The FASEB Journal • Research Communication

Rab5a is required for spindle length control and - attachment during in oocytes

Rujun Ma,*,‡,1 Xiaojing Hou,‡,1 Liang Zhang,†,‡ Shao-Chen Sun,† Tim Schedl,§ Kelle Moley,§ and Qiang Wang‡,2 *College of Veterinary Medicine and †College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, China; ‡State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China; and §Washington University School of Medicine, St. Louis, Missouri, USA

ABSTRACT Rab GTPases are highly conserved com- this process can lead to the generation of aneuploid ponents of vesicle trafficking pathways. Rab5, as a eggs. Fertilization of aneuploid eggs in humans is a master regulator of endocytic trafficking, has been major cause of pregnancy loss, and if there is survival to shown to function in membrane tethering and docking. term, it will result in developmental disabilities (2). However, the function of Rab5 in meiosis has not been Although various molecules have been proposed to addressed. Here, we report elongated spindles and contribute to spindle/ defects in oocyte misaligned , with kinetochore-microtu- meiosis, the pathways and mechanisms that modulate bule misattachments, on specific depletion of Rab5a in the meiotic apparatus remain to be discovered. mouse oocytes. Moreover, the localization and levels of Rab GTPases, an extended family of small GTPases, F (CENPF), a component of the are highly conserved components of vesicle trafficking nuclear matrix, are severely reduced at in pathways. They function in determining vesicles iden- metaphase oocytes following Rab5a knockdown. Con- tity and control vesicle budding, uncoating, motility, sistent with this finding, nuclear lamina disassembly in and fusion by recruiting a specific set of effector the transition from prophase arrest to meiosis I is also (3, 4). In addition to their roles in vesicles, Rab impaired in Rab5a-depleted oocytes. Notably, oocyte- GTPases, and their cofactors and adaptors, have been specific ablation of CENPF phenocopies the meiotic implicated in spindle-based cellular events. Epsin, an defects resulting from Rab5a knockdown. In summary, endocytic adaptor protein, is required for proper spin- our data support a model where Rab5a-positive vesi- dle morphogenesis, potentially through controlling cles, likely through interaction with nuclear lamina, membrane curvature (5). Rab6a regulates the dynamics modulate CENPF localization and levels at centrom- of the dynein/dynactin complex at the kinetochores eres, consequently ensuring proper spindle length and kinetochore-microtubule attachment in meiotic oocytes.— and, consequently, the inactivation of the Mad2-spindle Ma, R., Hou, X., Zhang, L., Sun, S.-C., Schedl, T., checkpoint (6). Furthermore, elegant work of Holub- Moley, K., Wang, Q. Rab5a is required for spindle cova et al. (7) revealed that vesicles positive for the length control and kinetochore-microtubule attach- Rab11a modulate an actin network for asymmetric ment during meiosis in oocytes. FASEB J. 28, spindle positioning in oocytes. Rab5, as the master 4026–4035 (2014). www.fasebj.org regulator of the endocytic trafficking, has been well recognized to involve in membrane tethering and docking (8–11). Three isoforms of Rab5 (a, b, and c) Key Words: CENPF ⅐ vesicles ⅐ chromosome share Ͼ90% of sequence identity yet can be function- ally different (12). Of note, recent findings have sug- gested that Rab5 GTPase participates in chromosome The oocyte is a central component of female fertil- congression in Drosophila and human mitotic cells (13, ity. Growth and maturation of the oocyte are highly coordinated processes involving both nuclear and cyto- 14). However, the potential functional involvement of plasmic events (1). Accurate control of spindle assem- Rab5 in meiosis has not been addressed yet. bly and chromosome organization is required for or- In this study, we set out to investigate the role of derly meiosis during oocyte maturation, where errors in Rab5a during mouse oocyte meiosis. We discovered a

1 These authors contributed equally to this work. Abbreviations: CENPF, centromere protein F; GV, germinal 2 Correspondence: State Key Laboratory of Reproductive vesicle; GVBD, germinal vesicle breakdown; K-MT, kineto- Medicine, Nanjing Medical University, 140 Hanzhong Rd., chore-microtubule; MI, metaphase I; MII, metaphase II; MO, Nanjing, Jiangsu, 210029 China. E-mail: qwang2012@njmu. morpholino; Pb1, first polar body; PI, propidium iodide; edu.cn PMSG, pregnant mare serum gonadotropin doi: 10.1096/fj.14-250886

4026 0892-6638/14/0028-4026 © FASEB novel function of Rab5a-containing vesicles: control of the times in milrinone-free M2 medium, and cultured for differ- spindle length and chromosome alignment through mod- ent times. ulation of centromere protein F (CENPF) localization to the centromere, as reported below. Western blotting

