BRCA1 Is Required for Meiotic Spindle Assembly and Spindle

BIOLOGY OF REPRODUCTION 79, 718–726 (2008) Published online before print 2 July 2008. DOI 10.1095/biolreprod.108.069641

BRCA1 Is Required for Meiotic Spindle Assembly and Spindle Assembly Checkpoint Activation in Mouse Oocytes1 Bo Xiong,3,4 Sen Li,3,4 Jun-Shu Ai,3,4 Shen Yin,3,4 Ying-Chun OuYang,3 Shao-Chen Sun,3,5 Da-Yuan Chen,3 and Qing-Yuan Sun2,3 State Key Laboratory of Reproductive Biology,3 Institute of Zoology; and Graduate School,4 Chinese Academy of Sciences, 100080 Beijing, China Animal Reproduction Institute,5 Gugangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization, Guangxi University, 530005 Nanning, China ing evidence has revealed the involvement of BRCA1 in a number of cellular processes, including DNA replication, DNA damage response, transcription regulation, translation regulation, centrosome duplication, maintenance of genome integrity, cell cycle checkpoints, and apoptosis [4–11]. Although much has been learned during the past decade about the functions of BRCA1 in mitosis, little is known about BRCA1 in meiosis. The spindle, mainly composed of microtubules and centrosomes, is one of the most essential cellular structures that are responsible for the accurate segregation of chromosomes, which is required for maintaining the integrity of the genome in both mitosis and meiosis. Segregation errors during mitosis in somatic cells contribute to the development and progression of cancer, and segregation errors during meiosis lead directly to birth defects [12, 13]. During mitosis, spindle assembly is directed to a large extent by the centrosomes, the main sites of microtubule polymerization. Oocytes, however, lack centriole-containing centrosomes, and the microtubules are polymerized at discrete sites in the cytoplasm called microtubule organizing centers (MTOCs) [14]. The latest finding proposes a new model of acentrosomal spindle assembly that relies on the self-organization of numerous acentriolar MTOCs, which functionally replace centrosomes [15]. Despite that, molecules involved in the acentrosomal spindle assembly during meiosis and their specific roles need further identification. Recently, BRCA1 has been reported to regulate the mitotic spindle assembly [16], but whether it participates in the meiotic spindle formation remains elusive. Accordingly, we attempt to examine the possible roles of BRCA1 in spindle organization during female meiosis. To prevent the missegregation of chromosomes, both mitotic and meiotic cells have developed a high-fidelity surveillance system to monitor the coordinated and precise operation of the segregation machinery, which is referred to as the spindle assembly checkpoint, ensuring the accurate chromosome segregation by sensing attachment to microtubules and tension on chromosomes [17–21]. The highly conserved checkpoint mechanisms and components have been widely studied in mitosis, whereas the functional roles and components of the spindle checkpoint in meiosis are still not fully clear. Recent studies by others and us have shown that the spindle checkpoint also operates in meiosis, and that many of its components are conserved between mitosis and meiosis; however, there are meiosis-specific molecules monitoring the segregation of homologous chromosomes at meiosis I and segregation of sister chromatids at meiosis II [22–31]. Especially, we also do not know much about the conserved differences between mitosis and meiosis, and even between the male and female meiosis in

ABSTRACT BRCA1 as a tumor suppressor has been widely investigated in mitosis, but its functions in meiosis are unclear. In the present study, we examined the expression, localization, and function of BRCA1 during mouse oocyte meiotic maturation. We found that expression level of BRCA1 was increased progressively from germinal vesicle to metaphase I stage, and then remained stable until metaphase II stage. Immunofluorescent analysis showed that BRCA1 was localized to the spindle poles at metaphase I and metaphase II stages, colocalizing with centrosomal protein gamma-tubulin. Taxol treatment resulted in the presence of BRCA1 onto the spindle microtubule fibers, whereas nocodazole treatment induced the localization of BRCA1 onto the chromosomes. Depletion of BRCA1 by both antibody injection and siRNA injection caused severely impaired spindles and misaligned chromosomes. Furthermore, BRCA1-depleted oocytes could not arrest at the metaphase I in the presence of low-dose nocodazole, suggesting that the spindle checkpoint is defective. Also, in BRCA1-depleted oocytes, gamma-tubulin dissociated from spindle poles and MAD2L1 failed to rebind to the kinetochores when exposed to nocodazole at metaphase I stage. Collectively, these data indicate that BRCA1 regulates not only meiotic spindle assembly, but also spindle assembly checkpoint, implying a link between BRCA1 deficiency and aneuploid embryos. aneuploid embryo, BRCA1, meiosis, mouse oocyte, spindle assembly, spindle assembly checkpoint

INTRODUCTION Inherited mutations of the breast cancer associated gene 1 (BRCA1), encoding a tumor suppressor, predispose women to breast, ovarian, and other cancers. BRCA1, mapped in 1990 [1] and then cloned in 1994 [2], is a large protein with multiple functional domains and interacts directly or indirectly with numerous molecules, such as tumor suppressors, oncogenes, DNA damage repair proteins, and cell cycle regulators, as well as transcriptional activators and repressors [3, 4]. Accumulat1

Supported by the National Basic Research Program of China (2006CB944001, 2006CB504004), National Natural Science Foundation of China (30430530, 30570944), and Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-R-52). 2 Correspondence: Qing-Yuan Sun, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing 100101, China. FAX: 86 10 64807099; e-mail: [email protected] Received: 2 April 2008. First decision: 5 May 2008. Accepted: 13 June 2008. Ó 2008 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

