WDR62 is a novel participator in spindle migration and asymmetric cytokinesis during mouse oocyte meiotic maturation

Journal Pre-proof WDR62 is a novel participator in spindle migration and asymmetric cytokinesis during mouse oocyte meiotic maturation Yong-Sheng Wang, Xiao-Fei Jiao, Fan Chen, Di Wu, Zhi-Ming Ding, Yi-Liang Miao, Li-Jun Huo PII:

S0014-4827(19)30658-5

DOI:

https://doi.org/10.1016/j.yexcr.2019.111773

Reference:

YEXCR 111773

To appear in:

Experimental Cell Research

Received Date: 26 August 2019 Revised Date:

8 December 2019

Accepted Date: 9 December 2019

Please cite this article as: Y.-S. Wang, X.-F. Jiao, F. Chen, D. Wu, Z.-M. Ding, Y.-L. Miao, L.-J. Huo, WDR62 is a novel participator in spindle migration and asymmetric cytokinesis during mouse oocyte meiotic maturation, Experimental Cell Research (2020), doi: https://doi.org/10.1016/j.yexcr.2019.111773. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

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Title: WDR62 is a novel participator in spindle migration and

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asymmetric cytokinesis during mouse oocyte meiotic maturation

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Authors: Yong-Sheng Wang1,2, Xiao-Fei Jiao1,2, Fan Chen1,2, Di Wu1,2, Zhi-Ming

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Ding1,2, Yi-Liang Miao1, and Li-Jun Huo1, 2*

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1

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Ministry of Education, College of Animal Science and Technology, Huazhong

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Agricultural University, Wuhan 430070, Hubei, China

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Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of

Department of Hubei Province Engineering Research Center in Buffalo Breeding and

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Products, Wuhan 430070, Hubei, China

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*Correspondence to: Li-Jun Huo ([email protected])

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Tel: +86 27 87281813

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Highlights

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division in mouse oocytes.

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WDR62 depletion compromised the first polar body extrusion and asymmetric

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WDR62 knockdown disrupted spindle organization and chromosome alignment in mouse oocytes.

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WDR62 participated in regulating meiotic spindle migration in mouse oocytes.

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WDR62 participated in regulating the distribution of cortical actin and Arp2/3

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complex in mouse oocytes.

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Abstract

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In female meiosis, oocyte meiotic maturation is a form of asymmetric cell

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division, producing the first polar body and a large oocyte, in which the asymmetry of

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oocyte meiotic division depends on spindle migration and positioning, and cortical

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polarization. In this study, we conclude that WDR62 (WD40-repeat protein 62) plays

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an important role for asymmetric meiotic division in mouse oocyte. Our initial study

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demonstrated that WDR62 mainly co-localized with chromosomes during mouse

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oocyte meiotic maturation. Interference of Wdr62 by siRNA microinjection did not

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affect germinal vesicle breakdown (GVBD) but compromised the first polar body

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extrusion (PBE) with the large polar bodies generated, which is coupled with a higher

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incidence of spindle abnormality and chromosome misalignment. Further analysis

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concluded that loss of WDR62 blocked asymmetric spindle positioning and actin cap

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formation, which should be responsible for large polar body extrusion. Moreover

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WDR62 decline intervened with the Arp2/3 complex, an upstream regulator for the

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cortical actin. Besides for p-MAPK, a critical regulator for the asymmetric division of

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oocyte, WDR62-depleted oocytes showed perturbation only in localization pattern but

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not expression level. In summary, our study defines WDR62 as an essential

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cytoskeletal regulator of spindle migration and asymmetric division during mouse

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oocyte meiotic maturation.

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Keywords: WDR62; oocyte; spindle migration; asymmetric cytokinesis

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1. Introduction

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In mammals, polar body emission, mainly involving nuclear division and

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cytoplasmic asymmetric division, is indispensable mandatory for oocytes to complete

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meiosis and confirm early embryonic development. The cytoplasmic asymmetric

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division requests precise and accurate meiotic spindle migration and positioning, and

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establishment of a cortical actomyosin domain overlying the spindle [1]. As germinal

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vesicle breakdown (GVBD), the bipolar spindle assembles close to the oocyte center

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[2], migrates to the cortex [3, 4] and activates the extrusion of a minuscule polar body

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with one half of homologous chromosomes [5]. Taken all together, confirm that

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oocyte has sufficient storage of maternally synthesized gene products and fuel to

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complete the first steps of embryonic development smoothly [6]. It is widely held that

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actin filaments mediate the spindle migration, but not the microtubules [7]. Actin is

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mandatory for oocytes to keep their shape for growth, polarization and replication [8].

