Astrin regulates meiotic spindle organization, spindle pole tethering ...

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Jun 25, 2009 - the formation of multipolar and highly disordered spindles.9,10. Phosphorylation of Astrin by phosphorylated by p34cdc2 kinase has been ...
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Cell Cycle 8:20, 3384-3395; October 15, 2009; © 2009 Landes Bioscience

Astrin regulates meiotic spindle organization, spindle pole tethering and cell cycle progression in mouse oocytes Ju Yuan,1,2 Mo Li,1,2 Liang Wei,1,2 Shen Yin,1,2 Bo Xiong,1,2 Sen Li,1,2 Sheng-Li Lin,1,2 Heide Schatten3 and Qing-Yuan Sun1,* State Key Laboratory of Reproductive Biology; Institute of Zoology; and 2Graduate School; Chinese Academy of Sciences; Beijing, China; 3Department of Veterinary Pathobiology; University of Missouri-Columbia; Columbia, MO USA

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Keywords: Astrin, meiotic maturation, spindle assembly, dominant negative, site-directed mutation

Astrin has been described as a microtubule and kinetochore protein required for the maintenance of sister chromatid cohesion and centrosome integrity in human mitosis. However, its role in mammalian oocyte meiosis is unclear. In this study, we find that Astrin is mainly associated with the meiotic spindle microtubules and concentrated on spindle poles at metaphase I and metaphase II stages. Taxol treatment and immunoprecipitation show that Astrin may interact with the centrosomal proteins Aurora-A or Plk1 to regulate microtubule organization and spindle pole integrity. Loss-of-function of Astrin by RNAi and overexpression of the coiled-coil domain results in spindle disorganization, chromosome misalignment and meiosis progression arrest. Thr24, Ser66 or Ser447 may be the potential phosphorylation sites of Astrin by Plk1, as site-directed mutation of these sites causes oocyte meiotic arrest at metaphase I with highly disordered spindles and disorganized chromosomes, although mutant Astrin localizes to the spindle apparatus. Taken together, these data strongly suggest that Astrin is critical for meiotic spindle assembly and maturation in mouse oocytes.

Introduction The spindle apparatus, mainly composed of chromosomes, microtubules and centrosomes, is one of the most essential cellular structures. The number and stability of microtubules nucleated from centrosomes change throughout the cell cycle, which is correlated with the assembly of the mitotic spindle.1,2 Spindle assembly involves coordinated activities of multiple proteins resulting in localized microtubule nucleation, dynamics and organization.3 These activities play critical roles in meiotic maturation which involves meiosis resumption of prophase I-arrested oocytes, completion of the first meiotic division and thereafter metaphase II (MII) arrest.4 Mouse oocytes lack centrioles but contain multiple microtubule-organizing centers (MTOCs)5,6 that participate in the assembly of a functional spindle which is critical for accurate chromosome segregation. Mammalian oocytes undergo two successive divisions (meiosis I and II) to form haploid gametes. During these divisions, meiotic spindles must ensure successively the segregation of homologous chromosomes and sister chromatids. Thus the precise regulation of the spindle apparatus, a bipolar array of highly dynamic microtubules (MTs), is indispensable for accurate chromosome segregation and genome stability. Human Astrin has been newly identified as a spindle associated non-motor protein involved in mitotic progression.7,8

Structurally, Astrin contains N-terminal globular domain and two predicted coiled-coil domains.9 During mitosis, Astrin also associates with microtubules, spindle poles and kinetochores but only at those chromosomes that have congressed.8,10 Silencing of Astrin in HeLa cells resulted in growth arrest and in the formation of multipolar and highly disordered spindles.9,10 Phosphorylation of Astrin by phosphorylated by p34cdc2 kinase has been determined in vitro,7 to serve as a substrate for GSK3β and it is specifically phosphorylated at Thr-111, Thr-937 (S/TP motif ) and Ser-974/Thr-978 (S/T-XXX-S/T motif ), suggesting that its association with spindle microtubules may be regulated by phosphorylation.11 Astrin also interacts with Aurora-A and regulates its localization in mitotic spindles.10 All of the previous studies on Astrin had mainly been focused on human mitotic cells, but its role in mammalian meiosis is not known. Here, we tested the roles of Astrin in mouse oocyte meiotic maturation. The results imply that Astrin is a meiotic spindle-associated protein and interacts with centrosome-related proteins Aurora-A or Plk1 to regulate microtubule organization and spindle pole integrity. Silencing of Astrin by RNA interference and microinjected coiled-coil domain mutants both results in the formation of highly disordered spindles with a low rate of polar body extrusion, and chromosomes failing to congress at the spindle equator while remaining dispersed.

*Correspondence to: Qing-Yuan Sun; Email: [email protected]; [email protected] Submitted: 06/25/09; Revised: 08/03/09; Accepted: 08/24/09 Previously published online: www.landesbioscience.com/journals/cc/article/9885 3384

