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Rab5a Is Required for Spindle Length Control and Kinetochore-Microtubule Attachment During Meiosis in Oocytes

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Rab5a Is Required for Spindle Length Control and Kinetochore-Microtubule Attachment During Meiosis in Oocytes The FASEB Journal • Research Communication Rab5a is required for spindle length control and kinetochore-microtubule attachment during meiosis 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/chromosome defects in oocyte misaligned chromosomes, 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, centromere protein F (CENPF), a component of the are highly conserved components of vesicle trafficking nuclear matrix, are severely reduced at kinetochores 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 proteins (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/
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