Spindles, mitochondria and redox potential in ageing oocytes

RBMOnline - Vol 8. No 1. 45-58 Reproductive BioMedicine Online; www.rbmonline.com/Article/1055 on web 17 October 2003

Symposium: Mitochondria and human conception

Spindles, mitochondria and redox potential in ageing oocytes Hang Yin received her PhD in the field of cytogenetics under the direction of Dr U Eichenlaub-Ritter at Bielefeld University, Germany in 1998. Afterwards, she started her career as an embryologist working in IVF clinics in Karlsruhe and Berlin. She finished postgraduate training in reproductive sciences at Toronto University in the group of Dr T Zenzes, and at McGill University, Canada, working with Dr R Gosden. Now she is Assistant Professor at the Jones Institute of Reproductive Medicine in Norfolk, Virginia, USA. Her research covers in-vitro growth and maturation of oocytes, embryo culture, and cryopreservation of embryos and oocytes.

Dr Hang Yin U Eichenlaub-Ritter1,3, E Vogt1, H Yin2, R Gosden2 1Institute of Gene Technology/Microbiology, Faculty of Biology, University of Bielefeld, D-33501 Bielefeld, Germany; 2The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, VA, USA 3Correspondence: e-mail: [email protected]

Abstract Studies of human oocytes obtained from women of advanced reproductive age revealed that spindles are frequently aberrant, with chromosomes sometimes failing to align properly at the equator during meiosis I and II. Chromosomal analyses of donated and spare human oocytes and cytogenetic and molecular studies on the origin of trisomies collectively suggest that errors in chromosome segregation during oogenesis increase with advancing maternal age and as the menopause approaches. Disturbances in the fidelity of chromosome segregation, especially at anaphase I, leading to aneuploidy are prime causes of reduced developmental competence of embryos in assisted reproduction, as well as being responsible for the genesis of genetic disease. This review provides an overview of spindle formation and chromosome behaviour in mammalian oocytes. Evidence of a link between abnormal mitochondrial function in oocytes and somatic follicular cells, and finally disturbances in chromosome cohesion and segregation, and cell cycle control in aged mammalian oocytes, are also discussed. Keywords: ageing oocytes, aneuploidy, chromosome segregation, mitochondria, redox potential

Introduction Spindles are essential in eukaryotic cells for normal chromosome segregation and genomic stability after mitosis and meiosis. In the past decade, much new information about cell cycle control, spindle formation and chromosome segregation during mitosis and meiosis has become available. However, for ethical reasons, only limited numbers of freshly retrieved, donated human oocytes have been available for research on the origin of meiotic errors. Predominantly spare oocytes with the lowest quality of cohorts obtained from individual patients, oocytes that failed to fertilize or immature oocytes from stimulated cycles, which developed to metaphase II in vitro have been studied for spindle aberrations. In most cases, oocytes were fixed and processed for fluorescent microscopic or confocal microscopic analysis (EichenlaubRitter et al., 1988b; Pickering et al., 1988; Battaglia et al., 1996a,b; George et al., 1996; Park et al., 1997; Sathananthan, 1997; Volarcik et al., 1998; Rawe et al., 2000; Cekleniak et al., 2001; Zenzes et al., 2001; Boiso et al., 2002), producing a more or less static picture of dynamic processes in the cell.

Recently, non-invasive analysis of oocytes using enhanced polarizing microscopy (reviewed by Wang and Keefe, 2002; Eichenlaub-Ritter et al., 2002; Keefe et al., 2003) or labelling of oocyte spindle proteins with fluorescent GFP (EichenlaubRitter and Peschke, 2002; Lefebvre et al., 2002) has provided more information about factors influencing dynamic processes during spindle formation. This paper discusses the elements of spindle formation and cell cycle regulation affecting chromosomal behaviour in mitosis, meiosis and oogenesis. The second part deals with the dependence of spindles on functionally intact mitochondria. The final part focuses on aberrations in oocytes and somatic cells, which may directly or indirectly create an environment adversely affecting fidelity of chromosome segregation, a hallmark of reproductive ageing. In view of the continuing trend in Western societies, and now appearing elsewhere, to delay childbearing to advanced parental ages, elucidation of factors that compromise spindle integrity and oocyte developmental capacity is of key importance in reproductive biology and medicine today.

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Molecular composition Spindles are composed of bundles of microtubules, which are non-covalent polar polymers of alpha/beta-tubulin heterodimers (Compton, 2000). Within each spindle, most microtubules are polarized and oriented with the faster polymerizing ‘plus’ end located at the centromeres of chromosomes and the more slowly assembling ‘minus’ end attached to the spindle pole (Figure 1A′, B′). GTP hydrolysis on beta-tubulin is needed for microtubule polymerization and to generate dynamic instability (Mitchinson and Kirschner, 1984; Desai and Mitchison, 1997). In-vitro and in-vivo tubulin polymerization kinetics are modulated by motor proteins, exchange factors, capping proteins, temperature, divalent cations and [H+] (e.g. Hunter and Wordemann, 2000; Wittman et al., 2001; Bamba et al., 2002; Dasso, 2002; Jordan, 2002).

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The relative stability of microtubules in ooplasm is governed by opposing activities, e.g. the stabilization by microtubuleassociated proteins (MAP), or the destabilization by other proteins, for instance the members of the kinesin-like microtubule motor protein family (Brunet and Vernos, 2001; Nedelec et al., 2003). The significance of post-translational modifications of alpha- and beta-tubulin in oocyte spindles (Asch et al., 1995; de Pennart et al., 1988; Vee et al., 2001) is largely unknown. Labelling and photobleaching experiments have revealed that the microtubules in the metaphase II spindle of mammalian oocytes turn over rapidly (Gorbsky et al., 1990), indicating that this organelle is highly dynamic and not stable as it appears from fixed cells. Studies of spindle shape in human and mouse oocytes exposed to low temperature or low concentrations of tubulin-depolymerizing agents revealed acute sensitivity, and the effects of drugs and temperature may be either reversible or

Figure 1. (A, A′) Astral bipolar spindle formation at spermatogenesis: Two MTOC with pairs of centrioles and pericentriolar material (two circles with cylinders) containing gamma-tubulin are present at the nuclear periphery already prior to nuclear membrane breakdown (A). At metaphase I (A′) they are at the centre of spindle poles, associated with the minus ends of spindle microtubules, while the plus ends are predominantly located at the centromeres (black ovals) of chromosomes. Dynamic de- and repolymerization is indicated by loss or integration of tubulin subunits (dots) at growing or shrinking microtubule ends, existing side by side. (B–B′′) In oogenesis multiple MTOC (grey ovals) with astral microtubules (lines) are recruited by interactions with microtubules attached to centromeres of chromosomes (dark ovals) during prometaphase I (B). At metaphase I (B′) a bipolar barrel-shaped spindle body is formed with several MTOC at poles and bivalent chromosomes all congressed to the spindle equator. Interactions of microtubules and kinetochores with MAP and motor proteins (indicated by double triangles) participate in establishing bipolarity and chromosome alignment (Modified from Eichenlaub-Ritter, 2003b). (C-C′′′) Characteristic disturbances in formation of a bipolar spindle in egg extracts after depletion of certain motor proteins or addition of antibodies to molecules like kinesin Eg5 (astral instead of bipolar microtubule array, C), kinesin XCTK2 (monopolar instead of bipolar spindle, C′), dynein (splayed instead of pointed poles, C′′), and kinesin Xklp2 (split spindle poles, C′′′) respectively, suggesting that Eg5 (or its human orthologue) is required for bundling, kinesin Xklp1 for microtubule-chromatin interactions and pole extension, and dynein and kinesin XCTK2 for focusing of spindle poles during spindle formation (modified from Walacik et al., 1998).

Symposium - Ageing oocytes - U Eichenlaub-Ritter et al.

irreversible (e.g. Pickering and Johnson, 1987; Wang et al., 2001; Zenzes et al., 2001; Eichenlaub-Ritter et al., 2002). Although microtubules can self-assemble in vitro from high concentrations of purified tubulin, microtubules nucleate in vivo at relatively low tubulin concentrations. Under these conditions, the initiation of new microtubule ends is kinetically limiting (reviewed in Desai and Mitchinson, 1997). In-vivo nucleation takes place primarily at morphologically distinct structures, termed microtubule-organizing centres (MTOC), allowing efficient polymerization under low concentrations of tubulin (reviewed in Kellogg et al., 1994). In most animals, the MTOC comprises a pair of centrioles surrounded by pericentriolar material (PCM) (Figure 1A, A′). While human oogonia still have distinct pairs of centrioles for mitosis (Sathananthan et al., 2000), oocytes of many vertebrates do not possess two major MTOC with pairs of

centrioles, as in most somatic cells. Instead, a large number of acentriolar centrosomal MTOC (Maro et al., 1985; Schatten et al., 1986; Battaglia et al., 1996a; George et al., 1996) are recruited by chromosomes after germinal vesicle breakdown (GVBD) (for review, see Carabatsos et al., 2000) (Figure 1B, B′, Figure 2). Accordingly, while most mitotic and male meiotic spindles have fusiform poles with astral microtubules radiating to the cell cortex, oocyte spindles are barrel-shaped with flat spindle poles (Figure 2) and fairly few astral microtubules. Gamma-tubulin is a constitutive centrosomal protein known to play a key role in microtubule patterning in a variety of cell systems and in oocytes (George et al., 1996). Gamma-tubulin is redistributed between cytoplasmic and nuclear compartments of mouse oocytes during meiotic progression, at times when demands for microtubule patterning shift from stabilization of the cortex to morphogenesis of the spindle (Combelles and Albertini, 2001).

