XY oocyte.

Analysis of oocytes from XYPOS females at both MI and MII metaphase revealed an extraordinarily high level of congression failure: >50% of the oocytes analysed after 6 h in culture had formed a bipolar spindle but exhibited no evidence of chromosome organization at the spindle equator (Table IeGo). The frequency of congression failure dropped somewhat among oocytes analysed after 10 h in culture; however, attempts to analyse oocytes at this timepoint were hampered by the fact that a large proportion of oocytes from XYPOS females had already initiated anaphase after 10 h in culture (see below). Further, the incidence of congression failure was also high among MII arrested oocytes (Table IeGo). Indeed, the incidence of aberrations at MII metaphase was considerably higher than evidenced by the frequency of congression failure since gross aberrations in spindle formation precluded the scoring of a significant number of oocytes (45/115). Thus, the high frequency of meiotic abnormalities observed among oocytes from XYPOS females suggests that a primary defect that affects oocyte–somatic cell communication can impact the meiotic process.

Does congression failure delay anaphase onset?
During mitotic cell division, the presence of a misaligned chromosome delays the onset of anaphase (Burke 2000Go; Shah and Cleveland, 2000Go). Hence, we wondered if abnormalities in chromosome congression during the first meiotic division would induce a similar delay. To assess this, we focused on the XYPOS female since the incidence of abnormalities at MI was high and, unlike the LHßCTP female, not complicated by phenotypic and genetic variability. We approached the question in two ways. First, we compared the kinetics of the first meiotic division in oocytes from XYPOS and control females for evidence of meiotic delay. Surprisingly, not only were we unable to detect a subset of cells that were delayed or arrested at MI, but comparison studies of the number of oocytes that had progressed beyond metaphase I after only 6 or 8 h in culture revealed a significant difference between oocytes from XYPOS and control females, suggesting an acceleration in anaphase onset among oocytes from XYPOS females (Figure 2Go). We next asked whether the incidence of congression failure was elevated among the latest subset of oocytes to initiate anaphase. Such an increase would suggest that defects in chromosome alignment interfered with the onset of anaphase, resulting in a `pile up' of such cells. As seen in Table IeGo, our data suggested the opposite; the number of cells exhibiting congression failure at metaphase I was lower among cells analysed after 10 h by comparison with cells analysed after 6 h in culture. Thus, the results of our analysis of oocytes from XYPOS females are consistent with the conclusion that alterations in oocyte growth predispose to congression failure at MI, and that this gross disturbance in meiotic chromosome behaviour does not affect the timing of anaphase onset.



View larger version (13K):
[in this window]
[in a new window]
 
Figure 2. Timing of anaphase onset in oocytes from XYPOS and sibling controls. Intact oocytes were fixed, immunostained with an antibody to acetylated tubulin and the meiotic stage of each oocyte classified on the basis of chromosome configuration and spindle morphology as previously described (LeMaire-Adkins et al., 1997Go). The percentage of oocytes that had completed anaphase after 6or 8 h in culture is compared for XY females (striped boxes) and control siblings (solid boxes). This graph shows representative results from an experiment using females stimulated with pregnant mare's serum gonadotropin 48 h prior to oocyte collection and culture; however, similar results were obtained in studies of oocytes from non-stimulated females. The vast majority of oocytes from control females initiated anaphase after >=9 h in culture. Although the difference in the number of oocytes that had initiated anaphase was not significant at the 6 h timepoint [{chi}2 = 3.64 (df = 1); 6/39 oocytes for XYPOS females and 10/152 for controls], after 8 h a highly significant difference was observed, with 23 of the 53 (43.4%) oocytes from XY (df = 1) females but only nine of the 121 (7.4%) oocytes from controls having progressed beyond metaphase I [{chi}2 = 31.77 (df = 1), P < 0.005].

