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• W2613590953 abstract "•Decreased oocyte cytoplasmic size allows spindles to have better-focused poles•Decreased oocyte cytoplasmic size enhances spindle checkpoint stringency•Increased oocyte cytoplasmic size confers the opposite effects•A large cytoplasmic size is linked to error-prone chromosome segregation in oocytes Chromosome segregation during meiosis in oocytes is error prone. The uniquely large cytoplasmic size of oocytes, which provides support for embryogenesis after fertilization, might be a predisposing factor for meiotic errors. However, this hypothesis remains unproven. Here, we show that cytoplasmic size affects the functionality of the acentrosomal spindle. Artificially decreasing the cytoplasmic size in mouse oocytes allows the acentrosomal spindle poles to have a better-focused distribution of microtubule-organizing centers and to biorient chromosomes more efficiently, whereas enlargement of the cytoplasmic size has the opposite effects. Moreover, we found that the cytoplasmic size-dependent dilution of nuclear factors, including anaphase inhibitors that are preformed at the nuclear membrane, limits the spindle's capacity to prevent anaphase entry with misaligned chromosomes. The present study defines a large cytoplasmic volume as a cell-intrinsic feature linked to the error-prone nature of oocytes. This may represent a trade-off between meiotic fidelity and post-fertilization developmental competence. Chromosome segregation during meiosis in oocytes is error prone. The uniquely large cytoplasmic size of oocytes, which provides support for embryogenesis after fertilization, might be a predisposing factor for meiotic errors. However, this hypothesis remains unproven. Here, we show that cytoplasmic size affects the functionality of the acentrosomal spindle. Artificially decreasing the cytoplasmic size in mouse oocytes allows the acentrosomal spindle poles to have a better-focused distribution of microtubule-organizing centers and to biorient chromosomes more efficiently, whereas enlargement of the cytoplasmic size has the opposite effects. Moreover, we found that the cytoplasmic size-dependent dilution of nuclear factors, including anaphase inhibitors that are preformed at the nuclear membrane, limits the spindle's capacity to prevent anaphase entry with misaligned chromosomes. The present study defines a large cytoplasmic volume as a cell-intrinsic feature linked to the error-prone nature of oocytes. This may represent a trade-off between meiotic fidelity and post-fertilization developmental competence. Correct chromosome segregation during cell division is essential for the viability of daughter cells. Most cells thus possess a robust system to ensure error-free chromosome segregation. However, oocytes are a clear exception. Meiotic chromosome segregation in oocytes is highly error prone and, thus, frequently results in the production of aneuploid eggs, which is a leading cause of pregnancy loss and congenital disorders, such as Down syndrome (Jones and Lane, 2013Jones K.T. Lane S.I. Molecular causes of aneuploidy in mammalian eggs.Development. 2013; 140: 3719-3730Crossref PubMed Scopus (147) Google Scholar, Nagaoka et al., 2012Nagaoka S. Hassold T. Hunt P. Human aneuploidy: mechanisms and new insights into an age-old problem.Nat. Rev. Genet. 2012; 13: 493-504Crossref PubMed Scopus (610) Google Scholar). The frequency of chromosome segregation errors is much higher in oocytes than in spermatocytes, even in young individuals, and increases with age (Chiang et al., 2012Chiang T. Schultz R. Lampson M. Meiotic origins of maternal age-related aneuploidy.Biol. Reprod. 2012; 86: 1-7Crossref PubMed Scopus (133) Google Scholar, Jones and Lane, 2013Jones K.T. Lane S.I. Molecular causes of aneuploidy in mammalian eggs.Development. 2013; 140: 3719-3730Crossref PubMed Scopus (147) Google Scholar, Nagaoka et al., 2012Nagaoka S. Hassold T. Hunt P. Human aneuploidy: mechanisms and new insights into an age-old problem.Nat. Rev. Genet. 