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Moreover, several actin nucleation factors have been shown that regulates polar body

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extrusion and cytokinesis by mediating actin-dependent spindle migration, such as

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Arp2/3 complex [9], Fmn2[10]

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stability are considered to be involved in regulation of asymmetric division, especially

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MAPK (mitogen-activated protein kinase). In Mos

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centrally but fail to migrate, which results in large polar body formation or symmetric

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division [12]. Recently increasing molecules have been reported to affect the spindle

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migration, yet the mechanism involved is not very clear and needs to be explored to

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understand asymmetric division.

and Spire1/2 [11]. Furthermore, regulators of spindle

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–/–

oocytes, the spindles form

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The absence of centrosomes is another feature of oocytes. Serving as the major

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microtubule organizing center (MTOC) in most animal cells, centrosome which

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comprises a pair of orthogonally oriented centrioles surrounded by pericentriolar

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material (PCM) [13] directs the assembly of the microtubule cytoskeleton during

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mitosis [14], and it is essential for several fundamental cellular processes, including

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cell polarity and division [15]. Moreover, increased studies have highlighted some

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complex links between centrosome defects and cancer [16], whereas mutations in

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centrosomal proteins have been genetically linked dwarfism and microcephaly

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(MCPH) [17]. Contradictory to the mitotic spindles, oocyte meiotic spindles in many

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animals totally lack centrioles, including humans and the laboratory models of mice,

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frogs, fruit flies, and nematodes [18-21] while the spindle assembly is orchestrated by

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the self-organization of over 80 microtubule organizing centers [22]. The

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aforementioned contradiction raises the question what functional role centrosomal

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proteins have during oocyte meiosis.

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WD40-repeat protein 62 (WDR62) was first characterized as involved in the

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assembly of signaling complexes, containing 13 annotated WD40 domain repeats

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which spanned the N-terminal half of the protein [23]. After finding homozygous

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missense and frame-shifting mutations in seven MCPH families [24], mutations in

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WDR62 as the second most common cause of primary microcephaly, together with

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ASPM, account for more than half of all cases [25]. Similar to Caenorhabditis elegans,

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the association between MCPH proteins and centrosome is evolutionarily conserved

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[26, 27]. The proteins, encoded by microcephaly related genes ,including WDR62 and

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ASPM (abnormal spindle-like, microcephaly-associated), are localized to the mitotic

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spindle poles, which suggest a common biological function [28]. Currently some

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studies have proposed that WDR62 and other MCPH proteins form a hierarchy in

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which each is required to localize another to the centrosome; besides, distinct

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centriolar satellite proteins facilitate to localize their cognate MCPH interactors to

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centrosomes and promote centriole duplication [29]. Moreover, it has been

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extensively shown that decline in WDR62 causes spindle instability, decreased

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integrity of centrosomes and dislocation of centriole from the spindle pole, spindle

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assembly checkpoint (SAC) activation, delayed mitotic progression and cell death [23,

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30, 31].

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Although WDR62 exerts regulatory function during mitosis, especially in

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centriole duplication, to our knowledge, the role of WDR62 during mouse oocyte

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meiotic maturation remains unknown. Here, we deciphered a novel role of WDR62 in

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spindle migration and asymmetric cytokinesis during mouse oocyte meiotic

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maturation by employing siRNA knockdown analysis.

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2. Methods and Materials

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2.1 Antibodies and reagents

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Rabbit anti-WDR62 polyclonal antibody (Cat# GTX119724) was purchased from

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GeneTex (Irvine, CA); mouse anti-Arp3 antibody (Cat# A5979), mouse

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anti-α-tubulin-FITC antibody (Cat# F2168) and FITC-phalloidin (Cat # P5282) was

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purchased from Sigma (St Louis, MO); rabbit anti-phospho-p44/42 MAPK

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monoclonal antibody (Cat# 4370) was purchased from CST (Danvers, MA).