 
The accelerated anaphase onset observed in oocytes from XYPOS females was unexpected. In retrospect, however, it is perhaps not surprising: the developmental regulation of the acquisition of meiotic competence is thought to be an oocyte-intrinsic program that is modulated by the granulosa cells (De La Fuente and Eppig, 2001Go). Thus, `fine tuning' of the process may well be defective in situations in which oocyte–somatic cell communication is compromised. Interestingly, similar changes in cell cycle progression have been reported previously in older female mice, with oocytes from 9–10 month old females showing a significant acceleration by comparison with 2–4 month old females (Eichenlaub-Ritter and Boll, 1989Go). Because the mouse also shows a modest age-related increase in aneuploidy, these differences have been interpreted as evidence that human age-related aneuploidy may result from oocyte-intrinsic changes that affect the cell cycle (Eichenlaub-Ritter, 1994Go). Thus, it may well be that these age-related changes—as well as the alterations we have observed in oocytes from XYPOS females—reflect abnormalities in oocyte–somatic cell communication.

Does congression failure result in increased aneuploidy?
Our previous observations on human oocytes from unstimulated ovaries led us to speculate that failure of normal chromosome alignment might be associated with human age-related non-disjunction (Volarcik et al., 1998Go). If so, we would predict that: (i) cells exhibiting congression failure are capable of initiating anaphase; and (ii) failure to properly align chromosomes at the spindle equator predisposes to non-disjunction at anaphase I. As detailed above, the results of our analyses of oocytes from XYPOS females provided no evidence for a delay in anaphase onset in the presence of congression failure. Thus, our observations are consistent with the first of the two predictions.

To test the second prediction, i.e. that congression failure predisposes to chromosome missegregation at anaphase, we analysed chromosome preparations of MII arrested oocytes from XYPOS females. Previous studies of MII arrested oocytes from XYPOS females suggested an increase in aneuploidy levels (Hunt and LeMaire, 1992Go). However, since the X and Y chromosomes frequently fail to recombine (Hunt and LeMaire, 1992Go), this could simply reflect sex chromosome non-disjunction or premature sister chromatid separation (PSCS) of a univalent X or Y chromosome at MI. To determine whether this was the cause of the increased aneuploidy in oocytes from XYPOS females or whether—as we would predict—all chromosomes are susceptible to meiotic non-disjunction, we initiated a cytogenetic analysis of MII arrested oocytes from XYPOS and control females.

Initially, we analysed DAPI-stained preparations, scoring for autosomal but not sex chromosome abnormalities and, to avoid artefactual loss, for hyperploidy but not hypoploidy. However, the analysis of oocytes from XYPOS females was complicated by the fact that several oocytes had multiple chromatid aberrations (e.g. Figure 3Go). Thus, we analysed an additional set of oocytes using spectral karyotyping (SKY) methodology. This allowed us to identify individual chromosomes and chromatids, and to distinguish single chromatids resulting from an MI segregation error (i.e. due to PSCS at MI) from chromosomes that had simply `fallen apart' at MII (i.e. due to the premature release of centromere cohesion between sister chromatids).



View larger version (15K):
[in this window]
[in a new window]
 
Figure 3. Analysis of meiosis I (MI) chromosome segregation. To analyse chromosome segregation at MI, air-dried chromosome preparations were made from MII arrested oocytes. This example from an XYPOS female shows both oocyte (top group) and polar body (bottom group) chromosomes. A cluster of five univalent chromosomes (dashed box) and a univalent Y chromosome (arrow) are present among the oocyte chromosomes. The colour inset illustrates the use of spectral karyotyping (SKY) to distinguish between premature sister chromatid segregation at MI and the precocious release of centromere cohesion at MII: in this instance, the close proximity of sister chromatids indicates the precocious release of sister centromere cohesion for two chromosomes (i.e. two yellow and two red chromatids). The remaining single chromatid (blue) has no obvious partner; however, the presence in the polar body of a homologue (not shown) but not a single chromatid suggests artefactual loss of the remaining single chromatid.