2012; 13: 493-504Crossref PubMed Scopus (610) Google Scholar, Webster and Schuh, 2017Webster A. Schuh M. Mechanisms of aneuploidy in human eggs.Trends Cell Biol. 2017; 27: 55-68Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). This suggests that not only age-related effects but also oocyte-intrinsic features contribute to the error-prone nature of chromosome segregation. However, which oocyte features are linked to the error-prone nature remains unclear. Moreover, the mechanism by which oocytes, which provide a critical basis for embryogenesis, are prone to errors remains unexplained. Chromosome biorientation, whereby spindle microtubules attach to the chromosome from opposite spindle poles, is critical for chromosome alignment and thus for correct chromosome segregation. In mouse oocytes, chromosome biorientation is error prone; a stable biorientation with correct attachments is established only after multiple rounds of error corrections (Kitajima et al., 2011Kitajima T. Ohsugi M. Ellenberg J. Complete kinetochore tracking reveals error-prone homologous chromosome biorientation in mammalian oocytes.Cell. 2011; 146: 568-581Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). This error-prone chromosome biorientation has been at least partly attributed to the acentrosomal nature of the spindle. Unlike somatic cells, in which centriole-containing centrosomes predefine the two spindle poles and allow biorientation of chromosomes, mammalian oocytes lack centrioles (Bennabi et al., 2016Bennabi I. Terret M.-E. Verlhac M.-H. Meiotic spindle assembly and chromosome segregation in oocytes.J. Cell Biol. 2016; 215: 611-619Crossref PubMed Scopus (116) Google Scholar, Dumont and Desai, 2012Dumont J. Desai A. Acentrosomal spindle assembly and chromosome segregation during oocyte meiosis.Trends Cell Biol. 2012; 22: 241-249Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, Howe and FitzHarris, 2013Howe K. FitzHarris G. Recent insights into spindle function in mammalian oocytes and early embryos.Biol. Reprod. 2013; 89: 71Crossref PubMed Scopus (68) Google Scholar). In mouse oocytes, acentriolar microtubule-organizing centers (MTOCs) initially form a ball-shaped apolar spindle. The MTOCs then relocate to two opposite poles, thus resulting in spindle bipolarization (Breuer et al., 2010Breuer M. Kolano A. Kwon M. Li C.-C. Tsai T.-F. Pellman D. Brunet S. Verlhac M.-H. HURP permits MTOC sorting for robust meiotic spindle bipolarity, similar to extra centrosome clustering in cancer cells.J. Cell Biol. 2010; 191: 1251-1260Crossref PubMed Scopus (81) Google Scholar, Clift and Schuh, 2015Clift D. Schuh M. A three-step MTOC fragmentation mechanism facilitates bipolar spindle assembly in mouse oocytes.Nat. Commun. 2015; 6: 7217Crossref PubMed Scopus (108) Google Scholar, Schuh and Ellenberg, 2007Schuh M. Ellenberg J. Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes.Cell. 2007; 130: 484-498Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). The bipolarized acentrosomal spindle exhibits loosely focused poles with a broad distribution of MTOCs, thereby allowing the microtubules to attach to chromosomes from various directions, thus providing an explanation for the error-prone chromosome biorientation (Breuer et al., 2010Breuer M. Kolano A. Kwon M. Li C.-C. Tsai T.-F. Pellman D. Brunet S. Verlhac M.-H. HURP permits MTOC sorting for robust meiotic spindle bipolarity, similar to extra centrosome clustering in cancer cells.J. Cell Biol. 2010; 191: 1251-1260Crossref PubMed Scopus (81) Google Scholar, Kitajima et al., 2011Kitajima T. Ohsugi M. Ellenberg J. Complete kinetochore tracking reveals error-prone homologous chromosome biorientation in mammalian oocytes.Cell. 2011; 146: 568-581Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, Schuh and Ellenberg, 2007Schuh M. Ellenberg J. Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes.Cell. 2007; 130: 484-498Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). The spindle with the loosely focused poles in oocytes resembles the somatic spindles with overamplified or fragmented centrosomes observed before the chromosome segregation errors in cancer cells (Breuer et al., 2010Breuer M. Kolano A. Kwon M. Li C.-C. Tsai T.-F. Pellman D. Brunet S. Verlhac M.-H. HURP permits MTOC sorting for robust meiotic spindle bipolarity, similar to extra centrosome clustering in cancer cells.J. Cell Biol. 2010; 191: 1251-1260Crossref PubMed Scopus (81) Google Scholar, Ganem et al., 2009Ganem N. Godinho S. Pellman D. A mechanism linking extra centrosomes to chromosomal instability.Nature. 