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Cy3-conjugated goat anti-rabbit IgG (H+L) and Cy3- conjugated goat anti-mouse IgG 6

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(H + L) were purchased from Boster Biotechnology Co., LTD (Wuhan; China). All

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other chemicals and culture media were purchased from Sigma Chemical Company

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(St Louis, MO) except for those specifically mentioned.

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2.2 Animals and ethics statement

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Wild-type Kunming strain (KM) mice were obtained from local Central Animal

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Laboratory and housed under a 12 h light/12 h dark regimen at 22 °C with water and

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food ad libitum. All experimental procedures of animals were performed in

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accordance with the rules stipulated by the Animal Care and Use Committee of

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Huazhong Agricultural University (HZAUSW-2017-005).

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2.3 Oocyte retrieval and culture

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Ovaries were isolated from 3-4 week-old KM mice sacrificed by cervical

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dislocation after intraperitoneal injection of 5 IU pregnant mare serum gonadotropin

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(PMSG) for 48 hours. Cumulus cells were removed by repeatedly pipetting and

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oocytes were collected in pre-warmed (37°C) M2 medium supplemented with 50 µM

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IBMX to arrest the oocytes at GV-stage.

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To resume meiosis, oocytes were washed out of IBMX and released into fresh

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M2 medium (Sigma) at 37 °C and 5% CO2 in air for 0, 2, 8, 9 and 14 h,

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corresponding to GV stage, germinal vesicle breakdown (GVBD) stage, metaphase I

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(MI), anaphase I (AI) and metaphase II (MII), respectively. Oocytes at a specific stage

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were collected according to the experiment purpose.

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2.4 Immunofluorescence and confocal microscopy

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Oocytes at the specific stage were fixed in 4% paraformaldehyde in PBS (pH 7.4)

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for 30 minutes and permeabilized in 0.5% Triton-X-100 for 30 min at room

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temperature. And then, oocytes were blocked in PBS containing 2% BSA and 0.05%

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Tween-20 for 1 h at room temperature and incubated with anti-WDR62 antibody

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(GeneTex, 1:100), anti-Arp3 antibody (Sigma, 1:100), or anti--phospho-p44/42

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MAPK antibody (CST, 1:100), at 4 °C overnight. After washing in PBS containing

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0.05% Tween-20 for 3 times and 10 min each, oocytes were incubated with Cy3-

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conjugated goat anti-rabbit (Boster; 1:100) or Cy3-conjugated goat anti-mouse

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(Boster; 1:100), for 1 h at 37°C. For double staining of spindle or actin, oocytes were

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stained with anti-α-tubulin-FITC antibody (Sigma, 1:100) or labeled with

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Phalloidin-FITC (Sigma, 1:100) for 1 h at 37°C. After washing three times, DNA was

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counterstained with DAPI (1 µg/ml) for 10 min at room temperature. Finally, oocytes

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were mounted on glass slides with DABCO and examined under a confocal laser

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scanning microscope (Carl Zeiss 800, Germany). Confocal images were further

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processed using Zeiss LSM Image Browser software and Adobe Photoshop (Adobe

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Systems Inc., San Jose, CA). Oocytes were incubated with fluorescence-labeled

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secondary antibodies as negative control and the primary antibody was replaced by

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non-immune IgG.

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2.5 Western blotting

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Samples containing about 200 oocytes each group were briefly washed in PBS

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and then lysed in 2 × SDS loading buffer and boiled for 5 min, and then stored at

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−80°C until use. The proteins were separated by SDS-PAGE and electrically

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transferred to PVDF membranes (Immobilon-P; Millipore). The membranes were

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washed briefly in TBST and then blocked in TBST containing 5% skim milk at room

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temperature for 1 h, followed by incubation overnight at 4 °C with WDR62 antibody

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(GeneTex, 1:1000), Arp3 antibody (Sigma, 1:1000), or phospho-p44/42 MAPK

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antibody (CST, 1:1000), at 4 °C overnight. After washing 3 times in TBST (10 min

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each), the membranes were incubated at 37 °C for 1 h with HRP conjugates secondary

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antibodies. Finally, the membranes were washed in TBST and the immunoblot bands

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were processed with ECL kit and visualized using the chemiluminescence system

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(Thermo Scientific). β-actin (Santa Cruz, 1:500), α-tubulin (Proteintech, 1:500) and

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GAPDH (Santa Cruz, 1:500) were served as a loading control. The relative signal

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intensity was assessed by Image J software (NIH, USA). Blank control oocytes were

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incubated with HRP-conjugated secondary antibody as negative control and the

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primary antibody was replaced by non-immune IgG.