A pool of ϳ100 oocytes was lysed in Laemmli sample buffer containing protease inhibitor and then subjected to 10% MATERIALS AND METHODS SDS-PAGE. The separated proteins were transferred to a PVDF membrane. Membranes were blocked in TBS contain- ing 0.1% Tween 20 and 5% low-fat dry milk for 1 h and then All chemicals and culture media were purchased from Sigma incubated with primary antibodies as follows: rabbit anti- (St. Louis, MO, USA) unless stated otherwise. ICR mice were Rab5a antibody (1:1000) or rabbit anti-CENPF antibody (1: used in this study. All experiments were approved by the 1500). After multiple washes in TBS containing 0.1% Tween Animal Care and Use Committee of Nanjing Medical Univer- 20 and incubation with horseradish peroxidase-conjugated sity and were performed in accordance with institutional secondary antibodies, the protein bands were visualized using guidelines. an ECL Plus Western Blotting Detection System (GE Health- care, Piscataway, NJ, USA). The membrane was then washed ␤ Antibodies and reblotted with anti- -actin (1:5,000) or anti-tubulin anti- body (1:3000) for loading control. Rabbit polyclonal anti-Rab5a (cat no. ab18211), rabbit poly- clonal anti-␤-actin (ab5441), and rabbit polyclonal anti- Immunofluorescence and confocal microscopy CENPF (ab5) antibodies were purchased from Abcam (Cam- ␣ bridge, MA, USA); mouse monoclonal anti- -tubulin-FITC Oocytes were fixed with 4% paraformaldehyde for 30 min and antibody was purchased from Sigma (76074); human anti- then permeabilized with 0.5% Triton X-100 for 20 min. centromere CREST antibody (09C-CS1058) was purchased Following blocking in 1% BSA-supplemented PBS for 1 h, from Fitzgerald Industries International (Concord, MA, samples were incubated overnight at 4°C with primary anti- USA); mouse monoclonal anti-NuMA antibody (610562) was bodies as follows: anti-Rab5a antibody, anti-lamin A/C anti- purchased from BD Transduction Laboratories (Lexington, body, anti-NuMA antibody, anti-CENPF antibody, and FITC- KY, USA); goat polyclonal anti-lamin A/C antibody (SC-6215) conjugated anti-tubulin antibody. To detect kinetochores, was obtained from Santa Cruz Biotechnology (San Jose, CA, oocytes were labeled with CREST (1:500) according to the USA); FITC-conjugated goat anti-rabbit IgG, FITC-conju- previous protocol (15). To examine the kinetochore-micro- gated donkey anti-goat IgG, and TRITC-conjugated goat tubule (K-MT) attachments, oocytes were briefly chilled at anti-rabbit IgG were purchased from Thermo Fisher Scien- 4°C to induce depolymerization of nonkinetochore microtu- tific (Rockford, IL, USA); and Cy5-conjugated goat anti- bules just before fixation. Chromosomes were stained with human IgG and Cy5-conjugated goat anti-rabbit IgG were propidium iodide (PI; red) or Hoechst 33342 (blue) for 10 purchased from Jackson ImmunoResearch Laboratory (West min. After washes with PBS, oocyte samples were mounted on Grove, PA, USA). antifade medium (Vectashield, Burlingame, CA, USA) and then examined under a laser-scanning confocal microscope (LSM 710; Zeiss, Oberkochen, Germany) equipped with the Oocyte collection and culture ϫ40 or ϫ63 oil objectives. To measure the fluorescent intensities of NuMA/CENPF, Z-stack images were used to Six- to 8-wk-old female mice were used for oocyte collection. generate a projection containing the sum of all the To collect fully grown germinal vesicle (GV) oocytes, mice CREST/CENPF signals. Fluorescent intensity of NuMA/ were superovulated with 5 IU pregnant mare serum gonado- CENPF was quantified by placing a small circle around the tropin (PMSG) by intraperitoneal injection, and 48 h later, signals. The average cytoplasmic fluorescence intensity was cumulus-enclosed oocytes were obtained by manual ruptur- subtracted as background. ImageJ software (U.S. National ing of antral ovarian follicles. Cumulus cells were removed by Institutes of Health, Bethesda, MD, USA) was used to quantify the spindle length and fluorescence intensity as repeatedly pipetting. For in vitro maturation, GV oocytes were described previously (16, 17). cultured in M2 medium under mineral oil at 37°C in a 5%

CO2 incubator. Chromosome spread Morpholino (MO) knockdown Chromosome preparations for metaphase II (MII) oocytes were as described previously (18). Oocytes were treated with Microinjection of MO, with a Narishige microinjector (Na- 1% sodium citrate for 20 min, individually transferred to a rishige Group, Tokyo, Japan), was used to knock down Rab5a glass slide, and then fixed with several drops of methanol/ and CENPF in mouse oocytes. Rab5a-MO 5=-TTGTTGCTC- acetic acid (3:1). After air drying and nuclear staining, the CTCGATTAGCCATGTC-3= and CENPF-MO 5=-GGCCCAGCTCA- chromosomes were observed by fluorescence microscopy. TCTTGTTTTATTTT-3= ( Tools, Philomath, OR, USA) targeting initiation of translation were diluted with water to give a stock concentration of 1 mM, and then a 2.5 pl MO Statistical analysis solution was injected into oocytes. A MO standard control was injected as control. Data are presented as means Ϯ sd, unless otherwise indi- After injections, oocytes were arrested at the GV stage in cated. Statistical comparisons were made with Student’s t test M2 medium supplemented with 2.5 ␮M milrinone for 20 h to and ANOVA when appropriate. Values of P Ͻ 0.05 were facilitate knockdown of mRNA translation, then washed 3 considered to be significant.

A ROLE FOR RAB5A IN OOCYTE MEIOSIS 4027 to be accumulated in the GV (Fig. 1, arrowheads). As the oocytes enter into metaphase, the vesicles become uni- formly localized in the cytoplasm. Of note, during ana- phase and , numerous vesicles become concen- trated around the spindle/chromosome region (Fig. 1, inset). Such a dynamic distribution pattern suggests that vesicular Rab5a may have a function in the formation or stability of meiotic spindle/chromosomes or in regulation of meiotic progression.