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ROLES OF BRCA1 IN MOUSE OOCYTES

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the regulation of the spindle assembly checkpoint [32]. Since BRCA1 is required for the spindle checkpoint in mitosis [33], we aim to explore whether BRCA1 takes part in the control of spindle assembly checkpoint and its regulatory mechanism during meiosis in mouse oocytes. Since BRCA1 mRNA is less abundant in aged compared with young oocytes [34], it is of high relevance to assess the function of BRCA1 in oocyte maturation. Therefore, we investigated the roles of BRCA1 during mouse oocyte meiotic maturation by spindle-perturbing drug treatment, antibody injection, and RNA interference. The results provide strong evidence showing that BRCA1 not only plays crucial roles in spindle assembly and chromosome alignment but also regulates the spindle checkpoint activation, which may be related to the aneuploid embryos causing birth defects. MATERIALS AND METHODS Antibodies Rabbit polyclonal anti-BRCA1 antibody was obtained from Signalway Antibody Co., Ltd. (Pearland, TX); mouse monoclonal anti-a-tubulinfluorescein isothiocyanate (FITC) antibody and mouse monoclonal anti-ctubulin antibody were obtained from Sigma-Aldrich Co. (St. Louis, MO); rabbit polyclonal anti-MAD2L1 antibody was purchased from Covance Inc. (Princeton, NJ); and mouse monoclonal anti-b-actin antibody was purchased from Proteintech Group Inc. (Chicago, IL).

Oocyte Collection and Culture 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 arrested at prophase of meiosis I were collected from ovaries of 6-wk-old female Kunmin White 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 378C in an atmosphere of 5% CO2 in air. At different times after culture, oocytes were collected for immunostaining, microinjection, or Western blot analysis.

Taxol and Nocodazole Treatment of Oocytes Oocytes at various stages were treated with taxol or nocodazole. For taxol treatment, 5 mM taxol (Sigma) in dimethylsulfoxide (DMSO) stock was diluted in M16 medium to give a final concentration of 10 lM, and oocytes were incubated for 45 min; for nocodazole treatment, 10 mg/ml nocodazole in DMSO stock (Sigma) was diluted in M16 medium to give a final concentration of 20 lg/ ml, and oocytes were incubated for 10 min. In the overriding metaphase I arrest experiment, siRNA-injected oocytes were incubated in the M16 medium containing 0.04 lg/ml nocodazole for 12 h. After treatment, oocytes were washed thoroughly and used for immunofluorescence. Control oocytes were treated with the same concentration of DMSO in the medium before examination.

FIG. 1. Expression and subcellular localization of BRCA1 during mouse oocyte meiotic maturation. A) Samples were collected after oocytes had been cultured for 0, 2, 4, 8, 9.5, and 12 h, corresponding to GV, GVBD, prometaphase I, metaphase I, anaphase I, and metaphase II stages, respectively. Proteins from a total of 400 oocytes were loaded for each sample. The molecular mass of BRCA1 is 220 kDa, and the molecular mass for b-actin is 42 kDa. B) Oocytes at various stages were double stained with antibodies against BRCA1 and a-tubulin. Green, a-Tubulin; red, BRCA1; blue, chromatin; yellow, overlapping of green and red; GV, oocytes at germinal vesicle; Pro-MI, oocytes at first prometaphase; M I, oocytes at first metaphase; AT I, oocytes at first anaphase and telophase; M II, oocytes at second metaphase. Each sample was counterstained with Hoechst 33258 to visualize DNA. Bar ¼ 20 lm.

Antibody Microinjection About 7 pl anti-BRCA1 (0.5 mg/ml) antibody was microinjected into the cytoplasm of a fully grown GV oocyte using a Nikon Diaphot ECLIPSE TE 300 (Nikon UK Ltd., Kingston upon Thames, Surrey, UK) inverted microscope equipped with Narishige MM0-202N hydraulic three-dimensional micromanipulators (Narishige Inc., Sea Cliff, NY). The oocytes were kept in M2 medium supplemented with 2.5 lM Milrinone (Sigma) to prevent GV breakdown during the injection period. After microinjection, the resumption of meiotic maturation was triggered by removal of the injected oocytes from Milrinone-containing medium. They were then cultured under paraffin oil at 378C, in an atmosphere of 5% CO2 in air, in fresh M16 medium. Control oocytes were microinjected with the same amount of rabbit immunoglobulin G (IgG). Finally, spindle phenotypes were examined.

RNA Interference Fully grown, GV-intact oocytes were microinjected in M2 medium containing 2.5 lM Milrinone with 5–10 pl of the control or BRCA1-specific siRNA (Ambion Inc., Austin, TX). The final concentration of the control or BRCA1-specific siRNA was 50 lM. Microinjected oocytes were incubated in M16 medium containing 2.5 lM Milrinone for 24 h, and then transferred to

Milrinone-free M16 medium to resume the meiosis. Oocytes in different stages were collected to run the subsequent experiments.