2009; 460: 278-282Crossref PubMed Scopus (1012) Google Scholar, Silkworth et al., 2009Silkworth W.T. Nardi I.K. Scholl L.M. Cimini D. Multipolar spindle pole coalescence is a major source of kinetochore mis-attachment and chromosome mis-segregation in cancer cells.PLoS One. 2009; 4: e6564Crossref PubMed Scopus (318) Google Scholar). These observations contrast with those of mouse embryos at the 8-cell and 16-cell stages, in which the acentrosomal spindles exhibit tightly focused poles (Courtois et al., 2012Courtois A. Schuh M. Ellenberg J. Hiiragi T. The transition from meiotic to mitotic spindle assembly is gradual during early mammalian development.J. Cell Biol. 2012; 198: 357-370Crossref PubMed Scopus (147) Google Scholar). Thus, the absence of centrosomes alone does not account for the loosely focused spindle poles during meiosis in oocytes. However, the oocyte-specific features that facilitate the loosening of the acentrosomal spindle poles remain unknown. The spindle checkpoint, a mechanism that prevents chromosome segregation errors by delaying anaphase onset when kinetochores are not attached to the microtubules (London and Biggins, 2014London N. Biggins S. Signalling dynamics in the spindle checkpoint response.Nat. Rev. Mol. Cell Biol. 2014; 15: 736-747Crossref PubMed Scopus (212) Google Scholar, Musacchio, 2015Musacchio A. The molecular biology of spindle assembly checkpoint signaling dynamics.Curr. Biol. 2015; 25: R1002-R1018Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar), exhibits several unique features in oocytes (Gui and Homer, 2012Gui L. Homer H. Spindle assembly checkpoint signalling is uncoupled from chromosomal position in mouse oocytes.Development. 2012; 139: 1941-1946Crossref PubMed Scopus (105) Google Scholar, Kolano et al., 2012Kolano A. Brunet S. Silk A. Cleveland D. Verlhac M.-H. Error-prone mammalian female meiosis from silencing the spindle assembly checkpoint without normal interkinetochore tension.Proc. Natl. Acad. Sci. USA. 2012; 109: E1858-E1867Crossref PubMed Scopus (113) Google Scholar, Lane and Jones, 2014Lane S. Jones K. Non-canonical function of spindle assembly checkpoint proteins after APC activation reduces aneuploidy in mouse oocytes.Nat. Commun. 2014; 5: 3444Crossref PubMed Scopus (39) Google Scholar, Lane et al., 2012Lane S.I. Yun Y. Jones K.T. Timing of anaphase-promoting complex activation in mouse oocytes is predicted by microtubule-kinetochore attachment but not by bivalent alignment or tension.Development. 2012; 139: 1947-1955Crossref PubMed Scopus (108) Google Scholar, Nagaoka et al., 2011Nagaoka S. Hodges C. Albertini D. Hunt P. Oocyte-specific differences in cell-cycle control create an innate susceptibility to meiotic errors.Curr. Biol. 2011; 21: 651-657Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, Sebestova et al., 2014Sebestova J. Danylevska A. Dobrucka L. Kubelka M. Anger M. Lack of response to unaligned chromosomes in mammalian female gametes.Cell Cycle. 2014; 11: 3011-3018Crossref Scopus (71) Google Scholar). The low stringency of the spindle checkpoint is one factor that contributes to the error-proneness of oocytes. Whereas one unattached kinetochore is sufficient to induce a checkpoint-dependent anaphase delay in normal somatic cells (Rieder et al., 1995Rieder C.L. Cole R.W. Khodjakov A. Sluder G. The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores.J. Cell Biol. 1995; 130: 941-948Crossref PubMed Scopus (580) Google Scholar), in oocytes this delay is triggered only when multiple kinetochores are unattached (Gui and Homer, 2012Gui L. Homer H. Spindle assembly checkpoint signalling is uncoupled from chromosomal position in mouse oocytes.Development. 2012; 139: 1941-1946Crossref PubMed Scopus (105) Google Scholar, Kolano et al., 2012Kolano A. Brunet S. Silk A. Cleveland D. Verlhac M.-H. Error-prone mammalian female meiosis from silencing the spindle assembly checkpoint without normal interkinetochore tension.Proc. Natl. Acad. Sci. USA. 2012; 109: E1858-E1867Crossref PubMed Scopus (113) Google Scholar, Lane et al., 2012Lane S.I. Yun Y. Jones K.T. Timing of anaphase-promoting complex activation in mouse oocytes is predicted by microtubule-kinetochore attachment but not by bivalent alignment or tension.Development. 