Depletion of Rab5a affects meiotic progression in oocytes

Next, to investigate the function of Rab5a in oocyte meiosis, we microinjected the Rab5a-targeting morpho- lino (Rab5a-MO) into fully grown oocytes. This led to a significant reduction of Rab5a protein and a corre- sponding loss of Rab5a-positive vesicles from oocytes, based on Western blot and immunostaining analysis (Fig. 2A, B). We then analyzed how the loss of Rab5a- positive vesicles affected oocyte maturation. Progression of oocyte maturation includes germinal vesicle breakdown (GVBD), organizing Figure 1. Cellular distribution of Rab5-positive vesicles during into the metaphase I (MI) spindle, chromosomes align- oocyte meiosis. Oocytes at GV, MI, and telophase I stages were ing at the MI metaphase plate, execution of the MI immunolabeled with Rab5a antibody (green) for vesicles and division, extruding the first polar body (Pb1), and then counterstained with PI for DNA (red) and visualized by confocal proceeding to arrest at MII until fertilization. Our microscopy. Arrowheads indicate examples of Rab5a-positive vesicles. Outlined regions are magnified in inset; 30 oocytes were results showed that Rab5a depletion appeared to affect analyzed for each group. Scale bar ϭ 25 ␮m. neither the morphology nor the fraction of oocytes that underwent GVBD (86.8Ϯ5.4 vs. 91.2Ϯ3.5% control; Fig. 2C). However, Pb1 extrusion was decreased in RESULTS Rab5a-MO oocytes compared with controls (68.6Ϯ7.2 vs. 87.7Ϯ5.3% control, PϽ0.05; Fig. 2D), indicative of Cellular distribution of vesicular Rab5a during oocyte the involvement of Rab5a in the meiotic process. meiosis Proper spindle length and chromosome alignment in We first examined Rab5a distribution during mouse oocytes requires Rab5a oocyte maturation. Immunostaining clearly showed the presence of Rab5a-containing vesicles in oocytes at differ- We then asked whether depletion of Rab5a also affects the ent developmental stages (Fig. 1), which are termed meiotic apparatus in oocytes. To this end, oocytes were Rab5a-positive vesicles in this study. These vesicles reside immunolabeled with anti-tubulin antibody to visualize the in the entire immature oocytes, and many of them appear spindle and costained with PI for chromosomes. Confocal

Figure 2. Effects of Rab5a depletion on oocyte maturation. Fully grown oocytes microinjected with Rab5a-MO were arrested at GV stage with milrinone for 20 h to block mRNA translation and then cultured in milrinone-free medium to evaluate the meiotic progression by phase-contrast microscopy. A sham MO standard was injected as control. A) Western blot showing partial knockdown of Rab5a after MO injection with actin as a loading control. Band intensity was calculated using ImageJ software, and the ratio of Rab5a/actin expression was normalized, and values are indicated. B) Immunostaining showing the loss of Rab5a-positive vesicles in oocytes with MO injection. Arrows indicate vesicles. Scale bar ϭ 25 ␮m. C, D) Quantitative analysis of GVBD (C) and Pb1 (D) extrusion in control (nϭ150) and Rab5a-MO (nϭ120) oocytes. Bars represent means Ϯ sd of results obtained in 3 independent experiments. *P Ͻ 0.05 vs. control.

4028 Vol. 28 September 2014 The FASEB Journal ⅐ www.fasebj.org MA ET AL. Figure 3. Proper spindle morphology and chromosome alignment in oocytes requires Rab5a. A) Control and Rab5a-MO oocytes were stained with ␣-tubulin antibody to visualize the spindle (green) and counterstained with PI to visualize chromosomes (red): control MII oocytes present a typical barrel-shaped spindle and well-aligned chromosomes on the metaphase plate (a); aberrant spindle (arrows) and misaligned chromosomes (arrowheads) were readily observed in Rab5a-MO MII oocytes (b–e). Representative confocal sections are shown. Scale bar ϭ 25 ␮m. B) Quantification of control and Rab5a-MO oocytes with chromosome misalignment. C) Quantitative analysis of spindle length in control and Rab5a-MO oocytes. D) Quantification of control and Rab5a-MO oocytes with spindle collapse. Data are expressed as mean Ϯ sd percentage from 3 independent experiments in which Ն100 oocytes were analyzed. *P Ͻ 0.05 vs. controls. microscopy revealed that Rab5a-MO oocytes displayed a type of matured oocytes. As postulated, loss of Rab5a high frequency of chromosome misalignment (20.5Ϯ6.8 resulted in ϳ3-fold increase in incidence of vs. 6.7Ϯ3.5% control, PϽ0.05; Fig. 3A, B, arrowheads) eggs compared with controls (Fig. 4A, B). Altogether, and obvious spindle morphology defects, remarkably in- these findings suggest that Rab5a-depleted oocytes are, cluding spindle elongation (Fig. 3Ac, d). Metaphase spin- in many cases, unable to properly assemble the meiotic dle lengths in Rab5a-MO oocytes were on average 40% spindle and align the meiotic chromosomes and thus longer than controls (Fig. 3A, C). An additional abnor- prone to produce aneuploid eggs. mality observed in Rab5a-MO oocytes was spindle collapse (10.5Ϯ2.8 vs. 3.7Ϯ1.5% control, PϽ0.05; Fig. 3Ae, D, Disrupted K-MT attachments in Rab5a-depleted oocytes arrow). These phenotypes contrasted sharply with meta- phases in control oocytes, which presented a typical Chromosome movement during meiosis involves a dy- barrel-shape spindle and well-aligned chromosomes at the namic interaction between spindle microtubules and equator (Fig. 3Aa). kinetochores, a macromolecular complex that localizes To determine whether the aberrant spindle and at the centromere of chromosomes. This interaction is misaligned chromosomes in Rab5a-MO oocytes would required for bipolar attachment and tension between act to generate aneuploid eggs, we analyzed the karyo- K-MT and subsequent alignment of chromosomes to