 
The combined results from the analysis of DAPI-stained and SKY preparations are provided in Table IIGo. Of the 34 modal or hyperploid cells analysed, three (9%) were hyperploid and five (15%) exhibited premature sister chromatid separation; considered together, these abnormalities were highly significantly increased by comparison with controls [{chi}2 = 8.25 (df = 1), P < 0.005]. Furthermore, in cells from XYPOS females in which we observed individual chromatids and in which SKY was applied, we were able to determine that some single chromatids were derived from segregation errors at MI. Thus, taken together, these results provide strong evidence that, at least in XYPOS females, the congression failure phenotype is `translated' into non-disjunctional or premature sister chromatid separation events at MI.


View this table:
[in this window]
[in a new window]
 
Table II. Chromosome analysis of metaphase II arrested oocytes
 
In addition to numerical abnormalities, we observed a surprisingly high incidence of structural abnormalities in oocytes from XYPOS females; specifically, six of the 34 oocytes (18%) contained chromosome fragments. Coupled with the accelerated onset of anaphase in oocytes from XYPOS females, this chromosome fragmentation phenotype indicates that there are extensive meiotic disturbances in XYPOS females, including failure to coordinate the events of anaphase. This suggests that the XYPOS female will be useful in unravelling the complex signals necessary to orchestrate the orderly and sequential events of normal anaphase, and further studies of these females are in progress. However, this unusual phenotype complicates interpretations relating congression failure to aneuploidy. Thus, we examined non-disjunction and premature sister chromatid segregation in the other congression failure-prone mutant, the LHßCTP female (Table IIGo). As in oocytes from XYPOS females, we observed a significant increase in cells that were hyperploid or exhibited premature sister chromatid segregation (8/44, or 18%) by comparison with controls [{chi}2 = 4.06 (df = 1), P < 0.05]. No structural abnormalities were identified, indicating that congression failure is not necessarily linked to chromosome fragmentation.

Thus, in two different situations in which we observed an increased frequency of congression failure at MI, the segregation of homologous chromosomes at anaphase I was also impaired. Further, the estimated incidence of such segregation errors (obtained by doubling the frequency of hyperploidy to estimate total non-disjunction, and adding to this the frequency of premature sister chromatid segregation events) was 32% for XYPOS females and 28% for LHßCTP females, values similar to the observed frequency of MI congression failure for the two types of females (Table Id,eGo). Thus, these data support the hypothesis that the congression failure phenotype is predictive of subsequent errors in chromosome segregation.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and interpretation
 Discussion
 Acknowledgements
 References
 
The control of oocyte growth is complex, requiring signals from the ovary, the pituitary, and the hypothalamus to produce the appropriate fluctuations in gonadotrophin levels necessary to sustain the final stages of oocyte maturation. In addition, the follicle itself is an intricate and highly compartmentalized structure, and both oocyte and follicle growth depend upon complex bidirectional signalling between the oocyte and its companion somatic cells (Albertini et al., 2001Go). Thus, disturbances in oocyte growth can result either from primary defects in intraovarian or extraovarian regulatory processes. The age-associated changes in the human ovary almost certainly include both intraovarian (e.g. a reduction in the number of oocytes recruited into the growing pool during each cycle) and extraovarian (e.g. increased FSH secretion, shortening of the cycles) changes. Hence, attempts to model the human age effect experimentally must be interpreted with caution. Nevertheless, mouse models can be used to ask basic questions about the influence of specific variables on the meiotic process. To this end, we utilized meiotically immature oocytes from control females and oocytes from a strain with defects in the acquisition of meiotic competence (LT/Sv females) to determine if oocyte immaturity influences meiotic chromosome behaviour. In addition, mutations in which the primary defect disrupts intraovarian (XYPOS females) and extraovarian (LHßCTP females) regulation allowed us to determine if alterations in the process of folliculogenesis increase the risk of meiotic aberrations. Finally, the recapitulation of the human age-associated congression failure phenotype in these mouse mutants provided a means of determining if defects in chromosome alignment at metaphase I are associated with a delay in anaphase onset or with an increased risk of aneuploidy.