2012; 139: 1947-1955Crossref PubMed Scopus (108) Google Scholar, Nagaoka et al., 2011Nagaoka S. Hodges C. Albertini D. Hunt P. Oocyte-specific differences in cell-cycle control create an innate susceptibility to meiotic errors.Curr. Biol. 2011; 21: 651-657Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, Sebestova et al., 2014Sebestova J. Danylevska A. Dobrucka L. Kubelka M. Anger M. Lack of response to unaligned chromosomes in mammalian female gametes.Cell Cycle. 2014; 11: 3011-3018Crossref Scopus (71) Google Scholar). This low stringency of the spindle checkpoint may be attributable to the large cytoplasmic volume (Gorbsky, 2015Gorbsky G.J. The spindle checkpoint and chromosome segregation in meiosis.FEBS J. 2015; 282: 2471-2487Crossref PubMed Scopus (60) Google Scholar, Jones and Lane, 2013Jones K.T. Lane S.I. Molecular causes of aneuploidy in mammalian eggs.Development. 2013; 140: 3719-3730Crossref PubMed Scopus (147) Google Scholar, Verlhac and Terret, 2016Verlhac M.-H. Terret M.-E. Oocyte maturation and development.F1000Res. 2016; 5https://doi.org/10.12688/f1000research.7892.1Crossref PubMed Scopus (27) Google Scholar), which is one of the most striking features of the oocyte (∼80 μm in diameter in mice and ∼120 μm in humans). The prevailing view is that a larger cytoplasmic volume dilutes the active checkpoint signal that is generated on kinetochores, thus potentially weakening the checkpoint response (Galli and Morgan, 2016Galli M. Morgan D. Cell size determines the strength of the spindle assembly checkpoint during embryonic development.Dev. Cell. 2016; 36: 344-352Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). This hypothesis is consistent with the observation that no spindle checkpoint response is detectable in Xenopus oocytes, which have a much larger cytoplasmic volume than do mouse oocytes (Shao et al., 2013Shao H. Li R. Ma C. Chen E. Liu J. Xenopus oocyte meiosis lacks spindle assembly checkpoint control.J. Cell Biol. 2013; 201: 191-200Crossref PubMed Scopus (29) Google Scholar). Moreover, when mouse oocytes are bisected, a single chromosome is sufficient to support the checkpoint-dependent anaphase delay (Hoffmann et al., 2011Hoffmann S. Maro B. Kubiak J.Z. Polanski Z. A single bivalent efficiently inhibits cyclin B1 degradation and polar body extrusion in mouse oocytes indicating robust SAC during female meiosis I.PLoS One. 2011; 6: e27143Crossref PubMed Scopus (26) Google Scholar). One recent study has shown that in Caenorhabditis elegans embryos, the strength of the spindle checkpoint activated on all kinetochores through the microtubule disruption by nocodazole depends on the kinetochore-to-cytoplasmic ratio (Galli and Morgan, 2016Galli M. Morgan D. Cell size determines the strength of the spindle assembly checkpoint during embryonic development.Dev. Cell. 2016; 36: 344-352Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). However, the influence of cytoplasmic volume on the checkpoint response in unperturbed oocytes remains unknown (Gerhold et al., 2016Gerhold A. Labbé J. Maddox P. Bigger isn’t always better: cell size and the spindle assembly checkpoint.Dev. Cell. 2016; 36: 244-246Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). In unperturbed somatic cells, in which on-kinetochore checkpoint activation is minimal, anaphase inhibitors that are preformed in the nucleus before nuclear envelope breakdown (NEBD) are rate limiting for the checkpoint-mediated anaphase delay (Rodriguez-Bravo et al., 2014Rodriguez-Bravo V. Maciejowski J. Corona J. Buch H. Collin P. Kanemaki M. Shah J. Jallepalli P. Nuclear pores protect genome integrity by assembling a premitotic and Mad1-dependent anaphase inhibitor.Cell. 2014; 156: 1017-1031Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The contribution, if any, of the preformed anaphase inhibitors to the stringency of the spindle checkpoint in oocytes remains unexplored. In the present study, we show that the uniquely large cytoplasmic volume of oocytes affects the functionality of the spindle through at least two distinct mechanisms. First, the large cytoplasmic volume allows acentrosomal spindle poles to form with a broad MTOC distribution, which