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2.6 Microinjection of Wdr62-targeted short interfering siRNA

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For WDR62 knockdown, GV-stage oocytes were collected in M2 medium

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containing 50 µM IBMX. After 1 h recovery 5–10 pL of 40 µM control siRNA

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(sc-37007; Santa Cruz, CA) or Wdr62 siRNA (siRNAPack1999; Ribobio, Guangzhou,

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China) was injected into the cytoplasm of oocytes. Following microinjection, the

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oocytes were arrested at the GV stage in M2 medium containing 50 µM IBMX

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(Sigma) for 24 h to efficiently achieve WDR62 knockdown. Next, oocytes were

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directly collected for western blotting or immunostaining or thoroughly washed out of

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IBMX and released into M16 medium for in vitro meiotic maturation or other

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experiments.

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2.7 Statistical analysis

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At least three independent replicates were used for each analysis. Data were

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presented as mean ± SEM and analyzed by paired-samples t-test using SPSS software

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(SPSS Inc, Chicago, IL) while P < 0.05 was considered to be statistically significant.

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Statistical difference was indicated by different superscripts.

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3. Results

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3.1. Subcellular localization and expression of WDR62 during mouse oocyte meiotic

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maturation

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To investigate the role of WDR62 in mouse oocyte maturation, we first examined

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the dynamic distribution and expression of WDR62 at different development stages.

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Oocytes were cultured in vitro for 0, 2, 8, 9 or 14 h until they reached the GV, GVBD,

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MI, AI, and MII stages, respectively. As shown in Fig. 1A, WDR62 co-localized with

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the chromosomes from GV to MII stages. The subcellular localization pattern of

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WDR62 associated with chromosomes during oocyte meiotic maturation, indicated

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that WDR62 might be involved in the regulation of meiotic progression in oocytes.

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Moreover, the relative protein level pattern of WDR62 during oocyte meiotic

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maturation was examined by Western blotting analysis. The result showed that the

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expression of WDR62 in oocytes was significantly reduced from GV to MII stage

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(Figure 1B). Based on these results, we speculated that WDR62 might have a

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previously unknown function in oocyte meiosis regulation.

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3.2. Knockdown of WDR62 affects the first polar body extrusion and asymmetric

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division

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To further explore its function during oocyte maturation, WDR62 was knocked

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down by microinjection of Wdr62 specific siRNA into GV-stage oocytes and then

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maintained at the GV stage for 24 h to achieve knockdown efficiency. Compared with

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oocytes microinjected with control siRNA, Western blot analysis revealed that the

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expression of WDR62 was significantly reduced in oocytes microinjected with Wdr62

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siRNA (Figure 2A). After WDR62 knockdown, oocytes were continuously cultured

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up to 14 h for maturation. After 2 h in culture, we found that knockdown of WDR62

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had no significant effect on the GVBD rate when compared with that in the control

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group (83.5 ± 1.6% vs. 91.8 ± 4.2%, P > 0.05, Fig. 2B). However, a high frequency of

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RNAi oocytes was unable to complete meiosis with no polar bodies or exhibited large

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polar body extrusion after 14 h in culture (Fig. 2C). Then, we analyzed the rate of the

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first polar body extrusion and large polar body extrusion in the control group and

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RNAi group, respectively. The rate of first polar body extrusion (PBE) was

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significantly reduced in RNAi group (39.3 ± 4.1% vs. 62.3 ± 3.6%, P < 0.05, Fig. 2D).

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Furthermore, the rate of large polar body extrusion of RNAi oocytes was also

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significantly higher than that of control group (16.5 ± 1.6% vs. 4.0 ± 0.8%, P < 0.01,

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Figure 2E). Taken together, these observations suggested that WDR62 had a critical

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role in oocyte meiotic maturation and asymmetric division.