Immunofluorescence and Confocal Microscopy For single staining of BRCA1, MAD2L1, and a-tubulin, oocytes were fixed in 4% paraformaldehyde in PBS (pH 7.4) for at least 30 min at room temperature. After being permeabilized with 0.5% Triton X-100 at room temperature for 20 min, oocytes were blocked in 1% BSA-supplemented PBS for 1 h and incubated overnight at 48C with 1:50 rabbit anti-BRCA1 antibody, 1:50 rabbit antiMAD2L1 antibody, and 1:200 anti-a-tubulin-FITC antibody, respectively. After three washes in PBS containing 0.1% Tween 20 and 0.01% Triton X-100 for 5 min each, the oocytes were labeled with 1:100 FITC-conjugated goat-anti-rabbit IgG for 1 h at room temperature (for staining of a-tubulin, this step was omitted). After three washes in PBS containing 0.1% Tween 20 and 0.01% Triton X-100, the oocytes were costained with propidium iodide (PI; 10 lg/ml in PBS). Finally, the oocytes were mounted on glass slides and examined with a confocal laser scanning microscope (Zeiss LSM 510 META, Germany).

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FIG. 2. Colocalization of BRCA1 and c-tubulin during meiotic maturation of mouse oocytes. A) Oocytes at various stages were double stained with antibodies against BRCA1 and c-tubulin. After GVBD, the signals of BRCA1 and c-tubulin were completely overlapped. Green, BRCA1; red, c-tubulin; blue, chromatin; yellow, overlapping of green and red; GV, oocytes at germinal vesicle; Pro-MI, oocytes at first prometaphase; M I, oocytes at first metaphase; AT I, oocytes at first anaphase and telophase; M II, oocytes at second metaphase. Each sample was counterstained with Hoechst 33258 to visualize DNA. B) Control stained with the secondary antibody and without primary antibody to BRCA1 and c-tubulin. Bars ¼ 20 lm. For double staining of BRCA1 and a-tubulin, after BRCA1 staining (the secondary antibody was 1:100 TRITC-conjugated goat-anti-rabbit IgG), the oocytes were again blocked in 1% BSA-supplemented PBS for 1 h at room temperature, followed by staining with 1:100 anti-a-tubulin-FITC antibody. Then, the oocytes were stained with Hoechst 33258 (10 lg/ml in PBS) for 20 min. For double staining of BRCA1 and c-tubulin, the same method mentioned above was employed, except that for the staining of c-tubulin, the primary antibody was 1:200 mouse anti-c-tubulin antibody and the secondary antibody was 1:100 TRITC-conjugated goat-anti-mouse IgG. Each experiment was repeated at least three times, and about 100 oocytes were examined in each group. The same instrument settings were used for each replicate.

Immunoblotting Analysis A total of 400 mouse oocytes at the appropriate stage of meiotic maturation were collected in SDS sample buffer and heated for 5 min at 1008C. Immunoblotting was based on the procedures reported by us previously [27]. The proteins were separated by SDS-PAGE and then electrically transferred to polyvinylidene fluoride membranes. Following transfer, the membrane were blocked in TBST (TBS containing 0.1% Tween 20) containing 5% skimmed milk for 2 h, followed by incubation overnight at 48C with 1:500 rabbit polyclonal anti-BRCA1 antibody, 1:500 rabbit polyclonal anti-MAD2L1 antibody, and 1:1000 mouse monoclonal anti-b-actin antibody. After washing three times in TBST, 10 min each, the membranes were incubated for 1 h at 378C with 1:1000 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).

Statistics All percentages from at least three repeated experiments were expressed as means 6 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.

RESULTS Expression of BRCA1 During Meiosis in Mouse Oocytes To examine the expression level of BRCA1 in mouse oocytes at the different stages of meiotic maturation, samples were collected after oocytes had been cultured for 0, 2, 4, 8, 9.5, and 12 h, corresponding to GV, germinal vesicle breakdown (GVBD), prometaphase I, metaphase I, anaphase/ telophase I, and metaphase II stages, respectively. The immunoblotting results showed that the expression level of BRCA1 was relatively low at GV stage, evidently increased from GVBD to prometaphase I stages, reached the maximal level at metaphase I stage, and then remained stable at anaphase/telophase I and metaphase II stages (Fig. 1A). Subcellular Localization of BRCA1 During Mouse Oocyte Meiotic Maturation To investigate the subcellular localization of BRCA1 during meiotic maturation, mouse oocytes were processed for the immunofluorescent staining at the different stages of maturation. As shown in Figure 1B, BRCA1 was only distributed in the GV of oocytes at GV stage. Shortly after GVBD, BRCA1 accumulated in the vicinity of the condensed chromosomes. By prometaphase I, chromosomes began to migrate to the equator of the spindle, and BRCA1 was gradually translocated to the