is prone to anisotropic deformation and contributes to the error-prone chromosome biorientation. Second, the large cytoplasmic volume dilutes the nuclear factors, including anaphase inhibitors, thus resulting in the failure of the spindle to induce a checkpoint arrest in response to a small number of misaligned chromosomes. These results demonstrate that the large cytoplasmic size is linked to the error-prone nature of oocytes. To investigate the role of the large cytoplasmic volume in meiosis I, we generated mouse oocytes carrying half the normal cytoplasmic volume (halved oocytes) and oocytes carrying twice the normal cytoplasmic volume (doubled oocytes) (Figure 1A). The “halved” oocytes were generated by removing half of the total cytoplasmic volume from an oocyte at the germinal vesicle (GV) stage (prophase of meiosis I) by using a micropipette (Figure S1A and Movie S1). The “doubled” oocytes were generated by the electrofusion of an intact GV oocyte with an enucleated oocytes (Figure S1B and Movie S2). The size measurements of the generated oocytes confirmed the high precision of this technique (Figures S1C and S1D). The induction of meiotic resumption in the halved and doubled oocytes resulted in normal frequencies of entry to and completion of meiosis I (Figure S1E). Quantitative 3D analysis of the spindle and chromosome dynamics, on the basis of EGFP-MAP4 and H2B-mCherry labels, respectively (Kitajima et al., 2011Kitajima T. Ohsugi M. Ellenberg J. Complete kinetochore tracking reveals error-prone homologous chromosome biorientation in mammalian oocytes.Cell. 2011; 146: 568-581Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, Schuh and Ellenberg, 2007Schuh M. Ellenberg J. Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes.Cell. 2007; 130: 484-498Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar), by high-resolution live imaging revealed two major effects of the modified cytoplasmic sizes on meiosis I (Figure 1B and Movie S3). First, the spindle volume was decreased by approximately half in the halved oocytes throughout meiosis I, whereas the spindle volume increased by approximately 2-fold in the doubled oocytes (Figures 1B and 1C). Nevertheless the spindle shape was preserved, because both the length and width of the spindle were similarly affected (Figure S2A). We observed no significant differences in the timing of the establishment of the bipolar spindle shape (Figure S2B) or the density of the microtubules that were labeled with EGFP-MAP4 (Figure S2C). Second, the onset of anaphase was significantly delayed in the halved oocytes and accelerated in the doubled oocytes (Figures 1B and 1D). These results indicate that the cytoplasmic volume affects the scale of the spindle and the timing of anaphase onset. We considered three possibilities to explain how changes in cytoplasmic size before NEBD affected the spindle scale after NEBD. The pre-NEBD cytoplasm might provide finite amounts of spindle components, thus scaling the spindle through a limiting component mechanism (Levy and Heald, 2012Levy D. Heald R. Mechanisms of intracellular scaling.Annu. Rev. Cell Dev. Biol. 2012; 28: 113-135Crossref PubMed Scopus (97) Google Scholar, Marshall, 2015Marshall W. Subcellular size.Cold Spring Harb. Perspect. Biol. 2015; 7: a019059Crossref Scopus (19) Google Scholar), as observed in in vitro experiments using encapsulated Xenopus egg extracts (Good et al., 2013Good M. Vahey M. Skandarajah A. Fletcher D. Heald R. Cytoplasmic volume modulates spindle size during embryogenesis.Science. 2013; 342: 856-860Crossref PubMed Scopus (183) Google Scholar, Hazel et al., 2013Hazel J. Krutkramelis K. Mooney P. Tomschik M. Gerow K. Oakey J. Gatlin J.C. Changes in cytoplasmic volume are sufficient to drive spindle scaling.Science. 2013; 342: 853-856Crossref PubMed Scopus (141) Google Scholar). Alternatively, factors liberated from the nucleus through NEBD might negatively control the spindle size in a concentration-dependent manner, in agreement with the hypothesis that the nuclear-to-cytoplasmic ratio is critical for the spindle size (Cui et al., 2005Cui L.-B. Huang X.-Y. Sun F.-Z. Nucleocytoplasmic ratio of fully grown germinal vesicle oocytes is essential for mouse meiotic chromosome segregation and alignment, spindle shape and early embryonic development.Hum. Reprod. 