A ROLE FOR RAB5A IN OOCYTE MEIOSIS 4029 Figure 4. Increased aneuploidy in oocytes de- pleted of Rab5a. A) Chromosome spread of control and Rab5a-MO MII oocytes. Represen- tative fluorescence images of euploid and ane- uploid oocytes are shown. B) Quantification of aneuploidy in control and Rab5a-MO oocytes. Data are expressed as mean Ϯ sd percentage; 30 oocytes/group were analyzed. *P Ͻ 0.05 vs. controls.

the metaphase plate (19). The frequently observed and categorized as amphitelic attachment (the predom- spindle defects and chromosome misalignment in inant form in normal cells; each kinetochore attached Rab5a-MO oocytes suggested that K-MT attachments to one of the poles; Fig. 5B, chromosomes 1 and 2), loss are defective. To directly analyze K-MT attachment attachment (kinetochores unattached to either pole; status, we immunostained kinetochores with CREST Fig. 5B, chromosomes 3 and 4), merotelic/lateral at- and microtubules with anti-tubulin antibody following tachment (1 kinetochore attached to both poles; Fig. destabilizing dynamic nonkinetochore microtubules 5B, chromosomes 5–7), as well as undefined attach- through a brief cold treatment (Fig. 5). Four types of ment (Fig. 5B, ), indicated by different K-MT attachments were detected by confocal scanning color. Merotelic and loss attachments are regarded as

Figure 5. Correct K-MT attachments depend on Rab5a. A) Control and Rab5a-MO MI oocytes were stained with anti-tubulin (microtubules, green), CREST (kineto- chore, purple), and Hoechst 33342 (chromosomes, blue). Representative confocal sections are shown. B) Magnified views for the K-MT attachments in the oocytes shown in A. Kinetochore-microtubule attach- ments are classified into 4 categories: amphitelic (chro- mosomes 1 and 2), loss ( and 4), mero- telic/lateral (–7), and undefined (chromosome 8) attachment. Image border colors cor- respond to the categories. C) Quantitative analysis of K-MT attachments in oocytes as indicated. Attachment of kinetochores to microtubules was assessed through examination of the full series of Z-axis focal planes. Kinetochores in regions where fibers were not easily visualized were not included in the analysis; 10 control oocytes and 8 Rab5a-MO oocytes were analyzed. Scale bars ϭ 10 ␮m. *P Ͻ 0.05 vs. controls.

4030 Vol. 28 September 2014 The FASEB Journal ⅐ www.fasebj.org MA ET AL. Figure 6. Depletion of Rab5a impairs nuclear lamina disassembly in oocytes. A) Control and Rab5a-MO oocytes were labeled with anti-lamin A/C antibody (green) and counterstained with PI to visualize chromosomes (red). Representa- tive images of GV and MI oocytes are shown. Arrowhead in Rab5a-MO MI oocyte shows re- sidual nuclear lamina as judged by lamin A/C staining. Scale bar ϭ 25 ␮m. B) Quantification of control and Rab5a-MO oocytes showing per- sistent lamin at metaphase stage. At least 50 oocytes were counted for each group. Bars represent means Ϯ sd.*P Ͻ 0.05 vs. controls.