The combined data from our studies are consistent with the conclusion that congression failure is not a symptom of meiotic immaturity but, rather, a reflection of oocyte growth in an altered environment. Importantly, this abnormal meiotic phenotype can be provoked by different mechanisms: e.g. defects in oocyte–somatic cell communication in the XYPOS female and the altered endocrine status of the LHßCTP female both result in a high incidence of congression failure at MI. Moreover, the degree of meiotic disturbance appears to be correlated with the progression of the abnormal endocrine phenotype in the LHßCTP mouse. Thus, the available evidence suggests that, although the primary defect may vary, congression failure results from changes that influence the final stages of oocyte growth and maturation. Accordingly, we predict that meiotic studies of several additional currently available mouse mutants [e.g. knockouts for 5{alpha}-reductase type I (Mahendroo and Russell, 1999Go) and Bmp1 (Yan et al., 2001Go)] will also reveal an increased level of congression failure.

The ability to recapitulate congression failure in mouse mutants has allowed us to determine if cells exhibiting this phenotype are capable of initiating anaphase. Our results provide no evidence that gross aberrations in the alignment of the chromosomes on the MI spindle delay the onset of anaphase. This is in striking contrast to mitotic cells (Burke, 2000Go; Shah and Cleveland, 2000Go) and male meiotic cells (Eaker et al., 2001Go) where the actions of the spindle assembly checkpoint mechanism cause the delay or arrest of cells with unaligned chromosomes at metaphase. More importantly, our data provide evidence that the failure to delay or arrest such cells leads to aneuploidy. We previously hypothesized that this difference in cell cycle control is a feature of mammalian female meiosis and that it contributes to the high incidence of human female-derived aneuploidy (LeMaire-Adkins et al., 1997Go). Our current data strongly support this conclusion, and suggest that changes in the endocrine environment—such as those that occur in the human female in the decade preceding menopause—may influence meiotic chromosome behaviour, creating age-related increase in aneuploidy.

If this interpretation is correct, understanding the molecular basis of congression failure will provide important insight to human age-related non-disjunction. Obviously a complex effect that has multiple aetiologies and involves both the somatic and germ cell component of the developing follicle poses a number of questions. Of these, one of the most interesting is the reason why chromosomes fail to congress on the meiotic spindle.

The first meiotic division is unique in many respects; however, the formation of the MI spindle and the alignment of chromosomes is particularly unusual. In mammals, the female meiotic spindle is formed through the action of multiple microtubule organizing centres rather than from a pair centrosomes and, as a result, the MI spindle has a characteristic barrel shape. Previous studies have suggested that bivalents become oriented on the meiotic spindle very early, such that the movement of the chromosomes to the equator has already commenced by the time that organized spindle poles become evident (Woods et al., 1999Go). However, although the chromosomes rapidly congress to the vicinity of the spindle equator, stable microtubule/kinetochore connections are not formed immediately, and a classic tight metaphase configuration is not achieved until just prior to anaphase onset (Brunet et al., 1998Go, 1999Go). Hence, the configuration that we describe as `normal metaphase' with chromosomes loosely aligned at the spindle equator is, in actuality, an extended prometaphase. Given these features, it seems likely that the observation of one or two outlying chromosomes in an otherwise normal cell (e.g. Figure 1fGo) represents normal oscillation of the chromosomes near the equatorial plate. In contrast, the inability to loosely align the chromosomes at the spindle equator, the meiotic phenotype that we have termed congression failure, is diagnostic of meiotic abnormalities that predispose to non-disjunction.