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Results Astrin localizes to spindle microtubules and spindle poles in mouse meiotic oocytes. To investigate the subcellular localization of Astrin during meiotic maturation, mouse oocytes were processed for immunofluorescent staining at different stages of maturation. As shown in Figure 1A, no evident signal of Astrin was detected at the GV stage. Shortly after GVBD, Astrin and microtubules began to accumulate at the vicinity of the condensed chromosomes. By prometaphase I, chromosomes began to migrate to the equator of the spindle and Astrin was gradually translocated to the spindle. When oocytes progressed to metaphase I, chromosomes aligned at the equatorial plate, and Astrin distributed at the spindle and became more concentrated on spindles poles. At telophase I, Astrin was localized in the region around the separating homologous chromosomes. At metaphase II, Astrin was again translocated to microtubules and spindle poles. To examine the expression level of Astrin in mouse oocytes at different stages of meiotic maturation, 150 oocytes were collected at 0, 2, 4, 8 and 12 h of culture corresponding to GV, germinal vesicle breakdown (GVBD), prometaphase I, metaphase I and metaphase II stages, respectively. Immunoblotting results showed that the expression level of Astrin was similar at all stages (Fig. 1B). To further define Astrin’s localization, γ-tubulin was used to determine centrosome position. During metaphase I and metaphase II, Astrin is diffusely localized to microtubules, mainly at the poles, overlapping with but distinct from the areas stained with antibodies against γ-tubulin (Fig. 1C). To further ensure the reliability of the antibody and physiological Astrin localization, Myc-Astrin mRNA was injected and expressed at microtubules and spindle poles (Fig. 1D). Because localization of Astrin in mouse oocyte meiosis is different from that reported for human mitotic cells,10 we also determined Astrin localization in mouse mitotic cells. Mouse NIH/3T3 cells grown on coverslips were fixed and simultaneously immunostained with antibodies against Astrin and α-tubulin. The result showed that Astrin was present in mitotic spindle microtubules during metaphase, similar to meiosis (Fig. 1E). The dual localization of Astrin to both spindles and centrosomes indicates that it may be required for microtubule organization and spindle pole tethering. Localization of Astrin in mouse oocytes treated with taxol and nocodazole. To further clarify the correlation between Astrin and microtubule dynamics, spindle-perturbing drugs were employed. First, we used taxol, a microtubule-stabilizing drug, to treat the oocytes. After oocyte culture for 16 h, corresponding to metaphase II stages, the microtubule fibers in taxol-treated oocytes were excessively polymerized, leading to significantly enlarged spindles and numerous asters in the cytoplasm. In this case, Astrin signals were detected at the microtubule-organizing centers (MTOCs) of the abnormal spindles and cytoplasmic asters which differs from its normal localization (Fig. 2A). Next, metaphase II oocytes were treated with low concentration nocodazole (0.04 μg/ ml), a microtubule-depolymerizing drug. After treatment with this concentration, microtubules were partly disassembled and

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abnormal; the localization of Astrin was altered and correlated to partly disassembled spindle microtubules (Fig. 2B). In order to determine whether localization of Astrin depends on spindle microtubules, we next applied high concentration nocodazole (20 μg/ml) to treat oocytes for 10 min; we then washed thoroughly to allow microtubule re-assembly. The results clearly showed that the localization of Astrin was consistently dependent on spindle microtubules (Fig. 2C). Astrin is complexed with Aurora-A and Plk1 during spindle formation. Since Astrin is localized to microtubules, there may be specific interacting proteins required to associate Astrin with spindles. Therefore, we further investigated the interaction of Astrin with two kinases, Aurora-A and Plk1, that we previously have shown to play a role in meiotic spindle assembly.12-15 At metaphase I and metaphase II stages, as shown in Figure 3A and B, Astrin distribution was correlated with but distinct from the areas stained with antibodies against Aurora-A and Plk1. Aurora-A was localized to microtubules and spindle poles (Fig. 3A) and Plk1 was localized to spindle poles and kinetochores (Fig. 3B), while Astrin was diffusely localized to microtubules and mainly localized to spindle poles. Myc-Astrin was also expressed and double stained with Aurora-A and Plk1, revealing the same distribution patterns as the endogenous proteins (Fig. 3C). To better understand functional relationships of Astrin, Aurora-A and Plk1, co-immunoprecipitation was performed with Aurora-A, Plk1 and control IgG antibodies with about 1000 MII stage oocyte extracts. Astrin was detected by immunoblot analysis using anti-Astrin antibody. The results showed that Astrin was detected in the immunoprecipitates and appeared at the same positions corresponding to the mouse extract lane but not with anti-IgG antibody (Fig. 3D). Taken together, the data presented above imply that Astrin is complexed with Aurora-A and Plk1 to function together during the spindle formation. Astrin depletion causes abnormal spindles, chromosome mis-alignment and impairs meiosis progression. To investigate the function of Astrin during oocyte maturation, siRNAs were used to effectively deplete the target proteins. The efficiency of Astrin siRNAs was measured by both real-time quantitative PCR and western blot. The results indicated that the amount of Astrin mRNA was significantly reduced (23.48 ± 13.1% of Astrin-1 siRNA and 23 ± 1.4% Astrin-2 siRNA compared to control siRNA, p < 0.05; Fig. 4A). Based on these results, we choose Astrin-2 siRNA for Astrin depletion in subsequent experiments. As shown in Figure 4B, compared to the control group, the protein expression of Astrin was strikingly reduced (33.3 ± 6.3%, p < 0.05). SiRNA microinjected oocytes were cultured for 16 h after release from the inhibitory environment and then analyzed after staining. When siAstrin-injected oocytes were stained with antibodies against α-tubulin and Astrin, the Astrin protein expression levels were successfully downregulated by RNAi (Fig. 4C). The polar body extrusion rate was 36.78 ± 4.22% (n = 138) in the siAstrin-injected oocytes, which is significantly lower than that in the siControl-injected oocytes (81.28 ± 2.88% n = 92) (p < 0.05; Fig. 4D). Compared to control MI oocytes which displayed normal looking spindles, almost all (87.63%, n = 97) of the siAstrin-injected oocytes displayed abnormal spindles such

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Figure 1. Expression and subcellular localization of Astrin in meiotic mouse oocytes and mitotic mouse cells. (A) Oocytes at various stages were double stained with antibodies against Astrin (red) and α-tubulin (green); each sample was counterstained with Hoechst 33258 to visualize DNA (blue). GV, oocytes at germinal vesicle stage; PreMI, oocytes at first prometaphase; MI, oocytes at first metaphase; TI, oocytes at first telophase; MII, oocytes at second metaphase. Bar 20 μm. (B) Samples were collected after oocytes had been cultured for 0, 2, 4, 8 and 12 h, corresponding to GV, GVBD, prometaphase I, metaphase I and metaphase II stages, respectively. Proteins from a total of 150 oocytes were loaded for each sample. The molecular mass of Astrin is 133 kDa, and the molecular mass for β-actin is 42 kDa. (C) Oocytes cultured for 8 h (MI) and 16 h (MII) were fixed and stained for γ-tubulin (red), Astrin (green) and DNA (blue) as visualized with Hoechst 33258 staining. Bar 20 μm. (D) Myc-Astrin and Myc control mRNA injected oocytes grown for 16 h were fixed and stained with antibodies against α-tubulin (green) and MYC (red); DNA (blue) was visualized with Hoechst 33258 staining. Bar 20 μm. (E) Mouse NIH/3T3 cells grown on coverslips were immunostained with antibodies against Astrin (red), α-tubulin (green) and DNA (blue). Each sample was counterstained with Hoechst 33258 to visualize DNA. Bar 20 μm.