errors that must be corrected because they would cause oocytes (Fig. 6A). In contrast, persistent lamin around chromosome missegregation if they persisted until ana- the region of meiotic chromosomes, albeit not as intact phase (20). Quantitative analysis (Fig. 5C) demonstrated as controls, was observed in Ͼ15% of Rab5a-MO meta- that the loss/merotelic attachment in Rab5a-MO oocytes phase oocytes (Fig. 6A, B). The results suggest that was greatly increased in frequency relative to control cells Rab5a is essential for the properly disassembly of nu- (loss: 15.5Ϯ3.6 vs. 3.8Ϯ0.9% control, PϽ0.05; merotelic/ clear lamina during oocyte maturation. lateral: 19.9Ϯ4.6 vs. 4.6Ϯ0.8% control, PϽ0.05), whereas the proportion of amphitelic attachment was accord- ingly decreased (57.8Ϯ4.9 vs. 88.7Ϯ3.2% control, Loss of Rab5a does not affect NuMA localization or PϽ0.05). These K-MT misattachments, if uncorrected, levels in oocytes would lead to imbalanced force on the kinetochore NuMA has been implicated in spindle assembly and and, consequently, establish unstable chromosome chromosome congression in mitotic cells (25, 27). biorientation, which could contribute to the chromo- Kolano et al. (28) recently showed that without func- some alignment failure observed in Rab5a-depleted tional NuMA in mouse oocytes, microtubules lose con- oocytes. nection to spindle poles, resulting in highly disorga- nized early spindle assembly and long spindles with Depletion of Rab5 impairs oocyte nuclear lamina hyperfocused poles, phenotypes that are highly similar disassembly to what we observed following Rab5a knockdown. Thus, we assess the affect of Rab5a depletion on NuMA We then searched for a potential mechanism that localization and/or levels in oocytes. We observed would explain the requirement of Rab5a for maintain- nuclear NuMA puncta in GV oocytes and then concen- ing spindle morphology and chromosome alignment in trated at the spindle poles at metaphase in control oocytes. To date, a battery of Rab5 effector proteins oocytes (Fig. 7A), as previously reported (29). Double mediating diverse cellular events have been identified staining further revealed colocalization of NuMA and in different tissue and cell types (13, 14, 21, 22). Of Rab5a vesicles inside the GV (Fig. 7C). Confocal mi- note, recent findings in Caenorhabditis elegans and Dro- croscopy and quantitative analysis, however, demon- sophila suggest that Rab5 also promotes the disassembly strated that Rab5a depletion in oocytes had no signifi- of the nuclear lamina structure (13, 14). Lamin is the cant effects on NuMA localization or levels (Fig. 7A, B), major component of the nuclear lamina that is dis- suggesting that NuMA is unlikely to be an effector persed on mitotic entry. Among all the Rab5 targets, protein for Rab5a in oocytes. Mud (NuMA-like protein in Drosophila) and CENPF are of particular interest to us because they have been implicated to associate with nuclear matrix (23, 24) and Pronounced CENPF reduction in oocytes depleted affect spindle poles and kinetochore function in mi- of Rab5a totic cells (25, 26). Based on these data, we proposed that depletion of Rab5a might impair nuclear enve- CENPF is a component of the nuclear matrix in G2 lope/matrix disassembly, which in turn perturbs the phase and has been reported to be required for chro- functions of NuMA and/or CENPF, resulting in the mosome alignment and stable microtubule attach- defects of spindle morphology and chromosome align- ments at kinetochores in mitotic cells (23, 26, 30). ment in oocytes. Nonetheless, its function during meiosis remains un- To test this hypothesis, we first examined whether the known. To test whether the effect of Rab5a depletion nuclear envelope disassembly was disrupted during on spindles and chromosomes may be mediated Rab5a-MO oocyte maturation. Immunostaining with through CENPF, we first examined the localization/ anti-lamin A/C antibody showed the presence of intact levels of endogenous CENPF by immunofluorescence. nuclear lamina in control GV oocytes. With the resump- As indicated in Fig. 8A (arrowheads), CENPF puncta tion of meiosis, nuclear lamina was completely dis- were uniformly distributed in GV and then predomi- persed in most of prometaphase/metaphase of control nantly translocated to the kinetochores in metaphase of

A ROLE FOR RAB5A IN OOCYTE MEIOSIS 4031 Figure 7. Loss of Rab5a in oocytes has no effect on NuMA localization and levels. A) Control and Rab5a-MO oocytes were labeled with anti-NuMA antibody (green) and counterstained for chromosomes (red). Arrowheads indicate NuMA localization. Representative images of GV and MI oocytes are shown. Scale bar ϭ 25 ␮m. B) Quantification of NuMA staining in control and Rab5a-MO MI oocytes. At least 50 oocytes were analyzed for each experiment. C) Double staining of GV oocytes with anti-NuMA and anti-Rab5a antibodies. Error bars indicate Ϯ sd. control oocytes, consistent with a function for CENPF CENPF knockdown phenocopies the spindle/ in chromosome movement. Double staining was also chromosome defects resulting from Rab5a depletion performed in metaphase oocytes and confirmed that CENPF puncta colocalize with kinetochores, indicated Given the drastic effects of Rab5a depletion on CENPF by arrowheads (Fig. 8C). Notably, following Rab5a localization/levels, we reasoned that functional abla- knockdown, localization of CENPF to kinetochores was tion of CENPF may recapitulate at least some of the severely reduced and total CENPF was significantly meiotic defects observed in Rab5a-MO oocytes. To decreased, based on immunostaining and Western blot- investigate this possibility, fully grown oocytes were ting respectively (Fig. 8A, B, D). These data suggested microinjected with CENPF-targeting morpholino (CENPF- that CENPF localization/levels at kinetochores during MO) and then stained with anti-tubulin antibody to oocyte meiosis depend on Rab5a. evaluate spindle and counterstained with PI for chro-

Figure 8. Rab5a Depletion reduces the CENPF local- ization/levels on kinetochores in oocytes. A) Control and Rab5a-MO oocytes were labeled with anti-CENPF antibody (green) and counterstained for chromo- somes (red). Representative images of GV and MI oocytes are shown; arrowheads indicate CENPF local- ization. B) Quantification of CENPF staining in con- trol and Rab5a-MO oocytes; 30 oocytes/group were analyzed. C) Double staining of metaphase oocytes with CREST antibody (red) and CENPF antibody (green), and counterstaining of chromosome with Hoechst 33342 (blue), confirming the CENPF puncta colocalization with kinetochores (arrowheads). D) West- ern blot showing the dramatically reduced CENPF pro- tein levels in oocytes following Rab5a knockdown, with tubulin as a loading control. Bars represent means Ϯ sd.*P Ͻ 0.05 vs. controls.