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spindle poles. When oocytes progressed to metaphase I, chromosomes aligned at the equatorial plate, and BRCA1 concentrated at the spindle poles. At anaphase/telophase I, BRCA1 was localized in the region between the separating homologous chromosomes, and associated with the midbody between the oocyte and the first polar body. At metaphase II, BRCA1 was again translocated to the spindle poles. The polar localization has also been confirmed by double staining with BRCA1 and c-tubulin antibody. The c-tubulin is a well-studied component of pericentriolar material in vertebrate cells and locates exclusively at the centrosome throughout the cell cycle. As shown in Figure 2A, after GVBD, the signals of BRCA1 were overlapped by those of c-tubulin during the entire meiotic maturation of mouse oocytes, implying that BRCA1 may participate in the spindle formation through its interaction with the centrosomal proteins. The artifact of colocalization of BRCA1 and c-tubulin could be ruled out by the control with secondary antibody and without primary antibody to BRCA1 (Fig. 2B). Localization of BRCA1 in Mouse Oocytes Treated with the Spindle-Perturbing Agents To clarify the correlation between BRCA1 and microtubule dynamics, the spindle-perturbing drugs were employed. First, we used the taxol, a microtubule-stabilizing reagent, to treat the oocytes. After GVBD, when microtubule organization initiated, the microtubule fibers in taxol-treated oocytes were excessively polymerized, leading to the significantly enlarged spindles, together with numerous asters in the cytoplasm (Fig. 3A). In this case, BRCA1 signals were detected on the fibers of the abnormal spindles as well as cytoplasmic asters from prometaphase I to metaphase II stages, unlike its normal localization (Fig. 1B). Next, metaphase I oocytes were treated with the nocodazole, a microtubule-depolymerizing agent, to observe the BRCA1 localization. After treatment, the microtubules were completely disassembled, and no intact spindles were observed in the oocytes. Unexpectedly, BRCA1 disappeared from the spindle poles and emerged on both sides of the chromosomes (Fig. 3B). Since there were no visible stubs of microtubules detected on the chromosomes, we can rule out the possibility that BRCA1 accumulated at the minus ends of microtubules retained on the kinetochores. Instead, BRCA1 might correspond to kinetochores of the homologous chromosomes, which were still oriented toward opposite spindle poles in the metaphase I-blocked oocytes exposed to nocodazole. Disruption of BRCA1 Function Exhibits Severely Abnormal Spindles and Misaligned Chromosomes Next, we attempted to explore the functional roles of BRCA1 in the meiotic spindle assembly by antibody microinjection. In the control group injected with rabbit IgG, most of oocytes could organize a normal spindle, and only 13.4% 6 2.7% (n ¼ 236) were morphologically abnormal (Fig. 4B). However, in the antibody-injected group, a large amount of spindles exhibited various abnormalities and defects (54.0% 6 9.1%; n ¼ 194; P , 0.05; Fig. 4B), including monopolar spindles, multipolar spindles, elongated spindles, multiple spindle apparatuses, and irregularly dispersed spindle microtubules (Fig. 4A). In addition, BRCA1 antibody-injected oocytes displayed a severe defect in chromosome alignment, revealing lagging chromosomes and irregularly scattered chromosomes (Fig. 4A). The incidence of misaligned chromosomes in the antibody-injected group was up to 38.6% 6 10.2% (n ¼ 194),

FIG. 3. Localization of BRCA1 in mouse oocytes treated with spindleperturbing agents during meiotic maturation. A) Oocytes at various stages were incubated in M16 medium containing 10 lM taxol for 45 min and then double stained with antibodies against BRCA1 as well as a-tubulin. Green, a-Tubulin; red, BRCA1; blue, chromatin; yellow, overlapping of green and red; GV, oocytes at germinal vesicle; Pro-MI, oocytes at first prometaphase; M I, oocytes at first metaphase; AT I, oocytes at first anaphase and telophase; M II, oocytes at second metaphase. Each sample was counterstained with Hoechst 33258 to visualize DNA. Bar ¼ 20 lm. B) Oocytes at metaphase I stage were incubated in M16 medium containing 20 lg/ml nocodazole for 10 min and then double stained with antibodies against BRCA1 as well as a-tubulin. Green, a-Tubulin; red, BRCA1; blue, chromatin; yellow, overlapping of green and red. Each sample was counterstained with Hoechst 33258 to visualize DNA. Bar ¼ 10 lm.

much higher than that in the control group (5.0% 6 1.4%; n ¼ 236; P , 0.05; Fig. 4C). To further support the above results, RNAi was employed to perturb the function of BRCA1. As shown in Figure 4D,

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FIG. 4. Disruption of BRCA1 function impairs the spindle organization and chromosome alignment. A) Spindle morphologies and chromosome alignment in the oocytes injected with rabbit IgG and BRCA1 antibody. Injected oocytes were stained with a-tubulin antibody (green) and PI (red) to show the spindle and DNA. In the rabbit IgG-injected group, normal bipolar spindles formed in most of the oocytes. In the antibody-injected group, various morphologically aberrant spindles and misaligned chromosomes were present. B) The rate of abnormal spindles was recorded in the rabbit IgG-injected group and the antibody-injected group. C) The rate of misaligned chromosomes was recorded in the rabbit IgG-injected group and the antibody-injected group. D) Expression of BRCA1 in the siRNA-injected oocytes. Germinal vesicle oocytes were microinjected with the control