2005; 20: 2946-2953Crossref PubMed Scopus (11) Google Scholar, Novakova et al., 2016Novakova L. Kovacovicova K. Dang-Nguyen T. Sodek M. Skultety M. Anger M. A Balance between nuclear and cytoplasmic volumes controls spindle length.PLoS One. 2016; 11: e0149535Crossref Scopus (16) Google Scholar). Finally, a cell cortex mechanism that detects the distance to the spindle might control the spindle size, as has been observed in C. elegans embryo during anaphase (Hara and Kimura, 2009Hara Y. Kimura A. Cell-size-dependent spindle elongation in the Caenorhabditis elegans early embryo.Curr. Biol. 2009; 19: 1549-1554Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). We designed two experiments to determine which of these putative mechanisms is responsible for the proportional changes in the spindle size. First, we induced meiotic resumption in the presence of nocodazole, which prevents microtubule polymerization, and then removed half the cytoplasmic volume from oocytes <40 min after NEBD (“halved after NEBD”). These oocytes, which carried half the amount of cytoplasm-derived factors and the same concentration of nucleus-derived factors as those in control oocytes, were washed and monitored for spindle formation (Figure 2A, Halved after NEBD). In the oocytes halved after NEBD, the spindle size was significantly smaller than that in the controls but was not significantly different from that in oocytes that were halved before NEBD (Figure 2B). Thus, the concentration of the nucleus-derived factors did not significantly influence the spindle scaling. Second, we generated dumbbell-shaped doubled oocytes by interrupting cell rounding after the cell fusion by using the actin stabilizer jasplakinolide (Figure 2C). The spindle, which formed on one side of the dumbbell in the doubled oocytes, was significantly larger than that in the control oocytes but was not significantly different from that in the round doubled oocytes (Figure 2D). This finding suggested that the geometry of the cell cortex does not significantly influence the spindle scaling. Moreover, by exclusion, these results suggested that the amount of cytoplasm-derived factors plays a major role in the spindle scaling observed in our system, in agreement with the spindle scaling through a limiting component mechanism. We then addressed the integrity of the spindles scaled with cytoplasmic size. Live imaging of MTOCs with mNeonGreen-Cep192 confirmed that the MTOCs localized around the spindle poles from late prometaphase to metaphase (Figure 3A [“Side view”] and Movie S4) (Clift and Schuh, 2015Clift D. Schuh M. A three-step MTOC fragmentation mechanism facilitates bipolar spindle assembly in mouse oocytes.Nat. Commun. 2015; 6: 7217Crossref PubMed Scopus (108) Google Scholar). To more thoroughly investigate the local distribution of the MTOCs around the pole, we visualized the polar MTOCs along the spindle axis after a 3D reconstruction and surface rendering (Figure 3A, “Top view”). This view revealed a stochastic distribution of the MTOCs around the pole at the late prometaphase in the control oocytes (Figure 3A, “Top view”). Notably, this MTOC distribution was extended unilaterally and was perpendicular to the spindle axis by late metaphase (Figure 3A, “Top view”). For the quantitative visualization, the top-view images of the polar MTOCs from more than 20 oocytes were aligned and summed to generate an averaged image for each time point (Figure 3B). The averaged top-view images showed that the MTOCs were distributed stochastically around the spindle pole by late prometaphase. By late metaphase, the MTOCs showed an anisotropic, ellipse-like distribution (Figures 3C, S2D, and S2E). This finding suggested that the acentrosomal spindle poles composed of MTOCs were prone to anisotropic deformation, even in the control oocytes. In the doubled oocytes, the distribution of the polar MTOCs collapsed into a thread-like arrangement by late metaphase, thus resulting in an extremely broad and anisotropic spindle pole (Figures 3A–3C, S2D, and S2E). In contrast, the polar MTOCs in the halved oocytes maintained an isotropic ring-like arrangement, which was associated with focused spindle poles (Figures 