4032 Vol. 28 September 2014 The FASEB Journal ⅐ www.fasebj.org MA ET AL. Figure 9. CENPF knockdown phenocopies the spindle/chromosome defects resulting from Rab5a depletion. A) Reduced CENPF levels after CENPF-MO injection were confirmed by Western blot. Tubulin served as a loading control. Band intensity was calculated using ImageJ; ratio of CENPF/tubulin expression was normalized, and values are indicated. B) Control and CENPF-MO oocytes were stained with ␣-tubulin antibody to visualize the spindle (green) and counterstained with PI to observe chromosomes (red). Representative examples of spindle and chromosomes in control MII oocytes (a) and CENPF-MO oocytes (b–d). Arrow indicates the abnormal spindle; arrowheads indicate misaligned chromosomes. Scale bar ϭ 25 ␮m. C) Quantification of control (nϭ110) and CENPF-MO (nϭ100) oocytes with chromosome misalignment. D) Quantitative analysis of K-MT attachments in oocytes as indicated (10 control oocytes and 10 CENPF-MO oocytes). E) Quantitative analysis of spindle length in control (nϭ45) and CENPF-MO (nϭ50) oocytes. F) Quantification of control (nϭ110) and CENPF-MO (nϭ100) oocytes with spindle collapse. Data are expressed as mean Ϯ sd percentage. *P Ͻ 0.05 vs. controls. mosomes. Endogenous levels of CENPF were partially Rab5a and CENPF in oocytes was conducted. We found reduced as evidenced by Western blot (Fig. 9A). Con- that the aberrant meiotic phenotypes of the single knock- sistent with the hypothesis, CENPF-MO oocytes displayed downs were not exacerbated in the double knockdown similar phenotypes (Fig. 9B–F). Chromosome misalign- oocytes (data not shown), consistent with Rab5a and ment was readily observed in oocytes depleted of CENPF CENPF operating in the same pathway. Together, these (23.1Ϯ5.9 vs. 7.3Ϯ2.9% control, PϽ0.05; arrowheads). observations suggested that CENPF is a major, if not CENPF knockdown elevated the proportion of K-MT unique, target mediating the action of Rab5a on meiotic misattachments in oocytes, and the frequency of elon- apparatus during oocyte maturation. gated spindles and disorganized spindles was also mark- edly increased, in comparison to control oocytes (length: 35.3Ϯ4.2 vs. 24.1Ϯ1.4 ␮m control, PϽ 0.05; collapse: DISCUSSION 9.6Ϯ3.8 vs. 2.5Ϯ1.2% control, PϽ0.05). In addition, to test whether Rab5a and CENPF might function in a The cytoplasm of a typical eukaryotic cell is populated parallel or in a linear pathway, simultaneous depletion of with a variety of membranous organelles, and a vast