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the siAstrin injected oocytes that failed to extrude polar bodies were arrested at the metaphase I stage. To further examine spindle shape and interactions with Aurora-A and Plk1, siAstrin-injected oocytes were stained with antibodies against α-tubulin, Aurora-A and Plk1; we found that depletion of Astrin displaced Aurora-A from the spindle poles and it became localized at the aberrant spindle arrays in the siAstrin-injected oocytes (Fig. 4G) while Plk1 was localized to the aberrant spindle poles and dispersed chromosomes (Fig. 4H). Taken together, our observations show that siAstrin-injected oocytes display disorganized spindles, aberrant spindle poles, dispersed chromosomes, and maturation arrest, which strongly suggests that Astrin is essential for spindle integrity and meiotic cell cycle progression. The N-terminal domain and C-terminal coiled-coil domain of Astrin have different functions in spindle assembly. The human Astrin sequence contains an N-terminal globular domain and two predicted coiled-coil domains.9 However, the mouse Astrin amino-acid sequence is 66% identical to the human Astrin sequence.16 According to secondary structure predictions, the mouse Astrin sequence has two coiled coil domains (509–856 bp, 937–1146 bp) (Fig. 5A). To map the domain of Astrin required for spindle targeting, we microinjected the N-terminal globular domain (1–500 bp) and C-terminal coiled-coil domains (501–1165 bp) mRNA into mouse GV-stage oocytes and cultured the oocytes for 2 h in Milrinone-supplemented medium to allow protein expression; we then washed them thoroughly to allow meiosis progression for 16 hours. Immunoblot analysis of oocyte extracts confirmed that the protein of the expected size was expressed from each mRNA (Fig. 5B, lane 1, 2, 5). Figure 2. Localization of Astrin in mouse oocytes treated with taxol and nocodazole. To determine whether the truncating Astrin (A) Oocytes at metaphase II stage were incubated in M16 medium containing 10 μM mutations have dominant effects on meiosis protaxol for 45 min and then double stained with antibodies against Astrin (red), α-tubulin gression, the rate of the first polar body extru(green) and DNA (blue). (B) Oocytes at metaphase II stage were incubated in M16 medium containing 0.04 μg/ml nocodazole for 10 min and then double stained with antision was detected. When mRNA microinjected bodies against Astrin (red), α-tubulin (green) and DNA (blue). (C) Oocytes at metaphase oocytes were cultured for 16 h, the first polar body II stage were incubated in M16 medium containing 20 μg/ml nocodazole for 10 min and extrusion rate of Myc-Astrin head mRNA injected then washed thoroughly to recover. At 0 min, 5 min, 10 min, 15 min, 30 min, oocytes oocytes was inhibited in 65.83 ± 0.83% (n = were fixed and double stained with antibodies against Astrin (red), α-tubulin (green), and 110), compared to 85.5 ± 2% in the Myc-control DNA (blue). Bar 20 μm. injected oocytes (n = 103) (p > 0.05). In contrast, the first polar body extrusion rate of Myc-Astrin as non-polar, mono-polar, multi-polar or minute spindles with tail mRNA injected oocytes was inhibited in 51.45 ± 0.56% (n = dispersed chromosomes (Fig. 4E). However, there were 20 biva- 123), compared to 85.5 ± 2% in the myc-control injected oocytes lents in siAstrin-injected oocytes (without polar bodies) cultured (n = 103) (p < 0.05). The results showed that expression of the for 16 h compared to 20 univalents in control-injected oocytes, coiled-coil domain, to some extent, impaired meiosis progression, similar to control-injected oocytes cultured for 6 h after GVBD however, expression of the head domain only slightly influenced (approximately MI stage) (Fig. 4F, n = 60), which suggests that maturation.

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Figure 3. Analysis of Astrin interacting proteins in vivo. (A) Oocytes cultured for 8 h (MI) and 16 h (MII) were fixed and stained forAstrin (green), Aurora-A (red) and DNA (blue). Bar 20 μm. (B) Oocytes cultured for 8 h (MI) and 16 h (MII) were fixed and stained for Astrin (green), Plk1 (red) and DNA (blue). Bar 20 μm. (C) Myc-Astrin injected oocytes grown for 16 h were fixed and stained with antibodies against myc (green) and Aurora-A (red), or PLK1 (red). DNA (blue) was visualized with Hoechst 33258 staining. Bar 20 μm. (D) Astrin was immunoprecipitated from 1000 metaphase II oocyte extracts with Aurora-A monoclonal antibody (lane 1) or with control IgG (lane 2), or with PLK1 monoclonal antibody (lane 3). Lane 4 was loaded with about 10 μl oocyte extract sample. Astrin was immunodetected with a polyclonal antibody against Astrin (diluted 1/500).

To determine whether the truncating Astrin mutations have dominant effects on spindle assembly, mRNA injected oocytes were stained with anti-myc antibody to show its expression and localization. Immunofluorescence analysis of oocytes injected with the N-terminal globular domain showed that Myc staining was distributed throughout the spindle microtubules and spindle poles at metaphase II and did not impact other protein localizations; it was indistinguishable from the endogenous protein (Fig. 5C–F). However, the C-terminal coiled-coil domain could still localize to microtubules, although the spindles were abnormal (Fig. 5D). Moreover, overexpression of the coiled-coil domain caused Aurora-A localization on broadened spindles (Fig.  5E), and Plk1 on multipoles and disorganized chromosomes (Fig. 5F). Taken together, the C-terminal coiled-coil domain of Astrin displayed effects on polar body extrusion, chromosome congregation and spindle formation. The phenotype was similar to that found in Astrin-depleted oocytes. In contrast, oocytes microinjected with the empty vector myc mRNA showed only background staining. Exogenous expression of Astrin site-directed mutant causes abnormal spindles and meiotic arrest. According to the mouse Astrin sequence analysis, the N-terminal head domain (aa1-509) of Astrin is rich in proline (13%) together with serine (20%) and threonine (11%) residues, suggesting that Astrin’s function is