Our previous studies demonstrated that the meiotic chromosomes are not passive players, and that their ability to make bipolar attachments to the organizing spindle is essential for the formation of a stable MI spindle (Woods et al., 1999Go). Since the spindle appears normal in many cells exhibiting congression failure, it seems likely that the defect is not one of spindle formation, but rather of chromosome movement. The initial movement of the chromosomes to the vicinity of the equatorial plate of the spindle has been postulated to result from pushing forces exerted by motor proteins (Brunet et al., 1998Go, 1999Go). Interestingly, both microinjection of kinetochore antibodies into mouse oocytes (Simerly et al., 1990Go) and immunodepletion of the kinetochore-associated motor protein, CENP-E, in Xenopus egg extracts (Wood et al., 1997Go) produce a congression failure phenotype. Accordingly we postulate that the congression failure observed in oocytes from reproductively-aged human donors and in mouse oocytes from XYPOS and LHßCTP mutant females results from disturbances in the control of the final maturation phase of the oocyte that affect the transcription or post-translational modification of one or more microtubule motor proteins. The extended duration of MI in female mammals has been hypothesized to reflect a period of slow, progressive maturation of the kinetochores through post-translational modification (Brunet et al., 1999Go), hence it is possible that congression failure is a reflection of disturbances in this maturation process.

An important question not addressed by our studies is whether the congression failure defect is a meiotic `lethal', i.e. does the severity of the defect preclude such cells from forming functional oocytes capable of fertilization and development? Previous studies of human oocytes provide indirect evidence against this: age-related meiotic non-disjunction is clearly associated with an increase in chromosomally abnormal human conceptions. Thus, the finding that congression failure is an age-related phenotype in human oocytes argues that it is not merely a symptom of a degenerative oocyte.

Understanding a spindle defect with multiple aetiologies in a system as complex as the mammalian follicle is clearly a challenge. However, a recent model of paracrine signalling provides a mechanism whereby both intraovarian and extraovarian factors might affect oocyte–somatic cell communication (Albertini et al., 2001Go). Moreover, the role of microtubules in the signalling between the oocyte and its granulosa cells provides a potential link to spindle-specific defects. Thus, it seems likely to us that the oocyte–granulosa cell communication is a key component of the congression failure defect.

The fact that congression failure causes no obvious delay in anaphase onset and is correlated with increased non-disjunction indicates to us that cell cycle control mechanisms differ in the oocyte. It remains unclear, however, whether this is a generalized feature of female meiosis or whether congression failure occurs only when the normal cell cycle control mechanisms become non-functional. Future studies using these and other mouse models will be invaluable in understanding the control of female meiosis and, specifically, in understanding the mechanism responsible for the age-related disturbances in meiotic chromosome behaviour that we (Volarcik et al., 1998Go) and others (Battaglia et al., 1996Go) have observed in human oocytes.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and interpretation
 Discussion
 Acknowledgements
 References
 
We are grateful to E.Baart, R.LeMaire-Adkins, and L.Woods for their technical support and to T.Hassold for his comments on the manuscript. In addition, we thank E.Eicher for generously providing LT/Sv mice. These studies were supported by National Institutes of Health grant R01 HD31866 to P.A.H.


    Notes
 
3 Department of Genetics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4955, USA Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and interpretation
 Discussion
 Acknowledgements
 References
 
Albertini, D.F. and Eppig, J.J. (1995) Unusual cytoskeletal and chromatin configurations in mouse oocytes that are atypical in meiotic progression. Dev. Genet., 16, 13–19.[ISI][Medline]

Albertini, D.F., Combelles, C.M.H., Benecchi, E. and Carbatsos, M.J. (2001) Cellular basis for paracrine regulation of ovarian follicle development. Reproduction, 121, 647–653.[Abstract/Free Full Text]

Amleh, A. and Taketo, T. (1998) Live-borns from XX but not XY oocytes in the chimeric mouse ovary composed of B6.YTIR and XX cells. Biol. Reprod., 58, 574–582.[Abstract]

Amleh, A., Smith, L., Chen, H. and Taketo, T. (2000) Both nuclear and cytoplasmic components are defective in oocytes of the B6.YTIR sex-reversed female mouse. Dev. Biol., 219, 277–286.[ISI][Medline]

Battaglia, D.E., Goodwin, P. and Klein, N.A. (1996) Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum. Reprod., 11, 2217–2222.[Abstract]