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3.3. Knockdown of WDR62 disrupts spindle organization and chromosome

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alignment during mouse oocyte meiotic maturation

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Given that WDR62 co-localized with the chromosomes during oocyte meiosis

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and the proportion of meiotic maturation declined in RNAi oocytes, we analyzed the

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spindle morphology and chromosome alignment in oocytes after WDR62 knockdown.

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As shown in Fig. 3A, most oocytes had typical barrel-shaped spindles and

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well-aligned chromosomes on the metaphase plate at MI stage in control group.

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However, we found a high frequency of spindle organization defects (38.6 ± 4.6% vs.

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11.4 ± 3.7%, P < 0.01; Figure 3A and B) and chromosome alignment failure (46.3 ±

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1.0% vs. 13.4 ± 1.2%, P < 0.001; Figure 3A and C) in RNAi oocytes, displaying

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multipolar spindles with more than two poles, abnormal spindles with pointy poles,

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distorted spindles with one or several scattered chromosomes. These results suggested

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that depletion of WDR62 caused defective spindle morphogenesis and abnormal

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chromosome alignment, which indicated that WDR62 had a critical role in spindle

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architecture and chromosome alignment.

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3.4. Knockdown of WDR62 affects spindle migration during mouse oocyte meiotic

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maturation

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In mouse oocytes, asymmetric meiotic divisions are driven by the eccentric

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positioning of the spindle [32]. As the spindle positioning was the key step of the

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asymmetric division of oocytes [33], we next examined the spindle positioning to

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explore the reason for the large polar body extrusion after WDR62 knockdown. After

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culturing for 9 h, the spindle migrated to the oocyte cortex in most control oocytes,

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while the most spindles failed to migrate to cortex and stayed in the center of RNAi

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oocytes (Fig. 4A). Moreover, we analyzed the rate of the spindle localization pattern

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between the control group and RNAi group. The result showed that the rate of cortex

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located spindle in control group was significantly higher than that of RNAi group

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(74.0 ± 3.0% vs. 42.1 ± 4.3%, P < 0.01; Fig. 4B). While in RNAi oocytes, a higher

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proportion of spindles anchored to the center of cytoplasm compared with the control

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oocytes (57.9 ± 4.3% vs. 26.0 ± 4.3%, P < 0.01; Fig. 4B).

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3.5. Knockdown of WDR62 causes the failure formation of actin cap during mouse

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oocyte meiotic maturation

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The formation of actin cap is one of the predominant features of oocyte

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polarization [34], which is essential for the success of asymmetric division. Therefore,

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we examined the actin cap formation to investigate the effect of WDR62 on actin

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polymerization. Oocytes were collected after 8 h, 9 h or 14 h in culture, corresponding

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to MI, AI and MII stage, respectively. As shown in Fig. 5A, the actin caps were

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clearly observed on the membrane of control oocytes at these stages (arrowhead),

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proven by the fluorescence plot profiling. However, in sharp contrast, the clear actin

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caps were not found in RNAi oocytes. Moreover, the chromosomes localized under

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the region of the cortex with an actin cap in the control oocytes, while in RNAi

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oocytes, they segregated centrally with no actin cap. Furthermore, the actin cap

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formation of oocytes at AI stage was examined by quantitative analysis. The result

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showed that the proportion of oocytes with actin cap in RNAi oocytes was

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significantly lower than that in control group (40.2 ± 8.4% vs. 76.7 ± 5.0%, P < 0.001;

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Figure. 5B).

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3.6. Knockdown of WDR62 alters the distribution and expression of Arp3

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It is well known that Arp2/3-dependent actin dynamics are critical to leading edge

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protrusion in migrating cells [35]. Inhibition of Arp2/3 or its activator N-WASP

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diminished actin cap formation [36]. To explore the reason for the failure of actin cap

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formation in RNAi oocytes, Arp3 was examined. Significantly, Arp3 accumulated in

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the cortical region that overlaid the chromosomes, which was coincident with the

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actin cap in control oocytes, while in shape contrast, polarized Arp3 signals were

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undetectable in RNAi oocytes (Figure. 6A). Quantitative analysis demonstrated that

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Arp3 cap formation on cortex was significantly reduced in RNAi oocytes in

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comparison to control (32.1 ± 1.5% vs. 64.5 ± 4.3%, P < 0.01; Figure. 6B).