Figure 2. Spindles in mouse (A–C) and human (D–H) oocytes. Large spindles in rodent oocytes. (A) Typical barrel-shaped meiosis I spindle in mouse oocyte with bivalent chromosomes congressing to the spindle equator (A′). (B) Typical spindle in metaphase II-arrested mouse oocyte with all chromosomes assembled at the spindle equator (B′). (C) Metaphase II spindle in mouse oocyte with one unaligned chromosome (C′). Living human oocytes with (D) or without a birefringent (E) spindle as detected by non-invasive Polscope microscopy. The position of the first polar body (PB) is indicated. (D′) Typical metaphase II spindle (D′) in in-vitro matured, fixed human oocyte, which exhibited a birefringent spindle prior to fixation. Characteristically, the spindle is attached with one pole to the peripheral oolemma and has well aligned chromosomes (D′′). (E, E′′) Aberrant metaphase II spindle (E′) and unaligned chromosomes (E′′) in human oocyte, which failed to exhibit a birefringent structure by Polscope after retrieval from stimulated cycle in GV-stage from an antral follicle and maturation in vitro to meiosis II. (F–H) Spindles from metaphase II-aged human oocytes, which failed to become fertilized. (F, F′) Spindle with well-aligned chromosomes. (H, H′) Bipolar spindle with dispersed, unaligned chromosomes. (H, H′) Aberrant spindle with unordered chromosomes. (A–C, D′, E′, F, G, H) Tubulin immunofluorescence. (A′–C′, D′′, E′′, F′–H) Chromosomes stained by propidium iodide. Bar in (C′) for (A–C′) 15 μm. Bar in (E) for (D, E) 25 μm. Bar in (D′) for (D′–E′′) 10 μm. Bar in (H′) for (F–H′) 4 μm. [Images of human oocytes in (D–E′′) were kindly provided by Y Shen and H-R Tinneberg, Giessen University, Womens Hospital].

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Recruitment of gamma-tubulin and other centrosomal components such as NuMA (nuclear mitotic apparatus protein) by spindles and polar MTOC in oocytes is a hallmark of high quality and probably a prerequisite for normal spindle function in mammalian oocytes (George et al., 1996; Hewitson et al., 1999; Eichenlaub-Ritter and Peschke, 2002). Although centrioles in primates are introduced by the fertilizing spermatozoa, many maternal spindle components are also needed to promote spindle and cytoplasmic organization in the embryo after fertilization (e.g. Simerly et al., 1999; Hewitson and Schatten, 2002; Shin and Kim, 2003). Limiting amounts and specific distribution of such spindle components in the primate oocyte may reduce the likelihood that human cloning by somatic nuclear transfer will be successful (Simerly et al., 2003).

Dynamics of spindle function Resumption of oocyte maturation is accompanied by polymerization of multiple asters at MTOC and at the site for microtubule attachment on condensed chromosomes, the kinetochores. A gradual ‘sorting’, recruitment and alignment of MTOC at the flat poles of the spindle takes place throughout prometaphase I, resulting in the establishment of a bipolar

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spindle body (Figure 1B), initially with unaligned bivalent chromosomes. Chromosomes gradually congress to the spindle equator during prometaphase I, and the oocyte spindle attains a characteristic ‘anastral’, barrel-shape with only a few microtubules radiating from the poles (Figure 1B, B′; Figure 2). Characteristically, the spindle migrates to the cell periphery for an unequal division after GVBD. Shortly after alignment of the last chromosome at the equator in late prometaphase I, anaphase I begins (Brunet et al., 1999) and the first polar body is formed. Normal spindle formation and unequal cytoplasmic cleavage depend on expression of the c-mos kinase (Svoboda et al., 2000; Verlhac et al., 2000) and other cytoskeletal proteins (Leader et al., 2002) in mammalian oocytes. Without any intervening S-phase, reductional chromosome segregation at anaphase I is followed swiftly by formation of the second meiotic spindle. While a long period of oscillatory chromosome movement is typical for prometaphase I, metaphase II chromosomes rapidly align at the spindle equator during prometaphase II of oogenesis. In the presence of cytostatic factor (Heikinheimo and Gibbons, 1998), human oocytes, like those of most other mammalian species, arrest at metaphase II. During the constitutional oocyte-specific meiotic arrest chromosomes are aligned on the metaphase plate, a situation inducing rapid progression to anaphase in

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somatic cells. In contrast, oocytes remain blocked in meiosis II until they are fertilized or parthenogenetically activated. Meiotic arrest involves spindle proteins, which are posttranslationally modified by phosphorylation downstream in the c-mos kinase pathway (Lefebvre et al., 2002). Spindles of rodent oocytes attach with their long axes to cortical cytoplasm beneath a dense layer of actin filaments (Maro et al., 1986), while spindles in the primate oocyte attach to the cell cortex with only one slightly pointed pole, and there is no distinct actin layer (e.g. Pickering et al., 1988) (Figure 2). Spindle formation and chromosome behaviour depend on the expression and activity of motor proteins. Cytoplasmic dynein is a ubiquitously expressed, highly conserved minus-enddirected microtubule motor protein, whereas many motor proteins of the superfamily of kinesin-like motor proteins are plus-end directed. Microtubule motors use ATP to perform work, typically to move cargo in a directed manner along microtubules. Members of the kinesin superfamily have been implicated in spindle function during meiosis and mitosis as well as in microtubule destabilization (for review, see Heald, 2000; Hunter and Wordeman, 2000). Conventional kinesin is a highly processive motor that moves several microns along the microtubule before detaching (e.g. Svoboda et al., 1994).

Conventional kinesin hydrolyses on average 125 ATPs per microtubule-binding event, and therefore requires a local supply of high-energy substrate. In the last decade, ~100 eukaryotic proteins have been identified that contain a domain homologous to the ~300 amino acid catalytic ATPase domain of conventional kinesin (Hirokawa et al., 1998). Presumptive conserved microtubule motor and spindle proteins were identified by molecular screens with ovarian and oocyte libraries (e.g. Neilson et al., 2000; Stanton et al., 2002). The pivotal role of motor proteins in spindle organization has been demonstrated using cell-free egg extracts from nonprimate vertebrates (Heald and Walczak, 1999; Karsenti and Vernos, 2001). The self-organizing capacity of the spindle in ooplasm demonstrates the collective action of soluble and chromosome-bound motors in the absence of centrosomes, or even chromatin, when high-energy substrates are available (Brunet et al., 1998; Walczak et al., 1998). Microtubules randomly nucleate in oocyte extracts as the first step of spindle formation. They are progressively cross-linked and aligned by the activity of a homo-tetrameric kinesin motor protein, Eg5, thus generating an anti-parallel bundle of microtubules as a spindle template (Karsenti and Vernos, 2001). At the same time, microtubules are captured by chromatin-associated

Figure 3. (A′–A′′) Regulation of chromosome cohesion by APC/C. At prometaphase of mitosis (A) replicated sister chromatids (helices) are held together by cohesin complex of proteins (indicated by spheres and ring). Separin protease (scissors) is inactive by association with securin protein (black half moon), while MPF is active. At anaphase (A′) activated anaphase promoting complex/cyclosome (APC/C) triggers ubiquination and degradation of cyclin B (inactivation of MPF), and ubiquination of securin thus releasing and activating separase (scissors). Subsequently, separase may proteolytically cleave cohesin proteins (A′′) (e.g. the Scc1 protein in mitosis, the Rec8 protein or its human orthologue hRec8 in meiosis), thus releasing chromosome cohesion, such that sister chromatids can now separate (arrows) and cells enter interphase in absence of active MPF. (B–B′′) Sequential separation of sister chromatid arms and centromeres at mitosis. At prophase (B) sister chromatids are attached all along the chromosome arms by cohesin complex (open dots). At centromeres many cohesion complexes mediate especially tight binding (star). At late prometaphase (B′) Plk kinase phosphorylates some cohesin protein on chromosome arms, so that they might already detach from chromosome arms (B′), while centromeres stay tightly attached by cohesin proteins (star). At anaphase of mitosis (B′′) APC/C mediates degradation of securin and release of active separase that causes the proteolytic cleavage of cohesin proteins from the centromeres of chromosomes such that sister chromatids can segregate (B′′). (C–C′′) Loss of chromosome cohesion at meiosis. At metaphase I (C) sister chromatids in homologous chromosomes (grey/black chromosomes and dotted counterparts, respectively) are held together by cohesion complexes (open and grey dots, stars), while specific proteins at centromeres (open half moon structures) interacting with centromeric cohesin proteins (stars) mediate tight attachment and monopolar orientation of the two centromeres (checkered ovals) of sister chromatids. Kinases of the aurora kinase family (Aurora B) appear to associate with cohesin complexes at chromosomes at prometaphase I and modulate susceptibility to cleavage by separase at first anaphase of meiosis (C′). The centromeres remain attached and mono-oriented throughout first division (C′). At second meiosis (C′′) cohesin proteins from complexes at centromeres of sister chromatids (stars) are cleaved by separase, similar to mitotic anaphase (B′′). (D–D′) Simplified overview of the spindle checkpoint in mitosis and meiosis. In the presence of unattached centromeres and kinetochores without tension from spindle fibres (and thus in presence of uncongressed chromosomes or absence of a spindle) a cascade of molecular events involving protein phosphorylation and motor proteins like CENP-E, and checkpoint proteins like Bub1 and Mad2 at kinetochores is initiated, which causes formation of a complex called meiotic checkpoint complex (MCC) with several components like checkpoint proteins like Mad2 and Bub1/Bub1R, and p55 and APC/C (D). This renders APC/C inactive and thus arrests cells in prometaphase. In meiosis (D′) similar checkpoints exist, but may be inactive when proteins of the checkpoint are not available in sufficient concentrations (Mad2↓), or when proteins mediating tight binding and mono-orientation of centromeres of sister chromatids (stars and half moon structure) or cohesin proteins on sister chromatid arms (open dots) are precociously lost. In this case, the attachment of sisters may be bipolar (merotelic), as indicated by grey and black arrow, and the separation of sister chromatids may be precocious and random. Exchanges on chromosomes in consequence of recombination have not been marked.