compared with the control group, the protein expression of BRCA1 in specific siRNA-injected oocytes was strikingly reduced in spite of the more loading amount, revealing the successful BRCA1 downregulation by RNAi. Similar to the observation in the antibody injection experiment, knockdown of BRCA1 by siRNA injection also exhibited numerous spindle and chromosome defects (Fig. 4E). The abnormal rate of spindle formation in the control siRNA-injected group was 11.4% 6 3.0% (n ¼ 191), considerably lower than that in the BRCA1 siRNA-injected group (63.6% 6 7.8%; n ¼ 144; P , 0.05; Fig. 4F). Concomitantly, an obvious increase in misaligned chromosome rate was observed when the BRCA1-depleted group (44.8% 6 7.6%; n ¼ 144) was compared to the control group (5.2% 6 1.6%; n ¼ 191; P , 0.05; Fig. 4G). Depletion of BRCA1 Causes the Dissociation of c-Tubulin from Spindle Poles Attenuating the interaction between BRCA1 and c-tubulin may induce the accumulation of abnormal spindle formation during mitosis [35, 36]. Similarly, c-tubulin was not associated with the poles when spindle morphology was disrupted after knockdown of BRCA1 at the metaphase I stage in mouse oocytes. Instead, c-tubulin was found within the cytoplasm (Fig. 5). Depletion of BRCA1 Abrogates the Metaphase I Arrest Provoked by Low-Dose Nocodazole Since BRCA1 is required for the spindle assembly checkpoint during mitosis [33], we speculated that BRCA1 is most likely to participate in the regulation of spindle checkpoint during meiosis. To test this, oocytes were cultured for 12 h in the presence of low-dose nocodazole. The data showed that most of the oocytes were arrested at metaphase I in the control siRNA-injected group, and the rate of overriding metaphase I was only 8.2% 6 3.3% (n ¼ 258; Fig. 6A). In contrast, the BRCA1 siRNA-injected group exhibited a higher overriding percentage, and 37.1% 6 5.5% (n ¼ 211) of oocytes escaped the metaphase I arrest and then reached the metaphase II stage (Fig. 6A). The result that BRCA1-depleted oocytes failed to undergo the metaphase I arrest and continued to progress through the cell cycle suggests that spindle checkpoint is defective when perturbing the function of BRCA1. BRCA1 Is Required for Recruitment of MAD2L1 to Kinetochores after Meiotic Spindle Disruption To further investigate the roles of BRCA1 in the regulation of spindle checkpoint during meiosis, we studied MAD2L1, a component of the spindle assembly checkpoint. First, the expression of MAD2L1 was examined in siRNA-injected oocytes. As shown in Figure 6B, the expression level of MAD2L1 was not altered after knockdown of BRCA1 in metaphase I mouse oocytes, suggesting that it was not the reduction of MAD2L1 that led to the defective spindle

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FIG. 5. Dissociation of c-tubulin from spindle poles in BRCA1-depleted oocytes. In the control siRNA-injected group, c-tubulin was associated with the spindle poles at metaphase I stage, whereas in the BRCA1 siRNAinjected group, c-tubulin delocalized from abnormal spindle poles and dispersed into the cytoplasm. Green, a-Tubulin; red, c-tubulin; blue, chromatin; yellow, overlapping of green and red. Bar ¼ 20 lm.

checkpoint. Next, we checked the localization of MAD2L1 in siRNA-injected oocytes. As shown in Figure 6C, once spindle formed and chromosomes aligned at the spindle’s equator, MAD2L1 was detected on the spindle poles in metaphase I mouse oocytes injected with the control siRNA. However, in the presence of nocodazole, such control siRNA-injected oocytes remained arrested and had MAD2L1 relocate to the unattached kinetochores, and thus inhibited the onset of the anaphase. By contrast, in BRCA1 siRNA-injected oocytes, MAD2L1 was dispersed in the cytoplasm rather than accumulated at the spindle poles. After nocodazole treatment, the amount of MAD2L1 that rebound to the kinetochores decreased dramatically. Most of MAD2L1 seemed to be diffused in the cytoplasm, and only a few signals were present in the vicinity of chromosomes. DISCUSSION In this study, we demonstrate that BRCA1 plays an essential role in the control of spindle assembly during mouse oocyte meiotic maturation. Earlier reports have shown that BRCA1 is associated with the centrosome during mitosis [36], so we first examined the localization of BRCA1 during meiosis in mouse oocytes. The immunofluorescent results revealed that BRCA1 was localized at the spindle poles at the metaphase I and metaphase II stages, which is similar to many other proteins involved in the spindle formation in meiosis that we previously investigated, such as aurora-kinase A (AURKA), PKC, MDK (also known as MEK), and Polo-like kinase 1 (PLK1) [37–40]. Furthermore, BRCA1 was also associated with the centrosomal protein c-tubulin in mouse oocytes, consistent with the result during mitosis that BRCA1 has the c-tubulin-binding domain and can coimmunoprecipitate with c-tubulin [35, 36]. According to the centrosomal localization of BRCA1, together with the recent report that the BRCA1-BARD1 heterodimer modulates ran-dependent mitotic spindle assembly [16], we

3 siRNA and BRCA1-specific siRNA, respectively. After injection, oocytes were incubated in M16 medium containing 2.5 lM Milrinone for 24 h, and then transferred to Milrinone-free M16 for 8 h, followed by Western blotting. The molecular mass of BRCA1 is 220 kDa, and the molecular weight for b-actin is 42 kDa. E) Spindle morphologies and chromosome alignment in control siRNA-injected oocytes and BRCA1 siRNA-injected oocytes. After injection, oocytes were incubated in M16 medium containing 2.5 lM Milrinone for 24 h, and then transferred to Milrinone-free M16 for 8 h, followed by immunostaining with a-tubulin antibody (green) and PI (red). In the control siRNA-injected group, normal bipolar spindles formed, and chromosomes regularly aligned in the majority of oocytes (1). In BRCA1-specific siRNA-injected group, various morphologically aberrant spindles and misaligned chromosomes appeared (2–12). F) The rate of abnormal spindles was recorded in the control siRNA-injected group and BRCA1 siRNA-injected group. G) The rate of misaligned chromosomes was recorded in the control siRNA-injected group and the BRCA1 siRNA-injected group. Data were presented as mean percentage (mean 6 SEM) of at least three independent experiments. Different letters denote statistical difference at a P , 0.05 level of significance. Bar ¼ 20 lm.