3A–3C, S2D, and S2E). We confirmed these observations in late metaphase by immunostaining (Figure S2F). These results indicated that cytoplasmic size affects the proneness to the anisotropic deformation of the MTOC distribution at the acentrosomal spindle poles (Figure 3A, Diagram). The observed alterations in the MTOC distribution at the spindle poles suggested an altered spindle functionality. To examine the ability of the spindle to align chromosomes, we determined the fraction of aligned chromosomes on the basis of the spindle equator-chromosome distance, which was normalized to the spindle length in the 3D-reconstructed images of the oocytes expressing mNeonGreen-Cep192 and H2B-mCherry (Figure 4A and Movie S4). This analysis revealed that in the doubled oocytes, the chromosome alignment was significantly delayed compared with that in the control oocytes, whereas it was accelerated in the halved oocytes (Figure 4B). We confirmed these observations at early metaphase by immunostaining (Figure S3A). In agreement with these observations, the overall stability of the kinetochore-microtubule attachments was decreased in the doubled oocytes, whereas it was increased in the halved oocytes (Figures S3B and S3C). Moreover, during metaphase II, the aneuploidy was more frequent in the doubled oocytes compared with that in the control oocytes (Figures S3D and S3E), thus suggesting that chromosomes were missegregated during meiosis I. These cytoplasmic size-dependent effects were likely to be due to the altered functionality of the spindle rather than to the kinetochores, because the amounts of the kinetochore components (Hec1, CENP-C, and CENP-E) and the level of the regulatory phosphorylation (Aurora B/C-dependent phosphorylation of Hec1 at Ser55 and MCAK at Ser92) (Andrews et al., 2004Andrews P. Ovechkina Y. Morrice N. Wagenbach M. Duncan K. Wordeman L. Swedlow J. Aurora B regulates MCAK at the mitotic centromere.Dev. Cell. 2004; 6: 253-268Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, Cheeseman et al., 2006Cheeseman I. Chappie J. Wilson-Kubalek E. Desai A. The conserved KMN network constitutes the core microtubule-binding site of the kinetochore.Cell. 2006; 127: 983-997Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar, DeLuca et al., 2006DeLuca J. Gall W. Ciferri C. Cimini D. Musacchio A. Salmon E.D. Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.Cell. 2006; 127: 969-982Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, Yoshida et al., 2015Yoshida S. Kaido M. Kitajima T. Inherent instability of correct kinetochore-microtubule attachments during meiosis I in oocytes.Dev. Cell. 2015; 33: 589-602Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) were largely unaffected by cytoplasmic size (Figures S4A–S4F). These results suggest that cytoplasmic size affects the capacity of the spindle to efficiently align chromosomes at the spindle equator. To investigate the mechanism underlying the cytoplasmic size-dependent delay in chromosome alignment, we monitored chromosome biorientation by 3D tracking of the kinetochores (Figure 5A and Movie S5) (Kitajima et al., 2011Kitajima T. Ohsugi M. Ellenberg J. Complete kinetochore tracking reveals error-prone homologous chromosome biorientation in mammalian oocytes.Cell. 2011; 146: 568-581Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, Sakakibara et al., 2015Sakakibara Y. Hashimoto S. Nakaoka Y. Kouznetsova A. Höög C. Kitajima T. Bivalent separation into univalents precedes age-related meiosis I errors in oocytes.Nat. Commun. 2015; 6: 7550Crossref PubMed Scopus (83) Google Scholar). From 2.5 to 5.0 hr after NEBD, the majority of the chromosomes established a stable biorientation at the spindle equator (Figures 5A and 5B) as previously reported (Kitajim" @default.
• W2613590953 created "2017-05-19" @default.
• W2613590953 creator A5025126045 @default.
• W2613590953 creator A5091683043 @default.
• W2613590953 date "2017-05-01" @default.
• W2613590953 modified "2023-10-14" @default.
• W2613590953 title "Large Cytoplasm Is Linked to the Error-Prone Nature of Oocytes" @default.
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• W2613590953 doi "https://doi.org/10.1016/j.devcel.2017.04.009" @default.
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