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plus-end directed, kinesin-like microtubule motors like the conserved Xklp1 motor in Xenopus oocytes, capable of moving microtubule minus ends away from chromatin (Walczak et al., 1998). Dynein, which initiates aster formation in cell-free egg extracts, appears to be required to direct microtubule minus ends into the poles (Heald and Walczak, 1999). At the kinetochores of centromeres, active plus and minus end motors are expressed (Figure 3). The highly conserved centromere motor protein E (CENP-E) stabilizes microtubule capture at both sister centromeres in chromosomes at mitosis (e.g. Putkey et al., 2002), and maintains attachment of kinetochores to the end of a disassembling microtubule. Other kinetochore components, belonging to the kinesin-like family, like MCAK in mammals, are actually microtubule disassemblases involved in anaphase progression (e.g. Maney et al., 1998). Functional ablation of any specific spindle-associated protein causes distinct morphological aberrations (Figure 1C), which may interfere with spindle function and chromosome congression and distribution. Morphologically similar disturbances may be seen in oocytes of aged women (see below), although their origin is unknown. Cytoplasmic dynein (Carabatsos et al., 2000) and kinesin-like molecules have been functionally or immunologically identified in mammalian oocytes, where they are required for meiotic maturation. For instance, Mountain et al. (1999) stated that functional ablation of HSET, a kinesin-like motor protein expressed in mouse oocytes, did not result in gross defects in mitotic spindles but caused spindle disorganization in oocytes. CENP-E has been recognized at kinetochores of sister chromatids in meiotic chromosomes of mouse and pig oocytes (Duesbery et al., 1997; Lee et al. 2000a). Unlike in mitosis, where CENP-E is dissociated from kinetochores at anaphase, it remained attached to the kinetochores of chromosomes at anaphase I in pig oocytes. CENP-E also remained associated with kinetochores of aligned metaphase II chromosomes arrested in meiosis II (Duesbury et al., 1997), whereas it characteristically becomes dislodged from kinetochores of mitotic chromosomes after assembling at the spindle equator. These examples show that conserved motor and microtubulebinding proteins are expressed in oogenesis, although they may have distinct roles in formation of the anastral spindle, meiotic chromosome segregation and metaphase II arrest.

Meiotic kinases in spindle formation

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Regulation of spindle formation as well as progression of meiotic maturation requires the interaction of kinases and phosphatases with specific targets (e.g. Heikinheimo and Gibbons, 1998; Nigg, 2001), while M-phase progression requires active maturation promoting factor (MPF). MAP kinases, p90rsk ribosomal protein kinase, as well as checkpoint proteins (see below) are phosphorylated and activated downstream in the c-mos pathway at oogenesis, and account for activities of the ‘cytostatic factor’, which arrest oocytes at metaphase II (Kalab et al., 1996; Heikinheimo and Gibbons, 1998; Schwab et al., 2001). MAP kinases stably associate with the meiotic spindle in oogenesis and dictate microtubule characteristics (Verlhac et al., 1994, 1996). Accordingly, spindles in oocytes of mos –/– knockout mice are aberrant, polar bodies are larger than normal and oocytes

frequently fail to arrest at metaphase II (Hirao and Eppig, 1997; Verlhac et al., 2000; Wianny and Zernicka-Goetz, 2000). A protein associated with the second meiotic spindle, MISS, is a specific target of MAP kinases, downstream in the c-mos phosphorylation cascade, and participates in meiotic arrest at metaphase II (Lefebvre et al., 2002). NuMA, a microtubulebinding phosphoprotein, which is an essential component of functional MTOC, is transported to spindle poles by the activity of dynein/dynactin complexes in mitosis, and also associates with the spindle and becomes enriched at multiple MTOC at the spindle poles at meiosis I and II of oogenesis. It is also present at the spindle midzone in telophase I, as shown in mouse and pig oocytes (Eichenlaub-Ritter and Peschke, 2002; Lee et al., 2000b). Finally, polo-like kinases, a family of serine-threonine kinases, which regulate the dissociation of cohesin protein from chromosome arms during early mitosis, associate with the acentriolar spindle poles, meiotic chromosomes and the spindle mid-zone during oocyte maturation (Wianny et al., 1998; Pahlavan et al., 2000). Other cell cycle regulating proteins and kinases may perform specific meiotic functions, but are possibly not directly involved in spindle regulation (Viveiros et al., 2001; Spruck et al., 2003; Suzumori et al., 2003). In summary, comparative molecular biology and other strategies have revealed proteins that play important roles in spindle function in oogenesis. Evidently, sufficient highenergy substrates are required for microtubule polymerization, motor protein activity and cell cycle regulating kinases for timely and accurate segregation of chromosomes.

Characteristic features of spindles in aged or developmentally compromised oocytes Human oocytes frequently have small, asymmetric and aberrant spindles with unaligned chromosomes at metaphase II after ageing or in-vitro maturation following a hormone stimulated IVF cycle (Battaglia et al., 1996a; Volarcik et al., 1998). In contrast, ageing mouse oocytes more rarely have visible spindles abnormalities (Eichenlaub-Ritter and Boll, 1989). However, there is some evidence that spindle pole-topole distance decreases with post-ovulatory age as well as advancing maternal age in certain strains of mice (EichenlaubRitter et al., 1986, 1988a), while disturbances in chromosome alignment are associated with ovarian depletion, and tentative indications exist that aneuploidy is at least partly associated with the physiological age of the ovary rather than chronological maternal age (Brook et al., 1984; for recent discussion, see Eichenlaub-Ritter, 1998, 2002; te Velde and Pearson, 2002). Accordingly, it can be speculated that sustained meiotic arrest and the depletion of the primordial follicle pool may have differential effects on spindle and oocyte components. Bearing in mind the larger spindles in rodents compared with primates (Eichenlaub-Ritter et al., 2002) (Figure 2), small perturbations in concentration or activity of spindle components may have more dramatic effects on primate oogenesis than other species. What is more, some human oocytes are suspended in diplotene for decades before reaching maturity. Damage accumulating in human oocytes or their companion somatic cells may therefore be more severe than in short-lived animals. Whether species differences in hormonal regulation of the ovarian cycle can

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explain the large difference in oocyte aneuploidy requires further investigation, although there is evidence that oocyte quality is more compromised in young women at risk of an early menopause (Kline et al., 2000; van Montfrans et al., 2002).

Chromosome cohesion and chiasmata in aged oocytes Cohesion between sister chromatids is essential for normal chromosome segregation in mitosis and meiosis. It is mediated by a complex of proteins, including cohesins and condensins, which interact with chromatin in all eukaryotic cells (Petronczki et al., 2003). Homologous chromosomes pair at meiotic prophase I, but physical contact is maintained by one or more chiasmata and by cohesion between sister chromatid arms within each homologue (Figure 3C). Only at anaphase I, are the sister chromatid arms detached from each other and resolve the chiasmata (Figure 3C′). Exchange between homologous chromosomes and the presence of at least one chiasma and the mono-orientation of centromeres of sister chromatids are essential for bi-orientation and faithful segregation of homologues at meiosis I in oogenesis (e.g. Woods et al., 1999; Libby et al., 2002; Lipkin et al., 2002; Yuan et al., 2002). Accordingly, aneuploidy may increase dramatically when physically unattached univalents are present within the spindle, or after precocious loss of cohesion. Shifting bivalent chromosomes from a meiosis I spindle to a metaphase II spindle reveals the correct reductional segregation of homologues, and the control of chromosome behaviour by chromosome-intrinsic factors rather than the meiotic cytoplasm (Paliulis and Nicklas, 2000). Bivalent chromosomes are in fact not passive components of the female germ cell, but are mandatory for bipolar spindle organization at oogenesis (Woods et al., 1999). In consequence, transfer of mitotic chromatin to cytoplasm of a GV-stage enucleated oocyte may result in M-phase progression and execution of anaphase, but spindles appear aberrant (Fulka et al., 2002), and unreplicated chromosomes of a somatic G1-cell nucleus have a high risk of random segregation (reported for mouse by Tateno et al., 2003a; for human oocytes by V Galat, Reproductive Genetics Institute, Chicago, personal communication). Whether this is primarily the result of the introduction of an unreplicated centriolar MTOC from the somatic cell, or the presence of unreplicated non-exchange pairs of somatic chromosomes, is unknown. However, the observations carry a warning about attempting to haploidize somatic cells (Eichenlaub-Ritter, 2003a,b; Tateno et al., 2003b). Although univalents may be at high risk for random segregation at meiosis I in any oocyte, balancing forces of the spindle may reduce this risk for errors in segregation within the functional spindle of a young oocyte. However, the risks of non-disjunction for individual univalent chromosomes or such with distal exchange may increase significantly, but also differentially for each chromosome in aged oocytes (Robinson et al., 1998; Hassold and Hunt, 2001). Importantly, there appears to be no evidence of gross defects in meiotic recombination in aged human oocytes. It is conceivable that chromosomes with a single distal chiasma are particularly prone to errors in segregation, due to a precocious loss of

cohesion proteins during prolonged ageing. Studies in Drosophila suggest that compromised cohesion due to oocyte ageing could interfere with feedback mechanisms helping to separate non-exchange chromosomes (Jeffreys et al., 2003). For unknown reasons, some chromosomes, for example human chromosome 15, which possesses several chiasmata after meiotic recombination, appear also to have an increased risk for non-disjunction in aged ooplasm (Robinson et al., 1998). The mechanisms for differential susceptibility to errors in segregation at meiosis I and II of chromosomes, which possess no, or one distal, or even several exchanges, in an aged oocyte are still not well understood. Interestingly, meiosis I-derived aneuploidy can be associated with errors in segregation of a whole chromosome but also of a chromatid (see below). This suggests that disturbances in the actual timing of chromosome disjunction at meiotic divisions may be of importance (Eichenlaub-Ritter, 2002).