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regulated by phosphorylation, and this hypothesis is supported by previous findings of Chang et al.,7 who showed that Astrin is a substrate of cdk1 in vitro. It was also confirmed that Astrin is highly phosphorylated in mitosis; Plk1 interacts with Astrin via its polo-box binding domain, and the interaction of Astrin and Plk1 is dependent on phosphorylation at Thr111.10 The optimal phosphopeptide binding motif for PBD (polobox domain) docking the amino acid sequence S-[pS/pT]-P/X was identified, which was similar to conserved motifs recognized in human, Xenopus and yeast.17 Nevertheless, replacement of Ser at the pThr-1 position with Val, the amino acid showing the lowest selection in this position, was sufficient to eliminate peptide binding.18 Analysis of the Astrin amino acid sequence revealed three potential PBD docking sites conforming to this consensus motif (ST23/24, SS65/66, SS446/447) (Fig. 6A). So we site-directedly mutated ST23/24 SS65/66 and SS446/447 to AA, and cloned the mutant Astrin sequence into pCS2 + -myc plasmid. Then, we microinjected the Myc-Astrin mutant mRNA into mouse GV-stage oocytes and cultured the oocytes for 2 h in Milrinone-supplemented medium to allow protein expression; we finally washed thoroughly to allow meiosis resumption and progression for 16 hours. Immunoblot analysis of oocyte extracts confirmed that the mutant protein of the expected size was expressed from mRNA (Fig. 5B, lane 4). Myc-

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Figure 4. Astrin depletion results in formation of abnormal spindles and chromosome alignment defects. (A) The efficiency of siRNAs of Astrin. Analysis of relative gene expression was measured by using real-time quantitative PCR. The relative mRNA of Astrin compared with control (100%) is shown by mean ± SE. Different superscripts indicate statistical difference (p < 0.05). (B) 150 oocytes cultured for 16 h injected with siRNA directed against control or Astrin. Extracts were prepared and immunoblots were performed with antibodies against Astrin or β-actin. (C) Control or Astrin-depleted oocytes cultured for 16 h were stained with antibodies against α-tubulin (green) and Astrin (red). DNA (blue) was visualized with Hoechst 33258 staining. Bar 20 μm. (D) Percentages of polar body extrusion after siRNA treatment. Control and Astrin siRNA-injected oocytes were cultured for 16 h. The value expressed by each bar represents the mean ± SD (n = 3). Different superscripts indicate statistical difference (p < 0.05). (E) Spindle morphologies and chromosome alignment in control siRNA-injected oocytes (1) and Astrin siRNA-injected oocytes (2–8). After injection, oocytes were incubated for 16 h, followed by immunostaining with a-tubulin antibody (green) and PI (red). Bar 20 μm. (F) Chromosome spreading was performed of oocytes that had been cultured for 8 h (MI) or 16 h (MII) of maturation after control and Astrin siRNA injection. Representative images of each sample are shown (n = 35). Bar 10 μm. (G and H) Control or Astrin-depleted oocytes cultured for 16 h were stained with antibodies against α-tubulin (green) and Aurora-A (red; G) or PLK1 (red; H), respectively. DNA (blue) was visualized with Hoechst 33258 staining. Bar 20 μm. www.landesbioscience.com

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Figure 5. Coiled-coil domains have dominant effects on spindle assembly. (A) The schematic diagram shows the Astrin fragments. Domain structure shows predicted secondary structure and domain organization of Astrin. (B) Oocytes injected with the indicated Astrin mRNA fragments were cultured for 16 h and then lysed. Immunoblots were performed with antibodies against the myc epitope (myc), Astrin or β-actin. The Astrin fragments were as follows: Myc-Astrin head (lane 1; n = 150), Myc-Astrin Tail (lane 2; n = 176), Myc-Astrin (lane 3; n = 196), Myc-Astrin mutant (lane 4; n = 176), Myc control (lane 5; n = 150). (C) After the indicated Astrin mRNA fragments were injected, the percentage of arrested oocytes (without extruded polar body) or metaphase II (with extruded polar body) oocytes cultured for 16 h were observed for spindle abnormalities. Different superscripts indicate statistical difference (p < 0.05). (D–F) Myc control, Myc-Astrin head and Myc-Astrin Tail mRNA fragments were injected into the GV oocytes and cultured for 16 h. Oocytes were stained with antibodies against α-tubulin (green) and MYC (red; D) or MYC (green) and Aurora-A (red; E) or Plk1 (red; F), respectively. DNA (blue) was visualized with Hoechst 33258 staining. Bar 20 μm.

Astrin full length mRNA was microinjected and used as the control (Fig. 5B, lane 3). To determine whether Astrin mutant has dominant effects on spindle assembly and meiotic maturation progression, Astrin

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mutant and full length mRNA injected oocytes were stained with anti-myc antibody to determine its expression and localization, and double stained with α-tubulin and Plk1 antibody to determine the spindle microtubule structure and centrosome position.

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Figure 6. Site-directed mutation of potential Plk1 phosphorylation sites of Astrin. (A) The diagram shows predicted secondary structure and potential phosphorylation sites of Astrin (wild type) and the mutations of alanine substitution on ST23/24 SS65/66 and SS446/447 phosphorylation sites of mouse Astrin sequence (3A mutant). Numbers represent the positions for amino acid residues of Astrin. (B) After the indicated Astrin mRNA fragments were injected, the percentage of arrested oocytes (without extruded polar body) or metaphase II (with extruded polar body) oocytes were cultured for 16 h and their phenotypes were analyzed. Different superscripts indicate statistical difference (p < 0.05). (C and D) Myc-Astrin and myc-Astrin mutant mRNA fragments were injected into the GV stage oocytes and cultured for 16 h. Oocytes were stained with antibodies against α-tubulin (green) and MYC (red; C) or MYC (green) and Plk1 (red; D) respectively. DNA (blue) was visualized with Hoechst 33258 staining. Bar 20 μm.