Brunet, S., Polanski, Z., Verlhac, M.-H., Kubiak, J.Z. and Maro, B. (1998) Bipolar meiotic spindle formation without chromatin. Curr. Biol., 8, 1231–1234.[ISI][Medline]

Brunet, S., Maria, A.S., Guillaud, P., Dujardin, D., Kubiak, J.Z. and Maro, B. (1999) Kinetochore fibers are not involved in the formation of the first meiotic spindle in mouse oocytes, but control the exit from the first meiotic M phase. J. Cell Biol., 146, 1–12.[Abstract/Free Full Text]

Burke, D.J. (2000) Complexity in the spindle checkpoint. Curr. Opin. Genet. Dev., 10, 26–31.[ISI][Medline]

De La Fuente, R. and Eppig, J.J. (2001) Transcriptional activity of the mouse oocyte genome: companion granulosa cells modulate transcription and chromatin remodeling. Dev. Biol., 229, 224–236.[ISI][Medline]

Eaker, S., Pyle, A., Cobb, J. and Handel, M.A. (2001) Evidence for melotic spindle checkpoint from analysis of spermatocytes from Robertsonian-chromosome heterozygous mice. J. Cell Sci., 114, 2953–2965.[Abstract/Free Full Text]

Eichenlaub-Ritter, U. (1994) Mechanisms of nondisjunction in mammlian meiosis. Curr. Top. Dev. Biol., 29, 281–324.[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, 170–176.[ISI][Medline]

Eicher, E., Washburn, L., Whitney, B. and Morrow, K. (1982) Mus poschiavinus Y chromosome in the C57BL/6J murine genome causes sex reversal. Science, 217, 535–537.[ISI][Medline]

Eppig, J.J. (1994) Oocyte–somatic cell communication in the ovarian follicles of mammals. Semin. Dev. Biol., 5, 51–59.

Eppig, J.J., Wigglesworth, K., Varnum, D.S. and Nadeau, J.H. (1996) Genetic regulation of traits essential for spontaneous ovarian teratocarcinogenesis in strain LT/Sv mice: aberrant meiotic cell cycle, oocyte activation, and parthenogenetic development. Cancer Res., 56, 5047–5054.[Abstract]

Eppig, J.J., Wigglesworth, K. and Hirao, Y. (2000) Metaphase I arrest and spontaneous parthenogenetic activation of strain LTXBO oocytes: chimeric reaggregated ovaries establish primary lesion in oocytes. Dev. Biol., 224, 60–68.[ISI][Medline]

Hassold, T. and Chiu, D. (1985) Maternal age specific rates of numerical chromosome abnormalities with special reference to trisomy. Hum. Genet., 70, 11–17.[ISI][Medline]

Hassold, T. and Hunt, P. (2001) To err (meiotically) is human: the genesis of human aneuploidy. Nat. Rev. Genet., 2, 280–291.[ISI][Medline]

Hirao, Y. and Eppig, J.J. (1999) Analysis of the mechanism(s) of metaphase I-arrest in strain LT mouse oocytes: delay in the acquisition of competence to undergo the metaphase I/anaphase transition. Mol. Reprod. Dev., 54, 311–318.[ISI][Medline]

Hunt, P.A. and LeMaire, R. (1992) Sex-chromosome pairing: evidence that the behavior of the pseudoautosomal region differs during male and female meiosis. Am. J. Hum. Genet., 50, 1162–1170.[ISI][Medline]

Hunt, P.A., LeMaire, R., Embury, P., Mroz, K. and Sheean, L. (1995) Analysis of chromosome behavior in intact mammalian oocytes: monitoring the segregation of a univalent chromosome during mammalian female meiosis. Hum. Mol. Genet., 4, 2007–2012.[Abstract]

Lamb, N., Freeman, S.B., Savage-Austin, A., Pettay, D., Taft, D., Hersey, J., Gu, Y., Shen, J., Saker, D., May, K.M. et al. (1996) Non-disjunction of chromosome 21: evidence for initiation of all maternal errors during meiosis I. Nat. Genet., 14, 400–405.[ISI][Medline]