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FIG. 6. Expression and localization of MAD2L1 in siRNA-injected oocytes. A) BRCA1-depleted oocytes could not arrest at the metaphase I when exposed to nocodazole. The incidence of overriding metaphase I arrest by 0.04 lg/ml nocodazole was recorded in the control siRNAinjected group and the BRCA1 specific siRNA-injected group. Data were presented as mean percentage (mean 6 SEM) of at least three independent experiments. Different letters denote statistical difference at a P , 0.05 level of significance. B) Expression of MAD2L1 in the siRNA-injected oocytes. Germinal vesicle oocytes were microinjected with the control siRNA and BRCA1-specific siRNA, respectively. After injection, oocytes were incubated in M16 medium containing 2.5 lM Milrinone for 24 h, and then transferred to Milrinone-free M16 for 8 h, followed by Western blotting. In control group, the same number of oocytes without injection was collected to run the Western blotting. The molecular mass for MAD2L1 is 24 kDa, and the molecular mass for b-actin is 42 kDa. C) Recruitment and/or maintenance of MAD2L1 to kinetochores in BRCA1-

suggest that BRCA1 most likely contributes to the meiotic spindle assembly. The observation that BRCA1 was localized onto the microtubule fibers of the spindle and cytoplasmic asters after taxol treatment indicates that BRCA1 is probably transported along the microtubules via treadmilling and attachment to spindle components, like the dynein/dynactin complex. Loss of treadmilling would freeze the flow of BRCA1 to the spindle poles. This localization pattern of BRCA1 is different from the other proteins involved in spindle formation we previously investigated, such as MDK and Polo-like kinase 1, which are present in the center of MTOCs after taxol treatment, perhaps participating in microtubule nucleation or organization [39, 40]. Next, we examined the spindle morphology after blocking the functions of BRCA1 by antibody injection. The larger proportion of severely abnormal spindles and misaligned chromosomes in BRCA1-depleted oocytes compared with control oocytes indicates that BRCA1 is indispensable for the meiotic spindle assembly. This conclusion is further confirmed by the results of injecting the oocytes with BRCA1-specific siRNA. Furthermore, a recent report showing that mouse oocytes injected with BRCA1 double-stranded RNA exhibited a spectrum of abnormal chromosome configurations and spindle morphologies also supports our observations [34]. Since it has been demonstrated that BRCA1 contains the c-tubulin-binding domain, and the reduction of binding between these two proteins might contribute to the formation of abnormal spindles [35], we tried to explore the mechanism causing the spindle defects in BRCA1-depleted oocytes from the relationship between BRCA1 and c-tubulin. Therefore, we determined the distribution of c-tubulin in BRCA1-depleted oocytes. Delocalization of c-tubulin from spindle poles provides the evidence suggesting that the role of BRCA1 in the spindle assembly during meiosis in mouse oocytes might be mediated by the ctubulin, and that BRCA1 is critical for the maintenance of ctubulin in the spindle poles. Our report also reveals that BRCA1 is necessary for the recruitment of spindle checkpoint protein to the unattached kinetochores when spindle formation is disrupted. The most interesting finding in this study is that BRCA1 disappeared from spindle poles but emerged on the chromosomes after oocytes were treated with the nocodazole, which could result in the complete destruction of spindle structures and activate the spindle checkpoint. Since BRCA1 had no association with chromosomes at any normally developmental stages from GV to metaphase II during meiotic maturation in mouse oocytes, this unexpected localization of BRCA1 must predict its special functions on chromosomes during the activation of spindle checkpoint in the presence of spindle abnormalities. Combined with the previously reported spindle assembly checkpoint defect in cells expressing a mutant hypomorphic BRCA1 allele [33, 41], we raise a possibility that BRCA1 dysfunction could lead to the spindle checkpoint failure during meiosis. To test this, we checked the occurrence of overriding metaphase I arrest induced by low-dose nocodazole in BRCA1-depleted oocytes. The result that a high incidence of BRCA1-depleted oocytes could not 3 depleted oocytes with defective spindles. In control siRNA-injected oocytes, MAD2L1 was localized to spindle poles at metaphase I stage and then translocated to kinetochores when the spindle was destroyed by treatment with 20 lg/ml nocodazole (Noc) for 10 min. By contrast, in BRCA1-specific siRNA-injected oocytes, MAD2L1 dissociated from the spindle poles at metaphase I stage. After treatment with the same concentration of nocodazole, most of MAD2L1 diffused in the cytoplasm and could not rebind to the kinetochores. Green, MAD2L1; red, PI; yellow, overlapping of green and red. Bar ¼ 10 lm.