When mRNA microinjected oocytes were cultured for 16 h, the polar body extrusion rate of myc-Astrin mutant mRNA injected oocytes was 35.15 ± 3.86% (n = 134), which was significantly lower than that in the mycAstrin full length injected oocytes (82.49 ± 2.77%, n = 123) (p < 0.05). The results showed that the Astrin mutant was able to impair meiosis progression in contrast to the function of full-length Astrin (Fig. 6B). Furthermore, immunofluorescent staining showed that the mutant injected oocytes were still able to form a spindle apparatus, however, most of them arrested at the metaphase I stage with highly disordered spindles and disorganized chromosomes at 16 h of culture. In contrast, oocytes microinjected with the full-length myc-Astrin mRNA displayed normal looking spindles (Fig. 6C and D). Discussion In human mitosis, Astrin was reported to be a microtubule and kinetochore protein.10 However, our immunofluorescence observations show that Astrin localizes to microtubules and spindle poles in mouse mitosis and

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meiosis (Fig. 1). Furthermore, we did not find colocalization of Astrin and Plk1 at kinetochores (Fig. 3B), which indicates that Astrin is unlikely to serve as a kinetochore protein in the mouse, which is different from human mitosis. These results may be related to species differences since homology of the Astrin sequence between mouse and human is 66%.16 As proved by mouse Astrin myc-mRNA injection experiments (Figs. 1D and 3C), our newly developed antibody is reliable. However, there is also another possibility that kinetochore staining may be impaired if the peptide epitope is masked at this site, and the myc-Astrin expression may not be detected at the kinetochores for yet unknown reasons. Further studies are needed to clarify these possibilities. The colocalization or partial colocalization of Astrin with spindle-associated proteins such as α-tubulin and Aurora-A, and with centrosomal proteins such as γ-tubulin and Plk1 further provides support that Astrin participates in spindle organization and spindle pole formation during meiosis in mouse oocytes. Interestingly, in the presence of taxol which stabilizes microtubules and hence increases non-spindle microtubule structures, Astrin signals were found not only at the meiotic spindle poles but also as distinct dots at the centers of the cytoplasmic microtubule asters. The presence of Astrin in these “ectopic” microtubule organizing structures provides further support for its role as a component of microtubule organizing centers. This conclusion is also supported by the results from nocodazole-treated oocytes in which the spindle was significantly deformed or even completely destroyed. Astrin staining was irregularly dispersed around the deformed spindles, but not located at the spindles poles, indicating a correlation between Astrin and microtubule assembly. Furthermore, the Astrin-depleted oocytes display aberrant non-polar, mono-polar or multi-polar spindles, which strongly suggests that Astrin is required for microtubule organization and spindle pole integrity during meiosis. Moreover, Astrin is complexed with Aurora-A19-21 and Plk1,22 as revealed by immunoprecipitation; we infer that Astrin may interact with centrosomal proteins to regulate microtubule organization and spindle pole formation during meiosis in mouse oocytes.10,23 Silencing of Astrin in Hela cells resulted in growth arrest, with formation of multipolar and highly disordered spindles, eventually resulting in apoptotic cell death.9,10 Similarly, our results show that Astrin knockdown and loss-of-function by mutant mRNA injection both significantly reduced first polar body extrusion and the arrested oocytes displayed abnormal spindles, and chromosomes were not aligned properly at the spindle equator (Fig. 4D–F). We hypothesize that during normal meiosis Astrin at the spindle poles aids in the establishment of the bipolar spindle and monitors spindle formation and microtubule tension, which is required for correct chromosome alignment at metaphase and chromosome disjunction at anaphase.24-26 Abnormal levels of Astrin interferes with proper spindle assembly, as a consequence, the spindle assembly checkpoint (SAC) which monitors chromosome attachment to microtubules and microtubule tension,27,28 halts oocytes at the MI stage when unattached chromosomes and abnormal spindle tension are encountered. Similarly, TPX2 has multiple functions during

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mitosis including microtubule nucleation at chromosomes and targeting of Xklp2 and Aurora-A to the spindle;29 it controls acentriolar apindle pole integrity via phosphorylation of TACC3 at MTOCs in mouse oocytes, and overexpression and depletion both results in impaired spindle assembly, formation of multipolar structures and failure to extrude the polar body.30 Overexpression of the Astrin N-terminal globular domain, behaved like the endogenous protein, accumulated at microtubules and spindle poles of meiosis I and meiosis II spindles and it did not perturb meiotic maturation. Strikingly, overexpression of the C-terminal coiled-coil domain of Astrin disturbs meiotic spindle assembly, similar to the Astrin depletion phenotype, however, the coiled-coil domain was still expressed and localized to the abnormal spindles. Based on these results, we suggest that the C-terminal coiled coil domain may be the key domain to fulfill its function. We infer that the localization mechanism of the C-terminal coiled-coil domain on the spindle is different from the N-terminal. The N-terminal domain may be the activation domain which may need to interact with other proteins for activation, whereas the coiled-coil domain binds directly to the proteins located at the spindles to fulfill their functions.31,32 For example, hNinein interacts with Astrin through its coiledcoil domain, which in turn is required for maintenance of centrosome/spindle pole integrity.33 GSK3β interacts with and phosphorylates Astrin by the coiled-coil domain to target Astrin to the spindle microtubules and kinetochores.11 Therefore, the C-terminal coiled-coil domain is able to compete effectively with endogenous Astrin protein for binding to docking proteins, and has a dominant effect on spindle organization; such a mechanism is similar to Astrin depletion. Plk1 and Aurora-A play key roles in centrosome maturation, spindle assembly, and chromosome segregation during cell division,23 and Plk1 controls Aurora-A localization and function by regulating cellular levels of hBora.34 Studies by others and us show that Plk1 is localized to the meiotic spindle, and plays important roles in microtubule organization during oocyte meiotic maturation.13,35 It has been confirmed that Plk1 interacts with Astrin via its polo-box binding domain, and Astrin is highly phosphorylated at Thr111 in mitosis.10 According to the mouse Astrin sequence analysis, we mutated the potential phosphorylated sites (T24, S66, S447) to alanine. The mutant Astrin can also localize to the spindle apparatus, however, most of the oocytes arrested at the metaphase I stage displayed highly disordered spindles and disorganized chromosomes. Taken together, our observations strongly suggest that the three potential phosphorylated sites (T24, S66, S447), perhaps one or two or all, are the phosphorylation-sites responsible for the function of Astrin. Interestingly, we observed that the three potential phosphorylated sites all locate at the N-terminal domain; perhaps the functional mechanisms of the Astrin mutant and coiled-coil domain are the same. In summary, although the characteristics of cell cycle in meiosis are different from those in mitosis, several lines of evidence in our report demonstrate that Astrin, as a spindle-associated protein, exerts pivotal functions in spindle assembly, chromosome alignment and cell cycle progression in meiosis.