A ROLE FOR RAB5A IN OOCYTE MEIOSIS 4033 array of molecules traffic between these organelles by sembly during oocyte maturation. Since CENPF is vesicular transport. Rab GTPases are key regulatory known to be a nuclear matrix protein, impairment of factors that impinge on vesicle identity, coating, motility, lamina disassembly during oocyte meiosis perhaps in- and fusion, etc. (3, 31). In the present study, we showed terferes with the release and subsequent translocation that MO-mediated Rab5a ablation led to the pronounced of CENPF to kinetochores or leads to destabilization of loss of Rab5a-positive vesicles in mouse oocytes (Fig. 2). A CENPF. Due to the limitation of oocyte number and high frequency of chromosome misalignment was de- technical reason, we have not yet been able to directly tected in these oocytes depleted of Rab5a-positive vesi- analyze the dynamic interaction between nuclear enve- cles (Fig. 3), as described in mitotic cells (13, 14). lope disassembly and CENPF release in mouse oocytes. Furthermore, we provide evidence that such meiotic In summary, our data support a model where Rab5a- defects could dramatically increase aneuploidy in eggs positive vesicles, likely through interaction with nuclear (Fig. 4). Attachment between spindle microtubules and lamina, promote CENPF localization/levels at kineto- chromosomes depends on the kinetochore. The kinet- chores, consequently ensuring proper spindle mor- ochore is a hierarchical protein assembly of nearly 100 phology and K-MT attachment in meiotic oocytes. proteins that links centromeric DNA to spindle micro- Oocyte quality is a critical element dictating the tubules and thereby couples forces generated by micro- fertility of a female. This study reveals Rab5a-positive tubule dynamics to power chromosome movement. vesicles as cytoskeletal regulators that are required for The interplay between kinetochores and microtubules meiotic spindle morphology and meiotic chromosome leads to the establishment of biorientation (32, 33). alignment in oocytes. As any errors in this process result Defects in chromosome alignment may therefore arise in pregnancy loss and infertility, this vesicle-based path- from impairment of chromosome attachment to kinet- way is not only of fundamental scientific interest but ochores. In support of this notion, the K-MT attach- also vital for mammalian reproduction. ment errors were readily observed in metaphase oocytes depleted of Rab5a (Fig. 5). These observations The authors thank Dong Zhang (Nanjing Medical Univer- collectively suggest that loss of Rab5a-positive vesicles sity) for technical assistance. This work was supported by could disrupt the K-MT attachment, and thereby con- National Key Scientific Research Projects (2014CB943200), tribute to the chromosome congress failure in oocytes. National Natural Science Foundation (31301181), and Natu- ral Science Foundation of the Jiangsu Higher Education So far, the mechanisms by which spindle length is Institutions (13KJA310001) of China. The authors declare no restrained in mammalian oocytes remain poorly under- conflicts of interest. stood (34). Here we identified that, in addition to the chromosome misalignment, oocytes depleted of either Rab5a or CENPF presented a novel phenotype: spindle elongation (Fig. 3). This finding indicates that Rab5a REFERENCES and CENPF act in a process that functions in spindle 1. Krisher, R. L. (2004) The effect of oocyte quality on develop- length control in oocytes. Also noteworthy is that ment. J. Animal Sci. 82(E-Suppl.), E14–E23 Rab11a-positive vesicles have recently been identified as 2. Hassold, T., and Hunt, P. (2001) To err (meiotically) is human: a cytoskeletal regulator to affect the asymmetric spindle the genesis of human aneuploidy. Nat. Rev. Genet. 2, 280–291 positioning during mouse oocyte maturation (7). All 3. Barr, F. A. (2013) Review series: Rab GTPases and membrane identity: causal or inconsequential? J. Cell Biol. 202, 191–199 these findings support the concept that vesicle-based 4. Stenmark, H. (2009) Rab GTPases as coordinators of vesi- pathways play important roles in modulating the mei- cle traffic. Nat. Rev. Mol. Cell Biol. 10, 513–525 otic apparatus of oocytes, which deserve further study. 5. Liu, Z., and Zheng, Y. (2009) A requirement for epsin in mitotic Based on the literature (13, 14, 23–28), we reasoned membrane and spindle organization. J. Cell Biol. 186, 473–480 that NuMA and/or CENPF are likely to be major 6. Miserey-Lenkei, S., Couedel-Courteille, A., Del Nery, E., Bardin, S., Piel, M., Racine, V., Sibarita, J. B., Perez, F., Bornens, M., and targets mediating the effects of Rab5a-positive vesicles Goud, B. (2006) A role for the Rab6A’ GTPase in the inactiva- on spindle/chromosomes in oocytes. However, Rab5a tion of the Mad2-spindle checkpoint. EMBO J. 25, 278–289 depletion had little effects on NuMA distribution/ 7. Holubcova, Z., Howard, G., and Schuh, M. (2013) Vesicles levels (Fig. 7). Instead, we discovered that loss of Rab5a modulate an actin network for asymmetric spindle positioning. Nat. Cell Biol. 15, 937–947 caused considerable reduction of CENPF localization/ 8. Bucci, C., Lutcke, A., Steele-Mortimer, O., Olkkonen, V. M., levels at kinetochores in meiotic oocytes (Fig. 8). Pre- Dupree, P., Chiariello, M., Bruni, C. B., Simons, K., and Zerial, vious work has shown that CENPF is a large coiled-coil M. (1995) Co-operative regulation of endocytosis by three protein that transiently localizes to the outer kineto- Rab5 isoforms. FEBS Lett. 366, 65–71 9. Horiuchi, H., Lippe, R., McBride, H. M., Rubino, M., Wood- chore (32) and is required for stable microtubule man, P., Stenmark, H., Rybin, V., Wilm, M., Ashman, K., Mann, capture at kinetochores in mitotic cells (26, 30, 35). M., and Zerial, M. (1997) A novel Rab5 GDP/GTP exchange However, its function in meiosis was unknown. Here we factor complexed to Rabaptin-5 links nucleotide exchange to showed that CENPF knockdown results in spindle/ effector recruitment and function. Cell 90, 1149–1159 chromosome defects and K-MT misattachments in 10. Christoforidis, S., McBride, H. M., Burgoyne, R. D., and Zerial, M. (1999) The Rab5 effector EEA1 is a core component of oocytes, similar to the phenotypes of Rab5a knockdown endosome docking. Nature 397, 621–625 oocytes (Fig. 9), suggesting that CENPF is a critical 11. Simonsen, A., Gaullier, J. M., D’Arrigo, A., and Stenmark, H. target through which Rab5a-positive vesicles exert their (1999) The Rab5 effector EEA1 interacts directly with syn- effects on oocyte meiosis. We cannot rule out that other taxin-6. J. Biol. Chem. 274, 28857–28860 12. Chen, P. I., Kong, C., Su, X., and Stahl, P. D. (2009) Rab5 Rab5a targets also may mediate this process. As re- isoforms differentially regulate the trafficking and degradation ported in other systems (13, 14, 36), we also found that of epidermal growth factor receptors. J. Biol. Chem. 284, 30328– Rab5a-positive vesicles influence nuclear lamina disas- 30338