Anaphase progression in oogenesis Anaphase progression requires degradation of the regulatory subunit of maturation promoting factor (MPF), namely cyclin B (Ledan et al., 2001). Furthermore, anaphase depends on the concomitant loss of cohesion between sister chromatids mediated by proteolysis of cohesion proteins. Both events are initiated by ubiquitination of substrates by APC/C, the highly conserved anaphase-promoting complex/cyclosome. The latter targets proteins like cyclin B and a securin for proteolysis (for recent review, see Petronczki et al., 2003) (Figure 3A–A′′). Downstream of APC/C, meiosis-specific proteins are required in the degradation pathway of cyclin B (Spruck et al., 2003). By contrast, the general mechanisms triggering initially the loss of chromosome cohesion appear to be conserved between mitotic and meiotic divisions. Central to the loss of chromosome cohesion is the activity of a protease, separin, which may cleave cohesin proteins such as Scc1 (in meiosis: Rec8 proteins). Scc1 is present in a complex of proteins holding chromatids together. Prior to anaphase, the separin is inactive while it is associated with the protein securin (Figure 3A). Once securin becomes degraded by the APC/C pathway, the separin is released, becomes activated, and in the next step can proteolytically cleave cohesin proteins like Scc1 or its meiotic ortholog, Rec8. Unlike mitosis, meiotic bivalent chromosomes contain two sister chromatids in each of the paired, recombined homologous chromosomes (Figure 3C). As in mitosis, the cohesion proteins hold chromatids attached all along their length. The kinetochores of sister chromatids are especially closely paired at meiosis I. Normal meiotic segregation requires the expression of Rec8, a meiotic orthologue replacing the mitotic Scc1 cohesin within the cohesion complex (reviewed by Petronczki et al. 2003). Kinetochores of sister chromatids attach and migrate to the same spindle pole at metaphase I and anaphase I (monopolar, syntelic orientation) (Figure 3C, C′). In meiosis II, they orient and migrate to opposite spindle poles (amphitelic orientation) (Figure 3C′′), comparable to mitosis (Figure 3B′). Rec8 protein and expression of other specific proteins (e.g. monopolins) at centromeres appears to be necessary for monopolar orientation of sister chromatids at first meiosis. Unlike in mitosis, anaphase I of meiosis is initiated by the release of cohesion between arms of sister chromatids only up to the most proximal chiasma (Figure 3C′), while it is

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essential that the centromeres of the two sister chromatids retain their attachment until anaphase II of meiosis (Lee et al., 2003). Mechanisms controlling the differential loss of cohesion at the chromosome arms and the centromeres of sister chromatids in meiosis I and II are still under investigation. They may depend on the timed and coordinated activity of kinases like Aurora B that are essential to prevent precocious homologue and chromatid segregation (for discussion see Petronczki et al., 2003). Studies of both animal and human oocytes imply that non-disjunction and random segregation of homologues, as well as precocious separation of sister chromatids, contribute to aneuploidy in aged human oocytes (e.g. Clyde et al., 2001; Verlinski et al., 2001; Pellestor et al., 2002; Sandalinas et al., 2002), suggesting that a defect in timed loss of chromosome cohesion and in cell cycle control is at the basis of meiotic aneuploidy. It was recently found that granulosa cells may provide molecules protecting maturing oocytes from precocious loss of chromatid cohesion (Cukurcam et al., 2003), which implies that the somatic compartment may indirectly play a role in the aetiology of aneuploidy in oocytes. Support of full nuclear and cytoplasmic maturational competence of the oocyte by effective interactions with the somatic compartment prior to and at resumption of maturation may not only be of relevance for fidelity of chromosome segregation at meiosis, but possibly also affect chromosome disjunction during embryonic cleavage, before checkpoint and cohesion proteins are zygotically expressed. If the oocyte is unprepared to support early mitotic control, disturbances can give rise to mosaic and chaotic constitutions, which may impair implantation (Wilding et al., 2003).

Spindle checkpoint in oogenesis Regulation of the timing of the metaphase–anaphase transition is mediated by a checkpoint (the ‘spindle checkpoint’) (Waters et al., 1998; see this reference for a comprehensive list of additional names). In gametogenesis, such a checkpoint would be expected to play an important role in preventing aneuploidy. Abnormal chromosome number in spermatozoa and oocytes is the leading cause of spontaneous abortions and pregnancy loss in humans. The checkpoint monitors attachment to microtubules and tension at kinetochores. Unattached, misaligned chromosomes may initiate the checkpoint, which causes a cell cycle arrest and thus provides time for correction of defects (Figure 3D). The molecular identity of key checkpoint components was determined a decade ago through screening budding yeast, which identified Mad (‘mitotic arrest deficient’) (Li and Murray, 1991) and Bub (‘budding uninhibited by benzimidazole’) (Hoyt et al., 1991) mutations in checkpoint genes. Later, many checkpoint components were found to be well conserved from yeast to humans. Although the nature of the direct molecular interaction(s) between checkpoint proteins, kinetochores and other cell components have not been fully determined (Musacchio and Hardwick, 2002), the signalling cascade has been essentially established.

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Generally, it is assumed that checkpoint proteins are targeted to kinetochores by kinases (e.g. Ditchfield et al., 2003) or are expressed in the cytoplasm. Their major function is to prevent untimely activation of the APC/C. Bub1 and Mad2 proteins are central to the spindle checkpoint in mitosis and meiosis (Figure 3D). Mad2 and other checkpoint components can

form a complex with APC/C. This renders APC/C inactive (Sudakin et al., 2001). Regulation of binding to APC/C is dependent on complex phosphorylation events and also needs expression of the kinesin-like motor protein CENP-E protein at centromeres (Abrieu et al. 2000). In absence of Mad2 or Bub1 expression, chromatids segregate precociously at meiosis I in yeast (Bernard et al., 2001), suggesting a particular meiotic function of the checkpoint components. Bub1 appears also essential for the constitutional metaphase II arrest in oogenesis (Schwab et al., 2001). Loss of checkpoint control proteins like Mad2, Bub3, or Rae1 in mice is lethal early in embryogenesis, with cells accumulating mitotic errors and undergoing apoptosis by embryonic day 5 or 6 (Dobles et al., 2000; Kalitsis et al., 2000; Babu et al., 2003). Many previous studies in mice (e.g. Eichenlaub-Ritter and Boll, 1989; Kubiak et al., 1993; Soewarto et al., 1995; Brunet et al., 1998) showed that meiotic arrest can be induced by exposure of oocytes to chemicals interfering with microtubule dynamics at meiosis I and II, suggesting that a spindle checkpoint exists in mammalian oocytes. A high sensitivity to loose checkpoint control during female meiosis and a possibly rather permissive control in mammalian oocytes is therefore surprising (LeMaire-Adkins et al., 1997; Yin et al., 1998a; Hodges et al., 2002; Hunt et al., 2003). Sensitivity to loss of cell cycle checkpoints may be related to the large volume of the cell and the low expression of checkpoint proteins. For instance, in-vitro activation of the spindle checkpoint in response to microtubule inhibiting drugs is dependent upon the relative ratio of extract volume to chromatin content (Minshull et al., 1994). The lack of chromosome-mediated checkpoint control would be predicted to result in a higher meiotic error rate in female compared with male germ cells which are smaller and connected by cytoplasmic bridges. In humans this indeed appears to be the case. An estimated 20% of human conceptions are chromosomally abnormal and most errors are maternal in origin (Jacobs, 1992; Hassold et al., 1993). In the mouse, the error rate is also substantially higher in oogenesis than in spermatogenesis (Bond and Chandley, 1983). As may be expected with a labile checkpoint, aneuploidy is increased and maturation kinetics are advanced in aged mouse oocytes (Eichenlaub-Ritter and Boll, 1989), similar to conditions where cohesion is defective or improper chromosome congression may occur undetected (LeMaire-Adkins et al., 1997). Perhaps in oocytes of older women the number of unbound Mad2 molecules becomes reduced with advanced age below a critical threshold level, such that impaired recruitment of Mad2 to unattached kinetochores prevents checkpoint arrest in consequence of reduced checkpoint transcripts (Bub1, Mad2) (Steuerwald et al., 2001) and probably significantly reduced expression of protein. Since checkpoint control is intricately dependent on the activity of kinases and motor proteins, it is also plausible that critical reductions in the local supply of high energy substrates affect control mechanisms, allowing spindle formation to proceed but impairing the checkpoint monitoring chromosome behaviour. The turnover and stability of cohesin proteins is entirely unknown for oogenesis, but delayed expression past S-phase of the meiotic Rec8 protein in yeast interferes with control over timing and fidelity of chromosome segregation (Watanabe et al., 2001).

Symposium - Ageing oocytes - U Eichenlaub-Ritter et al.