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Materials and Methods Antibodies. The rabbit polyclonal antibody directed against the Astrin protein was produced by Bios (Beijing Biosynthesis Biotechnology Co., LTD) using the polypeptide HILEENLRRSD as an antigen. The following antibodies were obtained from commercial sources: mouse anti-α-tubulin-FITC, mouse anti-γ-tubulin, mouse anti-Aurora A, mouse anti-Plk1, rabbit anti-myc (Sigma, St. Louis, MO); mouse anti-myc-FITC (Invitrogen, Carlsbad, CA); rabbit anti-α-tubulin (Cell Signaling); mouse anti-β-actin (Santa Cruz Biotechnology Inc.,). Mouse oocyte collection, culture and drug treatment. Animal care and use were conducted in accordance with the Animal Research Committee guidelines of the Institute of Zoology, Chinese Academy of Sciences. Immature oocytes were collected from ovaries of 6-week-old female ICR mice in M2 medium (Sigma, St. Louis, MO). Only those immature oocytes displaying a germinal vesicle (GV) were cultured further in M16 medium under liquid paraffin oil at 37°C in an atmosphere of 5% CO2 in air. At different times of culture, oocytes were collected for immunostaining, drug treatment, microinjection or co-immunoprecipitation. For experiments in which oocytes were treated with taxol, 5 mM taxol (Sigma) in DMSO stock was diluted in M16 medium to give a final concentration of 10 μM and oocytes were incubated for 45 min; for experiments in which oocytes were treated with nocodazole, 10 mg/ml nocodazole in DMSO stock (Sigma) was diluted in M16 medium to give a final concentration of 0.04 μg/ml or 20 μg/ml and oocytes were incubated for 10 min. After treatment, oocytes were washed thoroughly and used for immunofluorescent staining.36,37 Control oocytes were treated with the same concentration of DMSO in the medium before examination. RNA interference. The GV-intact oocytes were microinjected in M2 medium containing 2.5 μM Milrinone with 5–10 pl of the negative control siRNA (Qiagen) and Astrin siRNAs (Ambion) (Astrin-1 siRNA, CUG UAA AGG UCA AAU CGA Att) or (Astrin-2 siRNA, GCG UGA AUC CGA UCC AAC Utt). The final concentration of the control or Astrin siRNA was 25 μM. Microinjected oocytes were incubated in M16 medium containing 2.5 μM Milrinone for 24 h, and then transferred to Milrinonefree M16 medium to resume meiosis. Oocytes were incubated for 16 h and collected for the subsequent experiments.38 Quantification of RNAi effects in oocytes by RT-PCR. Total RNA was extracted from 100 mouse GV oocytes using RNeasy micro purification kit (Qiagen). Single-stranded cDNAs were generated with cDNA synthesis kit (Takara), using poly (dT) primers according to the manufacturer’s instruction. The resultant cDNAs were used as templates for RT-PCR. cDNA fragment of Astrin and H2afz (H2A histone family, member Z, reference gene)39 was amplified by the following primers. Astrin, 5'-AGG CAC CAA GGA CAG TAC TTC AG-3' (forward) and 5'-CGG GAC AAG GTA GCC AGA TC-3' (reverse) H2afz, 5'-ACA GCG CAG CCA TCC TGG AGT A-3' (forward), 5'-TTC CCG ATC AGC GAT TTG TGG A-3' (reverse). Real-time PCR was

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conducted by using SYBR Premix Ex TaqTM kit (Takara) in ABI prism 7000 Sequence Detection System. The steps include 95°C 10 s, 40 cycles of 95°C 5 s and 60°C 31 s. Analysis of relative gene expression was measured by real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.40 Plasmid construction and synthesis of mRNA in vitro. Total RNA was extracted from 100 mouse GV oocytes using RNeasy micro purification kit (Qiagen), and the first strand cDNA was generated with cDNA synthesis kit (Takara), using poly (dT) primers. The full-length Astrin, N-terminal fragment of Astrin encoding amino acids 1–500 and C-terminal fragments of Astrin encoding amino acids 501–1165 were amplified with Pfu polymerase (Stratagene), cloned into pCS2 + -Myc6 plasmid and then sequenced. The Myc-Astrin, Myc-Astrin1-500 (myc-Astrin head) and Myc-Astrin501-1165 (myc-Astrin Tail) plasmids were linearized by SalI and purified by gel extraction kit (Qiagen). Sp6 mMESSAGE mMACHINE kit (Ambion Inc., Austin, TX, USA) was used for producing capped mRNA which was purified using the RNeasy cleanup kit (Qiagen) and dissolved in nuclease-free water to a final concentration of 2.0–3.0 mg/ml. Microinjected oocytes were incubated in M16 medium containing 2.5 μM Milrinone for 2 h, and then transferred to Milrinone-free M16 medium to resume meiosis. Oocytes were incubated for 16 h and collected for the subsequent experiments.41 Site directed mutagenesis. The mutant forms of Astrin were obtained by PCR amplification using QuikChange Site-directed Mutagenesis kit (Stratagene). The mutations were peformed by the first round of PCR in the presence of PBD1 primers using the pMD18-T-Astrin-Myc6 plasmid as the DNA template. The second PCR was performed with PBD2 primers using the first round of PCR as the DNA template. The third PCR was performed with PBD3 primers using the second round of PCR as the DNA template. PCR products (pMD18-T-myc-Astrin mutant) were cloned into pCS2 + and directly linearized by SalI and transcribed into synthetic RNA sequences in vitro as described above. PBD1-Sense