arrest at metaphase I in the presence of microtubule-depolymerizing drugs becomes one of the most important criteria supporting that BRCA1 belongs to the family of spindle checkpoint regulatory genes. Furthermore, we found that MAD2L1 failed to relocate to kinetochores after spindle checkpoint was activated by nocodazole treatment in BRCA1depleted oocytes. Although we cannot make sure whether BRCA1 regulates MAD2L1 directly or through other molecules, BRCA1 does participate in the recruitment or maintenance of MAD2L1 to kinetochores, and failure of this event might be a key cause resulting in the spindle checkpoint defect in mouse oocytes. During mitosis, Brca1D11/D11 cells displayed decreased expression of a number of genes that are involved in the spindle checkpoint, including Plk1, Bub1, Bub1b, Zw10, and Mad2l1 [6]. Further investigation found that BRCA1 positively regulates MAD2L1 expression by interacting with its promoter, so Mad2l1 was significantly downregulated in Brca1D11/D11 mutant embryos at developmental stages E13.5–E18.5. Also, overexpression of MAD2L1 in Brca1D11/D11 cells partially rescues the spindle checkpoint defect [33]. However, it is well known that there is no transcription activity during the final phase of mouse oocyte meiotic maturation, and the expression of MAD2L1 is not altered after knockdown of BRCA1, so the mechanism that BRCA1 regulates the MAD2L1 in the transcriptional level during mitosis is not available during meiosis. Therefore, another mechanism about BRCA1 participation in the regulation of spindle checkpoint is probably formed in meiosis: BRCA1 recruits or maintains MAD2L1 to kinetochores during the spindle checkpoint activation induced by the spindle disruption. It has been demonstrated that depletion of MAD2L1 in meiosis I mouse oocytes induced an increased incidence of aneuploidy [26], so it could be predicted that BRCA1 deficiency would also exhibit a higher number of chromosomal gains and losses. This is supported by the finding that BRCA1-associated breast cancer contains a higher percentage of aneuploidy than those without BRCA1 mutations [42]. In addition, it has recently been shown that reduced amounts of BRCA1 protein in old mouse oocytes could be a contributing factor underlying the age-associated increase in the incidence of aneuploidy [34]. In summary, several lines of evidence in our report demonstrate that BRCA1 exerts pivotal functions in spindle assembly, chromosome alignment, and spindle checkpoint regulation during mouse oocyte meiotic maturation. Once BRCA1 malfunctions, the oocytes harboring the abnormal spindles and misaligned chromosomes cannot arrest at metaphase I, but continue to progress to metaphase II stage and extrude the polar bodies, easily producing the aneuploid embryos and thus causing early embryo death, spontaneous abortion, or genetic diseases. Also, BRCA1 becomes dramatically reduced at the two- to three-cell stage in mouse embryos and then increases approximately 12-fold up to the 10-cell stage [43], suggesting that the initial supply appears entirely maternal, and reductions could therefore increase susceptibility to mitotic errors in preimplantation embryos. Taken together, BRCA1 perhaps helps to preserve the genomic stability through the regulation of spindle assembly and spindle checkpoint activation during the meiotic maturation in female mammals. ACKNOWLEDGMENT We thank S.W. Li for her technical assistance.

REFERENCES 1. Hall JM, Lee MK, Newman B, Morrow JE, Anderson LA, Huey B, King MC. Linkage of early-onset familial breast cancer to chromosome 17q21. Science 1990; 250:1684–1689.

725

2. Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM, Ding W, et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 1994; 266:66–71. 3. Brodie SG, Deng CX. BRCA1-associated tumorigenesis: what have we learned from knockout mice? Trends Genet 2001; 17:S18–S22. 4. Narod SA, Foulkes WD. BRCA1 and BRCA2: 1994 and beyond. Nat Rev Cancer 2004; 4:665–676. 5. Venkitaraman AR. Cancer susceptibility and the functions of BRCA1 and BRCA2. Cell 2002; 108:171–182. 6. Deng CX. BRCA1: cell cycle checkpoint, genetic instability, DNA damage response and cancer evolution. Nucleic Acids Res 2006; 34:1416– 1426. 7. Deng CX, Wang RH. Roles of BRCA1 in DNA damage repair: a link between development and cancer. Hum Mol Genet 2003; 12(spec no. 1): R113–R123. 8. Horwitz AA, Affar el B, Heine GF, Shi Y, Parvin JD. A mechanism for transcriptional repression dependent on the BRCA1 E3 ubiquitin ligase. Proc Natl Acad Sci U S A 2007; 104:6614–6619. 9. Dizin E, Gressier C, Magnard C, Ray H, Decimo D, Ohlmann T, Dalla Venezia N. BRCA1 interacts with poly(A)-binding protein: implication of BRCA1 in translation regulation. J Biol Chem 2006; 281:24236–24246. 10. Deng CX. Roles of BRCA1 in centrosome duplication. Oncogene 2002; 21:6222–6227. 11. Xu X, Wagner KU, Larson D, Weaver Z, Li C, Ried T, Hennighausen L, Wynshaw-Boris A, Deng CX. Conditional mutation of Brca1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation. Nat Genet 1999; 22:37–43. 12. Wang WH, Sun QY. Meiotic spindle, spindle checkpoint and embryonic aneuploidy. Front Biosci 2006; 11:620–636. 13. Compton DA. Spindle assembly in animal cells. Annu Rev Biochem 2000; 69:95–114. 14. Brunet S, Maro B. Cytoskeleton and cell cycle control during meiotic maturation of the mouse oocyte: integrating time and space. Reproduction 2005; 130:801–811. 15. Schuh M, Ellenberg J. Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes. Cell 2007; 130:484–498. 16. Joukov V, Groen AC, Prokhorova T, Gerson R, White E, Rodriguez A, Walter JC, Livingston DM. The BRCA1/BARD1 heterodimer modulates ran-dependent mitotic spindle assembly. Cell 2006; 127:539–552. 17. Hoyt MA. A new view of the spindle checkpoint. J Cell Biol 2001; 154: 909–911. 18. Amon A. The spindle checkpoint. Curr Opin Genet Dev 1999; 9:69–75. 19. Nicklas RB. How cells get the right chromosomes. Science 1997; 275: 632–637. 20. Musacchio A, Salmon ED. The spindle-assembly checkpoint in space and time. Nat Rev Mol Cell Biol 2007; 8:379–393. 21. 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. 22. Niault T, Hached K, Sotillo R, Sorger PK, Maro B, Benezra R, Wassmann K. Changing mad2 levels affects chromosome segregation and spindle assembly checkpoint control in female mouse meiosis I. PLoS ONE 2007; 2:e1165. 23. Malmanche N, Maia A, Sunkel CE. The spindle assembly checkpoint: preventing chromosome mis-segregation during mitosis and meiosis. FEBS Lett 2006; 580:2888–2895. 24. 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. 25. Zhang D, Li M, Ma W, Hou Y, Li YH, Li SW, Sun QY, Wang WH. Localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during the first meiosis and its functions as a spindle checkpoint protein. Biol Reprod 2005; 72:58–68. 26. Homer HA, McDougall A, Levasseur M, Yallop K, Murdoch AP, Herbert M. Mad2 prevents aneuploidy and premature proteolysis of cyclin B and securin during meiosis I in mouse oocytes. Genes Dev 2005; 19:202–207. 27. Zhang D, Ma W, Li YH, Hou Y, Li SW, Meng XQ, Sun XF, Sun QY, Wang WH. Intra-oocyte localization of MAD2 and its relationship with kinetochores, microtubules, and chromosomes in rat oocytes during meiosis. Biol Reprod 2004; 71:740–748. 28. Brunet S, Pahlavan G, Taylor S, Maro B. Functionality of the spindle checkpoint during the first meiotic division of mammalian oocytes. Reproduction 2003; 126:443–450.