Mitochondria in oogenesis and ageing Mitochondria are overwhelmingly maternal in origin in humans and other mammals. Sperm mitochondria enter the ooplasm, but are eliminated during embryonic cleavage stages, possibly due to ubiquitination of the sperm mid-piece (Sutovsky et al., 1999). There is a genetic bottleneck in primordial germ cells, which helps to guarantee homoplasmy and uniform quantity of organelles in oocytes, but this does not exist in somatic cells. Thus, mitochondrial mutations in granulosa cells as well as oocytes could play a role in agerelated non-disjunction, as could changes in vascular perfusion of follicles which impede mitochondrial function. Mature oocytes require pyruvate as an energy source during maturation (Downs et al., 2002) and embryos later switch to glycolysis before undergoing implantation. On the other hand, granulosa cells primarily use glycolytic metabolism to generate energy, perhaps because these cells are avascular and depend on diffusion of oxygen from theca cell layers. Glycolysis conserves oxygen for steroidogenesis and for oocyte growth prior to resumption of maturation (Boland et al., 1994). Insufficient perfusion has been suggested to contribute to the age-related decline of follicular function and oocyte quality (Van Blerkom, 2000). Oocytes may derive some of their ATP, as well as nutrients, directly from granulosa cells via gap junctions until junctional communication ceases when they re-enter meiosis. Thus, defective mitochondria in granulosa cells in aged women could be a factor for oocyte quality. Supporting this hypothesis, granulosa cells in women over the age of 38 years have recently been found to contain fewer normal mitochondria compared with women ≤34 years using Δ4977-bp as a marker (Seifer et al., 2002). Clearly, further studies are required to dissect the relevance of mitochondrial dysfunction in the oocyte and somatic cell compartment for oocyte maturation and quality. Most somatic cells have a few thousand mitochrondria containing several genomes per organelle, but human oocytes have only 1–2 genomes per mitochondrion and highly variable numbers of mitochondria (Barritt et al., 1999). The absence of genetic recombination, efficient mtDNA repair mechanisms, histones and introns plus exposure to free electrons leaking from the electron transport chain puts the mitochondrial genome at a higher risk of mutations, with implications for ageing (Wallace et al., 1987). Oocytes are amongst the largest cells in the body, and need sufficient ATP for transcription and translation during oocyte growth and preparation for nuclear and cytoplasmic maturation. Minor insufficiencies in high-energy sources could compromise oocyte quality long before meiosis is reinitiated. Many mitochondrial proteins are derived from transcription and translation of nuclear RNAs (Chinnery, 2003), and translocation into and out of the mitochondrial matrix is influenced by the redox potential (Pfanner and Wiedemann, 2002). Thus, redox potential and intracellular pH could also influence the mitochondrial status of a growing oocyte. If oocytes lack certain mitochondrial proteins spindle formation, checkpoint control and chromosome cohesion could be compromised, with implications for faithful chromosome segregation.

The mitochondrial hypothesis of somatic ageing predicts that post-mitotic cells accumulate mtDNA damage, leading to progressive failure of oxidative phosphorylation to generate enough ATP for cellular metabolism and maintenance (Beckman and Ames, 1999). Accordingly, an age-related increase in mutations and deletions in mitochondria has been implicated in maternal age-related non-disjunction in human oocytes (discussed by Eichenlaub-Ritter, 1998, 2003; Schon et al., 2000). The ‘common’ deletion (ΔmtDNA4977) has been used as a genetic marker of ageing (Cortopassi and Arnheim, 1992) and one study (Keefe et al., 1995) found that 93% of oocytes from patients aged >37 years undergoing IVF treatment contained detectable ΔmtDNA4977, compared with only 28% of oocytes from younger women. However, oocytes never contained more than a few percent of mtDNAs with the ΔmtDNA4977, which throws doubt on its pathophysiological significance. However, this deletion may represent the tip of an iceberg of damaged mtDNA. Using nested long and short PCR to amplify two-thirds of the mitochondrial genome, 23 novel mtDNA rearrangements have been found in human oocytes and embryos (Barritt et al., 2000), including deletions, insertions and duplications. Although there was a significant difference in the percentage of mtDNA rearrangements between oocytes and embryos (implying that selection might eliminate mutant mitochondria after fertilization), there was no significant relationship between maternal age and the percentage of human oocytes or embryos containing mtDNA rearrangements (Barritt et al., 2000). Neither point mutations of mtDNA (nt 3243, 8344) nor the common deletion were detected by other groups investigating mitochondria in aged human oocytes, although there was a substantial increase in mitochondrial volume fraction (Chen et al., 1995; Muller-Hocker et al., 1996). Furthermore, there is no evidence of selection against high levels of pathogenic mtDNA point mutations in oogenesis or development, suggesting that efficient respiratory chain function may not be especially critical until post-natal life (Shoubridge, 2000). In addition, clinical conditions in which oxidative phosphorylation activity is substantially reduced by mutations can be compatible with fertility and live birth (Chinnery et al., 2000; Shoubridge, 2000; Poulton and Marchington, 2002). No ideal animal model exists for testing correlations between mitochondrial mutations in the germ line and oocyte quality. Although mitochondrial dysfunction in senescence accelerated mouse (SAM) strains was associated with spindle defects and disturbances in chromosome alignment and behaviour, these phenotypes are complex and effects of oxidative stress and pathophysiological changes may act indirectly on germs cells (Nakahara et al., 1998; Liu and Keefe, 2002). Chloramphenicol, a potent inhibitor of peptidyl transferase during mitochondrial protein synthesis (e.g. Ramachandran et al., 2002), has limited value for this investigation because, although it interferes with mitochondrial function, administration to mice increases the numbers of diploid rather than aneuploid oocytes (Beermann and Hansmann, 1996). Mitochondria are functionally heterogenous and nonrandomly dispersed within the oocyte and embryo, and

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undergo complex stage-dependent intracellular translocation (Bavister and Squirrell, 2000; Van Blerkom et al., 2002). Diazepam is a tranquillizer (valium), anticonvulsant, muscle relaxant and psychostimulant acting on central benzodiazepine receptors in the brain (Byck, 1975). It can also bind with high affinity to peripheral benzodiazepine receptors on the mitochondrial membrane, which are involved in cholesterol transport and mitochondrial function (Papadopoulos et al., 1997; Casellas et al., 2002; Nieswender, 2002). In-vitro exposure of maturing mouse oocytes to diazepam not only interfered with mitochondrial distribution and their association with cell cortex and oocyte spindles, but also induced meiotic arrest, untimely homologue segregation, and aneuploidy (Yin et al., 1998b; Sun et al., 2001). It is not clear whether this effect was mediated by peripheral, mitochondrial benzodiazepine receptors (Hirsch et al., 1997). Even minor dysfunction of mitochondria could have pronounced effects on oogenesis if mitochondrial membrane potential and activity and local energy supply and distribution or calcium homeostasis were affected. In fact, chromosome nondisjunction leading to chaotic mosaicism in human preimplantation embryos is associated with low mitochondrial membrane potential through an effect on spindle formation in oocytes (Wilding et al., 2003). Mitochondrial reorganization and ATP concentrations differ between morphologically good and poor bovine oocytes, which could contribute to differences in developmental potential, possibly as a result of nondisjunction and/or disturbances in energy-dependent processes (Stojkovic et al., 2001). Once oocytes resume maturation, they become transcriptionally inactive and most protein synthesis ceases except for certain mRNAs, which become polyadenylated and expressed in a stage-specific and highly coordinated manner (Eichenlaub-Ritter and Peschke, 2002). Oocytes may not require a high production of ATP at this stage, except to support basal metabolism and spindle formation and motor activity. Mitochondrial clustering around the spindle and in the peripheral cytoplasm could be important for controlling local intracellular pH and influencing protein function and cytoskeletal and cytoplasmic organization. What is more, mitochondria play an essential role in regulation of intracellular calcium homeostasis. In an amplification loop commonly known as CICR (calcium-induced calcium release) that may involve other cell organelles like the endoplasmic reticulum, mitochondria can contribute to propagate calcium signals (for brief review, see Roderick et al., 2003). Although calcium oscillations are primarily of importance in oocyte activation, calcium/calmodulin-dependent protein kinase II has been implicated in the metaphase I to anaphase I transition in maturing oocytes (Su and Eppig, 2002). Finally, there appears to be an inter-relationship between the translocation of mitochondria during maturation and the dynamic microtubule network. Minor disturbances in the cytoskeleton at maturation may not only compromise spindle formation directly, but indirectly affect mitochondrial clustering and formation of ‘microdomains’. In consequence, even slight disturbances in mitochondrial distribution and in local regulation of intracellular pH can increase the risk of malsegregation of chromosomes.

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Conclusions Improving the quality of oocytes and identifying embryos of high implantation competence represents a most important challenge for improving assisted reproduction success rates and reducing multiple pregnancies. A high proportion of oocytes have defects arising during meiotic maturation in the oocyte. Furthermore, chromosomal analysis of preimplantation embryos suggests that mitotic errors are substantial, although this is not reflected in prenatal diagnosis at later stages. The causes of aneuploidy are complex, and there is evidence of involvement of metabolic, genetic and xenobiotic factors; one or more of these may underlie the rising incidence of chromosomal non-disjunction with maternal age. An understanding of nuclear maturation in oocytes and the oocyte’s contribution to secure high fidelity of chromosome segregation at embryogenesis is therefore one of the central goals in applied reproductive science. It will require investigation of developing cells when the organelles and molecules needed for timely and accurate spindle formation and maintenance and loss of chromosome cohesion are being expressed and localized. It is hoped that reliable, non-invasive methods of monitoring oocyte competence will eventually emerge from this new knowledge.