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Furthermore, we revealed Arp3 expression was increased in MI oocytes after WDR62

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knockdown by Western blot analysis (Fig. 6C).

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3.7. Knockdown of WDR62 has no effect on the expression of p-MAPK but perturbs

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its localization

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It has been reported that active mitogen-activated protein kinase (MAPK) is

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required for cortical reorganization [8]. Furthermore, live cell imaging showed that

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the spindle forms centrally but does not migrate in Mos –/– oocytes [37], similar to the

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phenomena observed in the oocytes when WDR62 was abated. To inspect the activity

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of p-MAPK, the control and RNAi oocytes were cultured in vitro for 8.5 h until they

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reached the post-MI stage for western blot and immunofluorescent staining to

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determine the expression and localization, respectively. As shown in Fig. 7A,

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p-MAPK was localized at the spindle poles in control oocyte, while the distribution of

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p-MAPK in RNAi oocyte was dispersed with separated chromosomes in the center of

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cytoplasm, which was coincided with the observed aberrant spindle morphogenesis in

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WDR62-depleted oocytes. However, the expression of p-MAPK was not affected

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after WDR62 knockdown (Figure. 7B). Therefore, we found that WDR62 participated

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in actin filament-mediated spindle migration during meiotic asymmetric division

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(Figure. 8) independent from the MAPK pathway.

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4. Discussion

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Among extensively expressed MCPH proteins, which have a centrosomal

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association related to part of cell cycle [38], WDR62 expresses in cytoplasm during

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interphase and localizes to the spindle pole in mitosis, showing strikingly cell

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cycle-dependent localization [39], whereas the cells with mutants of WDR62 lose the

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ability to restrict WDR62 localization to the mitotic spindle during cell division [24].

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Moreover, several studies have proposed that that WDR62 has varieties of functions

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in mitotic cells, especially in maintaining the integrity of centriole [40-42], while

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WDR62 depletion can cause mitotic delay with compromised centrosomal integrity

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and spindle abnormality. However, little is known regarding the regulation of WDR62

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in mouse oocyte which is absent in typical centrosomes.

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In this study, we discovered that WDR62 appeared to be co-localized with the

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chromosomes during mouse oocyte meiotic maturation, and disruption of PBE was

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observed with specific depletion of WDR62 in oocytes, moreover, a certain

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percentage of MII oocytes with large polar body were observed. To investigate the

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reason for decline in PBE rate, we examined the spindle assembly after WDR62

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knockdown. As expected, the abnormal spindle morphogenesis accompanied with

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defective chromosome alignment was observed through immunofluorescent analysis.

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Furthermore, observation of RNAi treated oocyte revealed that oocyte division to

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generate two MII oocytes was symmetric, resulted oocytes were of similar size

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instead of a large oocyte with small polar body.

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Asymmetric division is dependent on spindle migration mediated by actin, and

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dynamic changes in the cytoskeleton organization regulate the oocyte meiotic

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maturation [33]. Likewise, we found that the spindle migration and subsequent

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cortical reorganization were disturbed and characterized by an arrested spindle in the

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central cytoplasm, and a loss of actin cap in mouse oocyte with WDR62 knockdown.

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These findings suggests WDR62 might regulate actin filaments for spindle

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positioning, as actin filaments are the main power for spindle movement in

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mammalian oocyte [43]. The Arp2/3 complex is verified to be involved in multiple

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processes during mouse oocyte maturation. Disrupting the function of Arp2/3 by

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RNAi or specific inhibitor could interfere, not only with the spindle migration but also

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the cortical reorganization [44]. Furthermore, published data has validated that Arp2/3

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complex which localizes to the cortical cap is important for maintaining asymmetric

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meiotic spindle migration by generating an actin polymerization-driven cytoplasmic

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streaming [36]. Inline to these studies, we observed that the localization pattern of the

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Arp2/3 complex was disconcerted after WDR62 knockdown, which should be

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responsible for the disruption of actin polymerization in cortical region.