LeMaire-Adkins, R., Radke, K. and Hunt, P.A. (1997) Lack of checkpoint control at the metaphase–anaphase transition: a mechanism of meiotic non-disjunction in mammalian females. J. Cell Biol., 139, 1611–1619.[Abstract/Free Full Text]

Mahendroo, M.S. and Russell, D.W. (1999) Male and female isoenzymes of steroid 5alpha-reductase. Rev. Reprod., 4, 179–183.[Abstract/Free Full Text]

Mann, R.J., Keri, R.A. and Nilson, J.H. (1999) Transgenic mice with chronically elevated luteinizing hormone are infertile due to anovulation, defects in uterine receptivity, and midgestation pregnancy failure. Endocrinology, 140, 2592–2601.[Abstract/Free Full Text]

Merchant-Larios, H., Clarke, H.J. and Taketo, T. (1994) Developmental arrest of fertilized eggs from the B6.YDOM sex-reversed female mouse. Dev. Genet., 15, 435–442.[ISI][Medline]

Mroz, K., Carrel, L. and Hunt, P.A. (1999) Germ cell development in the XXY mouse: evidence that X chromosome reactivation is independent of sexual differentiation. Dev. Biol., 207, 229–238.[ISI][Medline]

Risma, K.A., Clay, C.M., Nett, T.M., Wagner, T., Yun, J. and Nilson, J.H. (1995) Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors. Proc. Natl Acad. Sci. USA, 92, 1322–1326.[Abstract]

Risma, K.A., Hirshfield, A.N. and Nilson, J.H. (1997) Elevated luteinizing hormone in prepubertal transgenic mice causes hyperandrogenemia, precocious puberty, and substantial ovarian pathology. Endocrinology, 138, 3540–3547.[Abstract/Free Full Text]

Shah, J.V. and Cleveland, D.W. (2000) Waiting for anaphase: Mad2 and the spindle assembly checkpoint. Cell, 103, 997–1000.[ISI][Medline]

Simerly, C., Balczon, R., Brinkley, B. and Shatten, G. (1990) Microinjected kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes. J. Cell Biol., 111, 1491–1504.[Abstract]

Tarkowski, A.K. (1966) An air drying method for chromosome preparations from mouse eggs. Cytogenetics, 5, 394–400.[ISI]

Vanderhyden, B.C., Macdonald, E.A., Merchant-Larios,H., Fernandez, A., Amleh, A., Nasseri, R. and Taketo, T. (1997) Interactions between the oocyte and cumulus cells in the ovary of the B6.YTIR sex-reversed female mouse. Biol. Reprod., 57, 641–646.[Abstract]

Volarcik, K., Sheean, L., Goldfarb, J., Woods, L., Abdul-Karim, F. and Hunt, P.A. (1998) The meiotic competence of in vitro matured human oocytes is influenced by donor age: evidence that folliculogenesis is compromised in the reproductively aged ovary. Hum. Reprod., 13, 154–160.[Abstract]

Wood, K.W., Sakowicz, R., Goldstein, L.S.B. and Cleveland, D.W. (1997) CENP-E is a plus end-directed kinetochore motor required for metaphase chromosome alignment. Cell, 91, 357–366.[ISI][Medline]

Woods, L.M., Hodges, C., Baart, E., Baker, S.M., Liskay, M. and Hunt, P.A. (1999) Chromosomal control of meiotic spindle assembly: abnormal meiosis I in female Mlh1 mutant mice. J. Cell Biol., 145, 1395–1406.[Abstract/Free Full Text]

Yan, C., Wang, P., DeMayo, J., DeMayo, F.J., Elvin, J.A., Carino, C., Prasad, S.V., Skinner, S.S., Dunbar, B.S., Dube, J.L. et al. (2001) Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Mol. Endocrinol., 15, 854–866.[Abstract/Free Full Text]

Submitted on August 13, 2001; accepted on January 10, 2002.