Acknowledgements The authors would like to thank Y Shen and H-R Tinneberg (Women´s Hospital, Justus-Liebig University of Giessen) and T Zenzes (University of Toronto) for sharing unpublished results. We apologize to colleagues whose work we could not cite due to space limitations. Work from the authors’ laboratories was supported by grants from the EU.

References Abrieu A, Kahana JA, Wood KW et al. 2000 CENP-E as an essential component of the mitotic checkpoint in vitro. Cell 102, 817–826. Asch R, Simerly C, Ord T et al. 1995 The stages at which human fertilization arrests: microtubule and chromosome configurations in inseminated oocytes which failed to complete fertilization and development in humans. Human Reproduction 10, 1897–1906. Babu JR, Jeganathan KB, Baker DJ et al. 2003 Rae1 is an essential mitotic checkpoint regulator that co-operates with Bub3 to prevent chromosome missegregation. Journal of Cell Biology 160, 341–353. Bamba C, Bobinnec Y, Fukuda M et al. 2002 The GTPase Ran regulates chromosome positioning and nuclear envelope assembly in vivo. Current Biology 12, 503–537. Barritt JA, Brenner CA, Cohen J et al. 1999 Mitochondrial DNA rearrangements in human oocytes and embryos. Molecular Human Reproduction 5, 927–933. Barritt JA, Brenner CA, Willadsen S et al. 2000 Spontaneous and artificial changes in human ooplasmic mitochondria. Human Reproduction 15, 207–217. Battaglia DE, Klein NA, Soules MR 1996a Changes in centrosomal domains during meiotic maturation in the human oocyte. Molecular Human Reproduction 2, 845–851. Battaglia DE, Goodwin P, Klein NA et al. 1996b Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Human Reproduction 11, 2217–2222. Bavister BD, Squirrell JM 2000 Mitochondrial distribution and function in oocytes and early embryos. Human Reproduction 15, 189–198. Beckman KB, Ames BN 1999 Endogenous oxidative damage of mtDNA. Mutation Research 424, 51–58.

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Beermann F, Hansmann I 1996 Follicular maturation, luteinization and first meiotic division in oocytes after inhibiting mitochondrial function in mice with chloramphenicol. Mutation Research 160, 47. Bernard P, Maure JF, Javerzat JP 2001 Fission yeast Bub1 is essential in setting up the meiotic pattern of chromosome segregation. Nature Cell Biology 3, 522–526. Boiso I, Marti M, Santalo J et al. 2002 A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage. Human Reproduction 17, 1885–1891. Boland NI, Humpherson PG, Leese HJ et al. 1994 The effect of glucose metabolism on murine follicle development and steroidogenesis in vitro. Human Reproduction 9, 617–623. Bond DJ, Chandley AC 1983 Aneuploidy. Oxford University Press, London and New York. Brook JD, Gosden RG, Chandley AC 1984 Maternal ageing and aneuploid embryos – evidence from the mouse that biological and not chronological age is the important influence. Human Genetics 66, 41–45. Brunet S, Vernos I 2001 Chromosome motors on the move. From motion to spindle checkpoint activity. EMBO Reports 2, 669–673. Brunet S, Polanski Z, Verlhac MH et al. 1998 Bipolar meiotic spindle formation without chromatin. Current Biology 8, 1231–1234. Brunet S, Maria AS, Guillaud P et al. 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. Journal of Cell Biology 146, 1–12. Byck R 1975 Drugs and the treatment of psychiatric disorders. In Goodman LS, Gilman A (eds) The Pharmacological Basis of Therapeutics, 5th edn. Macmillan, New York, pp. 189–192. Carabatsos MJ, Combelles CM, Messinger SM et al. 2000 Sorting and reorganization of centrosomes during oocyte maturation in the mouse. Microscopy Research and Technique 49, 435–444. Casellas P, Galiegue S, Basile AS 2002 Peripheral benzodiazepine receptors and mitochondrial function. Neurochemistry International 40, 475–486. Cekleniak NA, Combelles CM, Ganz DA et al. 2001 A novel system for in vitro maturation of human oocytes. Fertility and Sterility 75, 1185–1193. Chen X, Prosser R, Simonetti S et al. 1995 Rearranged mitochondrial genomes are present in human oocytes. American Journal of Human Genetics 57, 239–247. Chinnery PF 2003 Searching for nuclear-mitochondrial genes. Trends in Genetics 19, 60–62. Chinnery PF, Taylor DJ, Brown DT et al. 2000 Very low levels of the mtDNA A3243G mutation associated with mitochondrial dysfunction in vivo. Annals of Neurology 47, 381–384. Clyde JM, Gosden RG, Rutherford AJ et al. 2001 Demonstration of a mechanism of aneuploidy in human oocytes using Multifluor fluorescence in situ hybridization. Fertility and Sterility 76, 837–840. Combelles CM, Albertini DF 2001 Microtubule patterning during meiotic maturation in mouse oocytes is determined by cell cyclespecific sorting and redistribution of gamma-tubulin. Developmental Biology 239, 281–294. Compton DA 2000 Spindle assembly in animal cells. Annual Review of Biochemistry 69, 95–114. Cortopassi GA, Arnheim N 1992 Using the polymerase chain reaction to estimate mutation frequencies and rates in human cells. Mutation Research 277, 239–249. Cukurcam S, Hegele-Hartung C, Eichenlaub-Ritter U 2003 Meiosis activating sterol protects oocytes from precocious chromosome segregation. Human Reproduction 18, 1908–1917. Dasso M 2002 The Ran GTPase: theme and variations. Current Biology 12:R502–R508. de Pennart H, Houliston E, Maro B 1988 Post-translational modifications of tubulin and the dynamics of microtubules in mouse oocytes and zygotes. Biology of the Cell 64, 375–378. Desai A, Mitchison TJ 1997 Microtubule polymerization dynamics.

Annual Review of Cell and Developmental Biology 13, 83–117. Ditchfield C, Johnson VL, Tighe A et al. 2003 Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. Journal of Cell Biology 161, 267–280. Dobles M, Liberal V, Scott ML et al. 2000 Chromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2. Cell 101, 635–645. Downs SM, Humpherson PG, Leese HJ 2002 Pyruvate utilization by mouse oocytes is influenced by meiotic status and the cumulus oophorus. Molecular Reproduction and Development 62, 113–123. Duesbery NS, Choi T, Brown KD et al. 1997 CENP-E is an essential kinetochore motor in maturing oocytes and is masked during mos-dependent, cell cycle arrest at metaphase II. Proceedings of the National Academy of Sciences of the United States of America 94, 9165–9170. Eichenlaub-Ritter U 1998 Genetics of oocyte ageing. Maturitas 30, 143–169. Eichenlaub-Ritter U 2002 Ageing and aneuploidy in oocytes. Ernst Schering Research Foundation Workshop 41, 111–136. Eichenlaub-Ritter U 2003a Reproductive semi-cloning respecting biparental origin: reconstitution of gametes for assisted reproduction. Human Reproduction 18, 473–475. Eichenlaub-Ritter U 2003b Aneuploidy in ageing oocytes and after toxic insult. In: Trounson A, Gosden R (eds) The Biology and Pathology of the Oocyte. Cambridge University Press, Cambridge, pp. 220–257, in press. Eichenlaub-Ritter U, Boll I 1989 Nocodazole sensitivity, age-related aneuploidy, and alterations in the cell cycle during maturation of mouse oocytes. Cytogenetics and Cell Genetics 52, 170–176. Eichenlaub-Ritter U, Peschke M 2002 Expression of in-vivo and invitro growing and maturing oocytes: focus on regulation of regulation at the translational level. Human Reproduction Update 8, 21–41. Eichenlaub-Ritter U, Chandley AC, Gosden RG 1986 Alterations to the microtubular cytoskeleton and increased disorder of chromosome alignment in spontaneously ovulated mouse oocytes aged in vivo: an immunofluorescent study. Chromosoma 94, 337–345. Eichenlaub-Ritter U, Chandley AC, Gosden RG 1988a The CBA mouse as a model for age-related aneuploidy in man: studies of oocyte maturation, spindle formation and chromosome alignment during meiosis. Chromosoma 96, 220–226. Eichenlaub-Ritter U, Stahl A, Luciani JM 1988b The microtubular cytoskeleton and chromosomes of unfertilized human oocytes aged in vitro. Human Genetics 80, 259–264. Eichenlaub-Ritter U, Shen Y, Tinneberg HR 2002 Manipulation of the oocyte: possible damage to the spindle apparatus. Reproductive BioMedicine Online 6, 61–68. Fulka J Jr, Martinez F, Tepla O et al. 2002 Somatic and embryonic cell nucleus transfer into intact and enucleated immature mouse oocytes. Human Reproduction 17, 2160–2164. George MA, Pickering SJ, Braude PR et al. 1996 The distribution of alpha- and gamma-tubulin in fresh and aged human and mouse oocytes exposed to cryoprotectant. Molecular Human Reproduction 2, 445–456. Gorbsky GJ, Simerly C, Schatten G et al. 1990 Microtubules in the metaphase-arrested mouse oocyte turn over rapidly. Proceedings of the National Academy of Sciences USA 87, 6049–6053. Hassold T, Hunt P 2001 To err (meiotically) is human: the genesis of human aneuploidy. Nature Review Genetics 2, 280–291. Hassold T, Hunt PA, Sherman S 1993 Trisomy in humans: incidence, origin and etiology. Current Opinion in Genetics and Development 3, 398–403. Heald R 2000 Motor function in the mitotic spindle. Cell 102, 399–402. Heald R, Walczak CE 1999 Microtubule-based motor function in mitosis. Current Opinion in Structural Biology 9, 268–274. Heikinheimo O, Gibbons WE 1998 The molecular mechanisms of oocyte maturation and early embryonic development are