726

XIONG ET AL.

29. Wassmann K, Niault T, Maro B. Metaphase I arrest upon activation of the Mad2-dependent spindle checkpoint in mouse oocytes. Curr Biol 2003; 13:1596–1608. 30. Yin S, Wang Q, Liu JH, Ai JS, Liang CG, Hou Y, Chen DY, Schatten H, Sun QY. Bub1 prevents chromosome misalignment and precocious anaphase during mouse oocyte meiosis. Cell Cycle 2006; 5:2130–2137. 31. Zhang D, Yin S, Jiang MX, Ma W, Hou Y, Liang CG, Yu LZ, Wang WH, Sun QY. Cytoplasmic dynein participates in meiotic checkpoint inactivation in mouse oocytes by transporting cytoplasmic mitotic arrestdeficient (Mad) proteins from kinetochores to spindle poles. Reproduction 2007; 133:685–695. 32. Morelli MA, Cohen PE. Not all germ cells are created equal: aspects of sexual dimorphism in mammalian meiosis. Reproduction 2005; 130:761– 781. 33. Wang RH, Yu H, Deng CX. A requirement for breast-cancer-associated gene 1 (BRCA1) in the spindle checkpoint. Proc Natl Acad Sci U S A 2004; 101:17108–17113. 34. Pan H, Ma P, Zhu W, Schultz RM. Age-associated increase in aneuploidy and changes in gene expression in mouse eggs. Dev Biol 2008; 316:397– 407. 35. Hsu LC, Doan TP, White RL. Identification of a gamma-tubulin-binding domain in BRCA1. Cancer Res 2001; 61:7713–7718. 36. Hsu LC, White RL. BRCA1 is associated with the centrosome during mitosis. Proc Natl Acad Sci U S A 1998; 95:12983–12988. 37. 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

38.

39.

40.

41.

42.

43.

mouse oocytes, fertilized eggs, and early embryos. Biol Reprod 2004; 70: 1392–1399. Zheng ZY, Li QZ, Chen DY, Schatten H, Sun QY. Translocation of phospho-protein kinase Cs implies their roles in meiotic-spindle organization, polar-body emission and nuclear activity in mouse eggs. Reproduction 2005; 129:229–234. Yu LZ, Xiong B, Gao WX, Wang CM, Zhong ZS, Huo LJ, Wang Q, Hou Y, Liu K, Liu XJ, Schatten H, Chen DY, et al. MEK1/2 regulates microtubule organization, spindle pole tethering and asymmetric division during mouse oocyte meiotic maturation. Cell Cycle 2007; 6:330–338. Tong C, Fan HY, Lian L, Li SW, Chen DY, Schatten H, Sun QY. Pololike kinase-1 is a pivotal regulator of microtubule assembly during mouse oocyte meiotic maturation, fertilization, and early embryonic mitosis. Biol Reprod 2002; 67:546–554. Xu X, Weaver Z, Linke SP, Li C, Gotay J, Wang XW, Harris CC, Ried T, Deng CX. Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells. Mol Cell 1999; 3:389–395. Tirkkonen M, Johannsson O, Agnarsson BA, Olsson H, Ingvarsson S, Karhu R, Tanner M, Isola J, Barkardottir RB, Borg A, Kallioniemi OP. Distinct somatic genetic changes associated with tumor progression in carriers of BRCA1 and BRCA2 germ-line mutations. Cancer Res 1997; 57:1222–1227. Wells D, Bermudez MG, Steuerwald N, Thornhill AR, Walker DL, Malter H, Delhanty JD, Cohen J. Expression of genes regulating chromosome segregation, the cell cycle and apoptosis during human preimplantation development. Hum Reprod 2005; 20:1339–1348.