5'-GGG GAA GCC AGC TAT GGC TGC TCC TCT CCG AGA GCT-3'

PBD1-Antisense

5'-AGC TCT CGG AGA GGA GCA GCC ATA GCT GGC TTC CCC-3'

PBD2-Sense

5'-GGA AAG ATG CAA CAA CGC AGC TCC AGT GGA TTT TAT C-3'

PBD2-Antisense

5'-GAT AAA ATC CAC TGG AGC TGC GTT GTT GCA TCT TTC C -3'

PBD3-Sense

5'-CAG TAG TAC ACA GAC TGA CGC CGC TCC TTG TGG GGT CAC TAA-3'

PBD3-Antisense

5'-TTA GTG ACC CCA CAA GGA GCG GCG TCA GTC TGT GTA CTA CTG-3'

Immunoprecipitation and immunoblotting analysis. Immunoprecipitation was carried out according to the Instructions for ProFound Mammalian Co-Immunoprecipitation Kit (Pierce, Rockford, IL). The proteins were separated by SDSPAGE and then electrically transferred to polyvinylidene fluoride membranes. Following transfer, the membranes were blocked in

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TBST containing 5% skimmed milk for 2 h, followed by incubation overnight at 4°C with Astrin (rabbit 1:500), Aurora-A (mouse 1:500), Plk1 (mouse 1:500) and β-actin (mouse 1:1,000) antibodies, respectively. After washing three times in TBST, 10 min each, the membranes were incubated for 1 h at 37°C with 1:1,000 horseradish peroxidase-conjugated goat anti-rabbit IgG and horseradish peroxidase-conjugated goat anti-mouse IgG, respectively. Finally, the membranes were processed using the enhanced chemiluminescence detection system (Amersham, Piscataway, NJ). Immunofluorescence and confocal microscopy. For double staining of proteins, oocytes and NIH/3T3 cells grown on coverslips were fixed in 4% paraformaldehyde in PBS (pH 7.4) for at least 30 min at room temperature and processed for indirect immunofluorescence microscopy as described previously.42 The immunostained cells were mounted on glass slides and examined with a Confocal Laser-Scanning Microscope (Zeiss LSM 510 META, Germany). The following primary antibodies were used: Astrin (rabbit 1:50), Aurora-A (mouse 1:100), Plk1 (mouse 1:100), γ-tubulin (mouse 1:200), α-tubulin (rabbit 1:200), α-tubulin-FITC References 1.

Kline-Smith SL, Walczak CE. Mitotic spindle assembly and chromosome segregation: refocusing on microtubule dynamics. Mol Cell 2004; 15:317-27. 2. Piehl M, Tulu US, Wadsworth P, Cassimeris L. Centrosome maturation: measurement of microtubule nucleation throughout the cell cycle by using GFPtagged EB1. Proc Natl Acad Sci USA 2004; 101:15848. 3. Karsenti E, Vernos I. The mitotic spindle: a self-made machine. Science 2001; 294:543-7. 4. Dekel N. Cellular, biochemical and molecular mechanisms regulating oocyte maturation. Mol Cell Endocrinol 2005; 234:19-25. 5. Szollosi D, Calarco P, Donahue RP. Absence of centrioles in the first and second meiotic spindles of mouse oocytes. J Cell Sci 1972; 11:521-41. 6. Schuh M, Ellenberg J. Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes. Cell 2007; 130:484-98. 7. Chang MS, Huang CJ, Chen ML, Chen ST, Fan CC, Chu JM, et al. Cloning and characterization of hMAP126, a new member of mitotic spindle-associated proteins. Biochem Biophys Res Commun 2001; 287:116-21. 8. Mack GJ, Compton DA. Analysis of mitotic microtubule-associated proteins using mass spectrometry identifies astrin, a spindle-associated protein. Proc Natl Acad Sci USA 2001; 98:14434-9. 9. Gruber J, Harborth J, Schnabel J, Weber K, Hatzfeld M. The mitotic-spindle-associated protein astrin is essential for progression through mitosis. J Cell Sci 2002; 115:4053-9. 10. Thein KH, Kleylein-Sohn J, Nigg EA, Gruneberg U. Astrin is required for the maintenance of sister chromatid cohesion and centrosome integrity. J Cell Biol 2007; 178:345-54. 11. Cheng TS, Hsiao YL, Lin CC, Yu CT, Hsu CM, Chang MS, et al. Glycogen synthase kinase 3beta interacts with and phosphorylates the spindle-associated protein astrin. J Biol Chem 2008; 283:2454-64.

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(mouse 1:200), and myc (rabbit 1:200). Each experiment was repeated at least three times. Preparation of chromosome spreads from mouse oocytes. The Astrin RNAi injected oocytes and Myc-Astrin mutant injected oocytes were kept for 20 minutes in 1% sodium citrate in order to detect chromosomes clearly. Preparation of chromosome spreads from mouse oocytes was performed as previously described.43 Statistical analysis. All percentages from at least three repeated experiments were expressed as means ± SEM, and the number of oocytes observed was labeled in parentheses as (n=). Data were analyzed by paired-samples t-test. p < 0.05 was considered statistically significant. Acknowledgements

We are grateful to Ying-Chun OuYang and Yi Hou for their technical assistance. We thank Mau-Sun Chang for his gift of antibody. This work was supported by the National Basic Research Program of China (2006CB944001, 2006CB504004), National Natural Science Foundation of China (30570944) and Knowledge Innovation Program of the CAS (KSCX2-YW-R-52).