4034 Vol. 28 September 2014 The FASEB Journal ⅐ www.fasebj.org MA ET AL. 13. Capalbo, L., D’Avino, P. P., Archambault, V., and Glover, D. M. 25. Silk, A. D., Holland, A. J., and Cleveland, D. W. (2009) Require- (2011) Rab5 GTPase controls chromosome alignment through ments for NuMA in maintenance and establishment of mamma- lamin disassembly and relocation of the NuMA-like protein Mud lian spindle poles. J. Cell Biol. 184, 677–690 to the poles during . Proc. Natl. Acad. Sci. U. S. A. 108, 26. Holt, S. V., Vergnolle, M. A., Hussein, D., Wozniak, M. J., Allan, 17343–17348 V. J., and Taylor, S. S. (2005) Silencing Cenp-F weakens 14. Serio, G., Margaria, V., Jensen, S., Oldani, A., Bartek, J., centromeric cohesion, prevents chromosome alignment and Bussolino, F., and Lanzetti, L. (2011) Small GTPase Rab5 activates the spindle checkpoint. J. Cell Sci. 118, 4889–4900 participates in chromosome congression and regulates localiza- 27. Haren, L., Gnadt, N., Wright, M., and Merdes, A. (2009) NuMA tion of the centromere-associated protein CENP-F to kineto- is required for proper spindle assembly and chromosome chores. Proc. Natl. Acad. Sci. U. S. A. 108, 17337–17342 alignment in prometaphase. BMC Res. Notes 2, 64 15. Zhang, D., Ma, W., Li, Y. H., Hou, Y., Li, S. W., Meng, X. Q., Sun, 28. Kolano, A., Brunet, S., Silk, A. D., Cleveland, D. W., and Verlhac, X. F., Sun, Q. Y., and Wang, W. H. (2004) Intra-oocyte localiza- M. H. (2012) Error-prone mammalian female meiosis from tion of MAD2 and its relationship with kinetochores, microtu- silencing the spindle assembly checkpoint without normal in- bules, and chromosomes in rat oocytes during meiosis. Biol. terkinetochore tension. Proc. Natl. Acad. Sci. U. S. A. 109, Reprod. 71, 740–748 E1858–E1867 16. Wang, Q., Chi, M. M., and Moley, K. H. (2012) Live imaging 29. Yu, L. Z., Xiong, B., Gao, W. X., Wang, C. M., Zhong, Z. S., Huo, reveals the link between decreased glucose uptake in ovarian L. J., Wang, Q., Hou, Y., Liu, K., Liu, X. J., Schatten, H., Chen, cumulus cells and impaired oocyte quality in female dia- D. Y., and Sun, Q. Y. (2007) MEK1/2 regulates microtubule betic mice. Endocrinology 153, 1984–1989 organization, spindle pole tethering and asymmetric division 17. Fu, W., Chen, H., Wang, G., Luo, J., Deng, Z., Xin, G., Xu, N., during mouse oocyte meiotic maturation. 6, 330–338 Guo, X., Lei, J., Jiang, Q., and Zhang, C. (2013) Self-assembly 30. Bomont, P., Maddox, P., Shah, J. V., Desai, A. B., and Cleveland, and sorting of acentrosomal microtubules by TACC3 facilitate D. W. (2005) Unstable microtubule capture at kinetochores kinetochore capture during the mitotic spindle assembly. Proc. depleted of the centromere-associated protein CENP-F. EMBO J. Natl. Acad. Sci. U. S. A. 110, 15295–15300 24, 3927–3939 18. Hunt, P. A., Koehler, K. E., Susiarjo, M., Hodges, C. A., Ilagan, A., Voigt, R. C., Thomas, S., Thomas, B. F., and Hassold, T. J. 31. Hutagalung, A. H., and Novick, P. J. (2011) Role of Rab GTPases (2003) Bisphenol a exposure causes meiotic aneuploidy in the in membrane traffic and cell physiology. Physiol. Rev. 91, 119– female mouse. Curr. Biol. 13, 546–553 149 19. Tanaka, T. U., Clayton, L., and Natsume, T. (2013) Three wise 32. Tanaka, K. (2013) Regulatory mechanisms of kinetochore- centromere functions: see no error, hear no break, speak microtubule interaction in mitosis. Cell. Mol. Life Sci. 70, 559– no delay. EMBO Rep. 14, 1073–1083 579 20. Gregan, J., Polakova, S., Zhang, L., Tolic-Norrelykke, I. M., and 33. Foley, E. A., and Kapoor, T. M. (2013) Microtubule attachment Cimini, D. (2011) Merotelic kinetochore attachment: causes and spindle assembly checkpoint signalling at the kinetochore. and effects. Trends Cell Biol. 21, 374–381 Nat. Rev. Mol. Cell Biol. 14, 25–37 21. Galvez, T., Gilleron, J., Zerial, M., and O’Sullivan, G. A. (2012) 34. Howe, K., and FitzHarris, G. (2013) Recent insights into spindle SnapShot: mammalian Rab proteins in endocytic trafficking. function in mammalian oocytes and early embryos. Biol. Reprod. Cell 151, 234–234 e232 89, 71 22. Kawasaki, M., Nakayama, K., and Wakatsuki, S. (2005) Mem- 35. Yang, Z., Guo, J., Chen, Q., Ding, C., Du, J., and Zhu, X. (2005) brane recruitment of effector proteins by Arf and Rab GTPases. Silencing mitosin induces misaligned chromosomes, premature Curr. Opin. Struct. Biol. 15, 681–689 chromosome decondensation before onset, and mi- 23. Liao, H., Winkfein, R. J., Mack, G., Rattner, J. B., and Yen, T. J. totic cell death. Mol. Cell. Biol. 25, 4062–4074 (1995) CENP-F is a protein of the nuclear matrix that assembles 36. Audhya, A., Desai, A., and Oegema, K. (2007) A role for Rab5 in onto kinetochores at late G2 and is rapidly degraded af- structuring the endoplasmic reticulum. J. Cell Biol. 178, 43–56 ter mitosis. J. Cell Biol. 130, 507–518 24. Radulescu, A. E., and Cleveland, D. W. (2010) NuMA after 30 Received for publication February 9, 2014. years: the matrix revisited. Trends Cell Biol. 20, 214–222 Accepted for publication May 19, 2014.

A ROLE FOR RAB5A IN OOCYTE MEIOSIS 4035