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unveiling new insights into reproductive medicine. Molecular Human Reproduction 4, 745–756. Hewitson L, Schatten G 2002 The use of primates as models for assisted reproduction. Reproductive BioMedicine Online 5, 50–55. Hewitson L, Dominko T, Takahashi D et al. 1999 Unique checkpoints during the first cell cycle of fertilization after intracytoplasmic sperm injection in rhesus monkeys. Nature Medicine 5, 431–423. Hirao Y, Eppig JJ 1997 Parthenogenetic development of Mosdeficient mouse oocytes. Molecular Reproduction and Development 48, 391–396. Hirokawa N, Noda Y, Okada Y 1998 Kinesin and dynein superfamily proteins in organelle transport and cell division. Current Opinion in Cell Biology 10, 60–73. Hirsch T, Marchetti P, Susin SA et al. 1997 The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 15, 1573–1581. Hodges CA, Ilagan A, Jennings D et al. 2002 Experimental evidence that changes in oocyte growth influence meiotic chromosome segregation. Human Reproduction 17, 1171–1180. Hoyt MA, Totis L, Roberts BT 1991 S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 66, 507–517. Hunt PA, Koehler KE, Susiarjo M et al. 2003 Bisphenol A exposure causes meiotic aneuploidy in the female mouse. Current Biology 13, 546–553. Hunter AW, Wordeman L 2000 How motor proteins influence microtubule polymerization dynamics. Journal of Cell Science 113, 4379–4389. Jacobs PA 1992 The chromosome complement of human gametes. Oxford Reviews of Reproductive Biology 14, 47–72. Jeffreys CA, Burrage PS, Bickel SE 2003 A model system for increased meiotic nondisjunction in older oocytes. Current Biology 13, 498–503. Jordan MA 2002 Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Current Medicinal Chemistry/Anti-Cancer Agents 2, 1–17. Kalab P, Kubiak JZ, Verlhac MH et al. 1996 Activation of p90rsk during meiotic maturation and first mitosis in mouse oocytes and eggs: MAP kinase-independent and -dependent activation. Development 122, 1957–1964. Kalitsis P, Earle E, Fowler KJ et al. 2000 Bub3 gene disruption in mice reveals essential mitotic spindle checkpoint function during early embryogenesis. Genes and Development 14, 2277–2282. Karsenti E, Vernos I 2001 The mitotic spindle: a self-made machine. Science 294, 543–547. Keefe DL, Niven-Fairchild T, Powell S et al. 1995 Mitochondrial deoxyribonucleic acid deletions in oocytes and reproductive aging in women. Fertility and Sterility 64, 577–583. Keefe D, Liu L, Wang W et al. 2003 Imaging meiotic spindles by polarization light microscopy: principles and applications to IVF. Reproduction BioMedicine Online 7, 24–29. Kellogg DR, Moritz M, Alberts BM 1994 The centrosome and cellular organization. Annual Review of Biochemistry 63, 639–674. Kline J, Kinney A, Levin B et al. 2000 Trisomy pregnancy and earlier age at menopause. American Journal of Human Genetics 67, 395–404. Kubiak JZ, Weber M, de Pennart H et al. 1993 The metaphase II arrest in mouse oocytes is controlled through microtubuledependent destruction of cyclin B in the presence of CSF. EMBO Journal 12, 3773–3778. Leader B, Lim H, Carabatsos MJ et al. 2002 Formin-2, polyploidy, hypofertility and positioning of the meiotic spindle in mouse oocytes. Nature Cell Biology 4, 921–928. Ledan E, Polanski Z, Terret ME, Maro B 2001 Meiotic maturation of the mouse oocyte requires an equilibrium between cyclin B synthesis and degradation. Developmental Biology 232, 400–413. Lee J, Miyano T, Dai Y et al. 2000a Specific regulation of CENP-E

and kinetochores during meiosis I/meiosis II transition in pig oocytes. Molecular Reproduction and Development 56, 51–62. Lee J, Miyano T, Moor RM 2000b Spindle formation and dynamics of gamma-tubulin and nuclear mitotic apparatus protein distribution during meiosis in pig and mouse oocytes. Biology of Reproduction 62, 1184–1192. Lee J, Iwai T, Yokota T, Yamashita M 2003 Temporally and spatially selective loss of Rec8 protein from meiotic chromosomes during mammalian meiosis. Journal of Cell Science 116, 2781–2790. Lefebvre C, Terret ME, Djiane A et al. 2002 Meiotic spindle stability depends on MAPK-interacting and spindle-stabilizing protein (MISS), a new MAPK substrate. Journal of Cell Biology 157, 603–613. LeMaire-Adkins R, Radke K, Hunt PA 1997 Lack of checkpoint control at the metaphase/anaphase transition: a mechanism of meiotic nondisjunction in mammalian females. Journal of Cell Biology 139, 1611–1619. Li R, Murray AW 1991 Feedback control in mitosis in budding yeast. Cell 373, 519–531. Libby BJ, De La Fuente R, O’Brien MJ et al. 2002 The mouse meiotic mutation mei1 disrupts chromosome synapsis with sexually dimorphic consequences for meiotic progression. Developmental Biology 242, 174–187. Lipkin SM, Moens PB, Wang V et al. 2002 Meiotic arrest and aneuploidy in MLH3-deficient mice. Nature Genetics 31, 385–390. Liu L, Keefe DL 2002 Ageing-associated aberration in meiosis of oocytes from senescence-accelerated mice. Human Reproduction 17, 2678–2685. Maney T, Hunter AW, Wagenbach M et al. 1998 Mitotic centromereassociated kinesin is important for anaphase chromosome segregation. Journal of Cell Biology 142, 787–801. Maro B, Howlett SK, Webb M 1985 Non-spindle microtubule organizing centers in metaphase II-arrested mouse oocytes. Journal of Cell Biology 101, 1665–1672. Maro B, Howlett SK, Houliston E 1986 Cytoskeleton dynamics in the mouse egg. Journal of Cell Science Supplement 5, 343–359. Minshull J, Sun H, Tonks NK et al. 1994 A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts. Cell 79, 475–486. Mitchinson T, Kirschner M 1984 Dynamic instability of microtubule growth. Nature 312, 237–242. Mountain V, Simerly C, Howard L et al. 1999 The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle. Journal of Cell Biology 147, 351–366. Muller-Hocker J, Schafer S, Weis S et al. 1996 Morphological–cytochemical and molecular genetic analyses of mitochondria in isolated human oocytes in the reproductive age. Molecular Human Reproduction 2, 951–958. Musacchio A, Hardwick KG 2002 The spindle checkpoint: structural insights into dynamic signalling. Nature Reviews/Molecular Cell Biology 3, 731–741. Nakahara H, Kanno T, Inai Y et al. 1998 Mitochondrial dysfunction in the senescence accelerated mouse (SAM). Free Radical Biology and Medicine 24, 85–92. Nedelec F, Surrey T, Karsenti E 2003 Self-organisation and forces in the microtubule cytoskeleton. Current Opinion in Cell Biology 15, 118–124. Neilson L, Andalibi A, Kang D et al. 2000 Molecular phenotype of the human oocyte by PCR-SAGE. Genomics 63, 13–24. Nieswender GD 2002 Molecular control of luteal secretion of progesterone. Reproduction 123, 333–339. Nigg EA 2001 Mitotic kinases as regulators of cell division and its checkpoints. Nature Reviews/Molecular Cell Biology 2, 21–32. Pahlavan G, Polanski Z, Kalab P et al. 2000 Characterization of polo-like kinase 1 during meiotic maturation of the mouse oocyte. Developmental Biology 220, 392–400. Paliulis LV, Nicklas RB 2000 The reduction of chromosome number in meiosis is determined by properties built into the chromosomes. Journal of Cell Biology 150, 1223–1232.

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Papadopoulos V, Amri H, Boujrad N et al. 1997 Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis. Steroids 62, 21–28. Park SE, Son WY, Lee SH et al. 1997 Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage. Fertility and Sterility 68, 920–926. Pellestor F, Andreo B, Arnal F et al. 2002 Mechanisms of nondisjunction in human female meiosis: the co-existence of two modes of malsegregation evidenced by the karyotyping of 1397 in-vitro unfertilized oocytes. Human Reproduction 17, 2134–2145. Petronczki M, Siomos MF, Nasmyth K 2003 Un menage a quatre: the molecular biology of chromosome segregation in meiosis. Cell 112, 423–440 Pfanner N, Wiedemann N 2002 Mitochondrial protein import: two membranes, three translocases. Current Opinion in Cell Biology 14, 400–411. Pickering SJ, Johnson MH 1987 The influence of cooling on the organization of the meiotic spindle of the mouse oocyte. Human Reproduction 2, 207–216. Pickering SJ, Johnson MH, Braude PR et al. 1988 Cytoskeletal organization in fresh, aged and spontaneously activated human oocytes. Human Reproduction 3, 978–989. Poulton J, Marchington DR 2002 Segregation of mitochondrial DNA (mtDNA) in human oocytes and in animal models of mtDNA disease: clinical implications. Reproduction 123, 751–755. Putkey FR, Cramer T, Morphew MK et al. 2002 Unstable kinetochore-microtubule capture and chromosomal instability following deletion of CENP-E. Developmental Cell 3, 351–365. Ramachandran A, Moellering DR, Ceaser E et al. 2002 Inhibition of mitochondrial protein synthesis results in increased endothelial cell susceptibility to nitric oxide-induced apoptosis. Procedings of the National Academy of Sciences USA 99, 6643–6648. Rawe VY, Olmedo SB, Nodar FN et al. 2000 Cytoskeletal organization defects and abortive activation in human oocytes after IVF and ICSI failure. Molecular Human Reproduction 6, 510–516. Robinson WP, Kuchinka BD, Bernasconi F et al. 1998 Maternal meiosis I non-disjunction of chromosome 15: dependence of the maternal age effect on level of recombination. Human Molecular Genetics 7, 1011–1019. Roderick HL, Berridge MJ, Bootman MD 2003 Calcium-induced calcium release. Current Biology 13, R425. Sandalinas M, Marquez C, Munne S 2002 Spectral karyotyping of fresh, non-inseminated oocytes. Molecular Human Reproduction 8, 580–585. Sathananthan AH 1997 Ultrastructure of the human egg. Human Cell 10, 21–38. Sathananthan AH, Selvaraj K, Trounson A 2000 Fine structure of human oogonia in the foetal ovary. Molecular and Cellular Endocrinology 161, 3–8. Schatten H, Schatten G, Mazia D et al. 1986 Behavior of centrosomes during fertilization and cell division and mouse oocytes and in sea urchin eggs. Proceedings of the National Academy of Sciences USA 83, 105–109. Schon EA, Kim SH, Ferreira JC et al. 2000 Chromosomal nondisjunction in human oocytes: is there a mitochondrial connection? Human Reproduction 15, 160–172. Schwab MS, Roberts BT, Gross SD et al. 2001 Bub1 is activated by the protein kinase p90(Rsk) during Xenopus oocyte maturation. Current Biology 11, 141–150. Seifer DB, DeJesus V, Hubbard K 2002 Mitochondrial deletions in luteinized granulosa cells as a function of age in women undergoing in vitro fertilization. Fertility and Sterility 78, 1046–1048. Shin MR, Kim NH 2003 Maternal gamma-tubulin is involved in microtubule reorganization during bovine fertilization and parthenogenesis. Molecular Reproduction and Development 64, 438–445.