12. Yao LJ, Zhong ZS, Zhang LS, Chen DY, Schatten H, Sun QY. Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs and early embryos. Biol Reprod 2004; 70:1392-9. 13. Tong C, Fan HY, Lian L, Li SW, Chen DY, Schatten H, et al. Polo-like kinase-1 is a pivotal regulator of microtubule assembly during mouse oocyte meiotic maturation, fertilization and early embryonic mitosis. Biol Reprod 2002; 67:546-54. 14. Xiong B, Sun SC, Lin SL, Li M, Xu BZ, OuYang YC, et al. Involvement of Polo-like kinase 1 in MEK1/2regulated spindle formation during mouse oocyte meiosis. Cell Cycle 2008; 7:1804-9. 15. Wang LH, Yan M, Xu DZ, Cao JX, Zhu XF, Zeng YX, et al. Requirement of aurora-A kinase in astral microtubule polymerization and spindle microtubule flux. Cell Cycle 2008; 7:1104-11. 16. Chang MS, Chen CY, Huang CJ, Fan CC, Chu JM, Yang YC. Expression and promoter analysis of mouse mastrin gene. Biochem Biophys Res Commun 2003; 307:491-7. 17. Elia AE, Rellos P, Haire LF, Chao JW, Ivins FJ, Hoepker K, et al. The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain. Cell 2003; 115:83-95. 18. Elia AE, Cantley LC, Yaffe MB. Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates. Science 2003; 299:1228-31. 19. Glover DM, Leibowitz MH, McLean DA, Parry H. Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell 1995; 81:95-105. 20. Roghi C, Giet R, Uzbekov R, Morin N, Chartrain I, Le Guellec R, et al. The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly. J Cell Sci 1998; 111:557-72. 21. Cowley DO, Rivera-Perez JA, Schliekelman M, He YJ, Oliver TG, Lu L, et al. Aurora-A kinase is essential for bipolar spindle formation and early development. Mol Cell Biol 2009; 29:1059-71. 22. Hanisch A, Wehner A, Nigg EA, Sillje HH. Different Plk1 functions show distinct dependencies on PoloBox domain-mediated targeting. Mol Biol Cell 2006; 17:448-59.

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23. De Luca M, Lavia P, Guarguaglini G. A functional interplay between Aurora-A, Plk1 and TPX2 at spindle poles: Plk1 controls centrosomal localization of Aurora-A and TPX2 spindle association. Cell Cycle 2006; 5:296-303. 24. Gorbsky GJ, Kallio M, Daum JR, Topper LM. Protein dynamics at the kinetochore: cell cycle regulation of the metaphase to anaphase transition. FASEB J 1999; 13:231-4. 25. Pinsky BA, Biggins S. The spindle checkpoint: tension versus attachment. Trends Cell Biol 2005; 15:486-93. 26. Musacchio A, Salmon ED. The spindle-assembly checkpoint in space and time. Nat Rev Mol Cell Biol 2007; 8:379-93. 27. Vogt E, Kirsch-Volders M, Parry J, Eichenlaub-Ritter U. Spindle formation, chromosome segregation and the spindle checkpoint in mammalian oocytes and susceptibility to meiotic error. Mutat Res 2008; 651:14-29. 28. Yin S, Sun XF, Schatten H, Sun QY. Molecular insights into mechanisms regulating faithful chromosome separation in female meiosis. Cell Cycle 2008; 7:29973005. 29. Brunet S, Sardon T, Zimmerman T, Wittmann T, Pepperkok R, Karsenti E, et al. Characterization of the TPX2 domains involved in microtubule nucleation and spindle assembly in Xenopus egg extracts. Mol Biol Cell 2004; 15:5318-28. 30. Brunet S, Dumont J, Lee KW, Kinoshita K, Hikal P, Gruss OJ, et al. Meiotic regulation of TPX2 protein levels governs cell cycle progression in mouse oocytes. PLoS ONE 2008; 3:3338. 31. Giet R, Prigent C. The non-catalytic domain of the Xenopus laevis auroraA kinase localises the protein to the centrosome. J Cell Sci 2001; 114:2095-104. 32. Green RA, Wollman R, Kaplan KB. APC and EB1 function together in mitosis to regulate spindle dynamics and chromosome alignment. Mol Biol Cell 2005; 16:4609-22. 33. Cheng TS, Hsiao YL, Lin CC, Hsu CM, Chang MS, Lee CI, et al. hNinein is required for targeting spindleassociated protein Astrin to the centrosome during the S and G2 phases. Exp Cell Res 2007; 313:1710-21. 34. Chan EH, Santamaria A, Sillje HH, Nigg EA. Plk1 regulates mitotic Aurora A function through betaTrCPdependent degradation of hBora. Chromosoma 2008; 117:457-69.

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35. Pahlavan G, Polanski Z, Kalab P, Golsteyn R, Nigg EA, Maro B. Characterization of polo-like kinase 1 during meiotic maturation of the mouse oocyte. Dev Biol 2000; 220:392-400. 36. Yin S, Wang Q, Liu JH, Ai JS, Liang CG, Hou Y, et al. Bub1 prevents chromosome misalignment and precocious anaphase during mouse oocyte meiosis. Cell Cycle 2006; 5:2130-7. 37. Xiong B, Yu LZ, Wang Q, Ai JS, Yin S, Liu JH, et al. Regulation of intracellular MEK1/2 translocation in mouse oocytes: cytoplasmic dynein/dynactin-mediated poleward transport and cyclin B degradation-dependent release from spindle poles. Cell Cycle 2007; 6:1521-7.

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38. Yin S, Ai JS, Shi LH, Wei L, Yuan J, Ouyang YC, et al. Shugoshin1 may play important roles in separation of homologous chromosomes and sister chromatids during mouse oocyte meiosis. PLoS ONE 2008; 3:3516. 39. Mamo S, Gal AB, Bodo S, Dinnyes A. Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro. BMC Dev Biol 2007; 7:14. 40. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25:402-8.

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41. Li M, Yin S, Yuan J, Wei L, Ai JS, Hou Y, et al. Cdc25A promotes G2/M transition in oocytes. Cell Cycle 2008; 7:1301-2. 42. Suzuki H, Yagi M, Suzuki K. Duplicated insertion mutation in the microtubule-associated protein Spag5 (astrin/MAP126) and defective proliferation of immature Sertoli cells in rat hypogonadic (hgn/hgn) testes. Reproduction 2006; 132:79-93. 43. Hodges CA, Hunt PA. Simultaneous analysis of chromosomes and chromosome-associated proteins in mammalian oocytes and embryos. Chromosoma 2002; 111:165-9.

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