Shoubridge EA 2000 Mitochondrial DNA segregation in the developing embryo. Human Reproduction 15, 229–234. Simerly C, Zoran SS, Payne C et al. 1999 Biparental inheritance of gamma-tubulin during human fertilization: molecular reconstitution of functional zygotic centrosomes in inseminated human oocytes and in cell-free extracts nucleated by human sperm. Molecular Biology of the Cell 10, 2955–2969. Simerly C, Dominko T, Navara C et al. 2003 Molecular correlates of primate nuclear transfer failures. Science 300, 297. Soewarto D, Schmiady H, Eichenlaub-Ritter U 1995 Consequences of non-extrusion of the first polar body and control of the sequential segregation of homologues and chromatids in mammalian oocytes. Human Reproduction 10, 2350–2360. Spruck CH, de Miguel MP, Smith AP et al. 2003 Requirement of Cks2 for the first metaphase/anaphase transition of mammalian meiosis. Science 300, 647–650. Stanton JL, Bascand M, Fisher L et al. 2002 Gene expression profiling of human GV oocytes: an analysis of a profile obtained by serial analysis of gene expression (SAGE). Journal of Reproductive Immunology 53, 193–201. Steuerwald N, Cohen J, Herrera RJ et al. 2001 Association between spindle assembly checkpoint expression and maternal age in human oocytes. Molecular Human Reproduction 7, 49–55. Stojkovic M, Machado SA, Stojkovic P et al. 2001 Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture. Biology of Reproduction 64, 904–909. Su YQ, Eppig JJ 2002 Evidence that multifunctional calcium/calmodulin-dependent protein kinase II (CaM KII) participates in the meiotic maturation of mouse oocytes. Molecular Reproductive Development 61, 560–569. Sudakin V, Chan GK, Yen TJ 2001 Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2. Journal of Cell Biology 154, 925–936. Sun F, Yin H, Eichenlaub-Ritter U 2001 Differential chromosome behaviour in mammalian oocytes exposed to the tranquilizer diazepam in vitro. Mutagenesis 16, 407–417. Sutovsky P, Moreno RD, Ramalho-Santos J et al. 1999 Ubiquitin tag for sperm mitochondria. Nature 402, 371–372. Suzumori N, Burns KH, Yan W, Matzuk MM 2003 RFPL4 interacts with oocyte proteins of the ubiquitin–proteasome degradation pathway. Proceedings of the National Academy of Sciences USA 100, 550–555. Svoboda K, Mitra PP, Block SM 1994 Fluctuation analysis of motor protein movement and single enzyme kinetics. Proceedings of the National Academy of Sciences USA 91, 11782–11786. Svoboda P, Stein P, Hayashi H, Schultz RM 2000 Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development 127, 4147–4156. Tateno H, Akutsu H, Kamiguchi Y et al. 2003a Inability of mature oocytes to create functional haploid genomes from somatic cell nuclei. Fertility and Sterility 79, 216–218. Tateno H, Latham KE, Yanagimachi R 2003b Reproductive semicloning respecting biparental origin. A biologically unsound principle. Human Reproduction 18, 472–473. te Velde ER, Pearson PL 2002 The variability of female reproductive ageing. Human Reproduction Update 8, 141–154. Van Blerkom J 2000 Intrafollicular influences on human oocyte developmental competence: perifollicular vascularity, oocyte metabolism and mitochondrial function. Human Reproduction 15, 173–188. Van Blerkom J, Davis P, Mathwig V et al. 2002 Domains of highpolarized and low-polarized mitochondria may occur in mouse and human oocytes and early embryos. Human Reproduction 17, 393–406. van Montfrans JM, van Hooff MH, Martens F et al. 2002 Basal FSH, estradiol and inhibin B concentrations in women with a previous Down’s syndrome affected pregnancy. Human Reproduction 17,

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44–47. Vee S, Lafanechere L, Fisher D et al. 2001 Evidence for a role of the (alpha)-tubulin C terminus in the regulation of cyclin B synthesis in developing oocytes. Journal of Cell Science 114, 887–898. Verlhac MH, Kubiak JZ, Clarke HJ et al. 1994 Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes. Development 120, 1017–1025. Verlhac MH, Kubiak JZ, Weber M et al. 1996 Mos is required for MAP kinase activation and is involved in microtubule organization during meiotic maturation in the mouse. Development 122, 815–822. Verlhac MH, Lefebvre C, Guillaud P et al. 2000 Asymmetric division in mouse oocytes: with or without Mos. Current Biology 10, 1303–1306. Verlinsky Y, Cieslak J, Ivakhnenko V et al. 2001 Chromosomal abnormalities in the first and second polar body. Molecular and Cellular Endocrinology 183, S47–49. Viveiros MM, Hirao Y, Eppig JJ 2001 Evidence that protein kinase C (PKC) participates in the meiosis I to meiosis II transition in mouse oocytes. Developmental Biology 235, 330–342. Volarcik K, Sheean L, Goldfarb J et al. 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. Human Reproduction 13, 154–160. Walczak CE, Vernos I, Mitchison TJ et al. 1998 A model for the proposed roles of different microtubule-based motor proteins in establishing spindle bipolarity. Current Biology 8, 903–913. Wallace DC, Ye JH, Neckelmann SN et al. 1987 Sequence analysis of cDNAs for the human and bovine ATP synthase beta subunit: mitochondrial DNA genes sustain seventeen times more mutations. Current Genetics 12, 81–90. Wang WH, Keefe DL 2002 Spindle observation in living mammalian oocytes with the polarization microscope and its practical use. Cloning Stem Cells 4, 269–276. Wang WH, Meng L, Hackett RJ et al. 2001 Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy. Human Reproduction 16, 2374–2378. Watanabe Y, Yokobayashi S, Yamamoto M, Nurse P 2001 Pre-

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meiotic S phase is linked to reductional chromosome segregation and recombination. Nature 409, 359–363. Waters JC, Chen RH, Murray AW et al. 1998 Localization of Mad2 to kinetochores depends on microtubule attachment, not tension. Journal of Cell Biology 141, 1181–1191. Wianny F, Zernicka-Goetz M 2000 Specific interference with gene function by double-stranded RNA in early mouse development. Nature Cell Biology 2, 70–75. Wianny F, Tavares A, Evans MJ et al. 1998 Mouse polo-like kinase I associates with the acentriolar spindle poles, meiotic chromosomes and spindle midzone during oocyte maturation. Chromosoma 107, 430–439. Wilding M, De Placido G, De Matteo L et al. 2003 Chaotic mosaicism in human preimplantation embryos is correlated with a low mitochondrial membrane potential. Fertility and Sterility 79, 340–346. Wittmann T, Hyman A, Desai A 2001 The spindle: a dynamic assembly of microtubules and motors. Nature Cell Biology 3, E28–E34. Woods LM, Hodges CA, Baart E et al. 1999 Chromosomal influence on meiotic spindle assembly: abnormal meiosis I in female Mlh1 mutant mice. Journal of Cell Biology 145, 1395–1406. Yin H, Cukurcam S, Betzendahl I et al. 1998a Trichlorfon exposure, spindle aberrations and nondisjunction in mammalian oocytes. Chromosoma 107, 514–522. Yin H, Baart E, Betzendahl I et al. 1998b Diazepam induces meiotic delay, aneuploidy and predivision of homologues and chromatids in mammalian oocytes. Mutagenesis 13, 567–580. Yuan L, Liu JG, Hoja MR et al. 2002 Female germ cell aneuploidy and embryo death in mice lacking the meiosis-specific protein SCP3. Science 296, 1115–1118. Zenzes MT, Bielecki R, Casper RF et al. 2001 Effects of chilling to 0 degrees C on the morphology of meiotic spindles in human metaphase II oocytes. Fertility and Sterility 75, 769–77.

Received 26 June 2003; refereed 22 July 2003; accepted 10 August 2003.