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Previous studies have shown that the Mos/MAPK pathway regulates oocyte

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asymmetric division. The establishment of cortical polarity is critical for asymmetric

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meiotic cell division, and the subcortically positioned chromatin induces cortical

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reorganization in a MOS-dependent manner [45]. During the polar body extrusion in

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starfish oocytes, MAPK is required for spindle attachment to the cortex and the

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formation of cleavage furrow [46]. Moreover, in the oocytes, microinjected of Arf1

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dominant-negative mutant, the meiotic spindles do not position at the cortex during

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meiosis I stage and result in extrusion of large polar bodies, supposed to be regulated

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by decline in expression of p-MAPK [47]. The C-terminal region of WDR62 contains

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serine/threonine phosphorylation motifs, which interact with proline-directed kinases

361

such as MAPKs and cyclin-dependent kinases (CDKs) [48]. WDR62 specifically

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binds components of the JNK pathway to potentiate stress-stimulated signal

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transduction [23] and interacts with MKK7 (MAPK kinase 7) via direct

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protein-protein interactions in somatic cells [49, 50]. Therefore, we speculated that the

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formation of large polar body in WDR62-depleted oocytes might be connected with

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the MAPK pathway. To confirm our hypothesis, the localization and expression of

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p-MAPK in anaphase oocytes were examined. Nonetheless, our results showed that

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the activation of MAPK was not perturbed, whereas the localization was disrupted

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following WDR62 knockdown, which strengthen our finding that knockdown of

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WDR62 induced abnormal spindle microtubule organization. Hence we verified that

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WDR62 is involved in the regulation of asymmetric cell division independent of

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MAPK pathway.

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WDR62 contains WD40 repeat (WDR) domain, which is a typically seven bladed

374

β-propeller domain of donut shape [51]. As one of the most abundant and interacting

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domains in the eukaryotic genome[52], WDR domains typically serve as interaction

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platforms for multiple proteins, and let them ideally fit into the cellular interaction

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networks [53]. Some studies have revealed that WDR62 can scaffold kinases that are

378

important mitotic regulators such as JNK and AURKA (Aurora kinase A) in somatic

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cells [54]. Thus WDR62 may serves as a scaffold protein to orchestrate specific

380

transmission of signaling. Recently, to explain the mechanism underlying spindle

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migration, a model posits that MI spindle migration is biphasic. This model has

382

indicated that following Fmn2-mediated initial movement, the spindle experiences a

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fast phase of migration via cortical Arp2/3-orchestrated cytoplasmic streaming [1]. In

384

this model, asymmetry breaking and polarity maintenance depend on a positive

385

feedback loop between cortical polarization and asymmetric spindle movement,

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which contact these two well-known characteristics of oocyte maturation. When the

387

chromosomes migrate to the vicinity of cortex within a distance the chromatin signal

388

transmitted by Ran guanosine triphosphate (RanGTP) gradient stimulates the polarized

389

accumulation of the cortical N-WASP-Arp2/3 machinery to nucleate actin

390

polymerization [32], thereby driving the formation of the polarized actin cap and

391

promoting cytoplasmic streaming to maintain spindle positioning [55]. Furthermore,

392

the subcortically positioned chromatin or DNA-coated beads can induce the

393

establishment of cortical polarity and assembly of a myosin II-based ring, and the Ran

394

GTPase plays a critical role in this process [56]. Similar to the localization of RCC1,

395

the exchange factor of the RanGTP gradient, WDR62 co-localized with the

396

chromosome. Based on the localization of WDR62 during mouse oocyte maturation

397

and the larger polar body extrusion when WDR62 abated, we hypothesized that

18

398

WDR62 might cooperate with RCC1 to participate in transmitting chromosome signal,

399

for accumulating cortical Arp2/3 complex to regulate actin nucleation during meiotic

400

asymmetric division. Therefore, when the expression of WDR62 declined, the

401

activation of RCC1 would be increased for compensation, so that the expression of

402

Arp2/3 complex was increased as shown in Figure 6C. However, Arp2/3 complex was

403

not the direct downstream of RCC1, thus when the transmission of chromosome

404

signal was affected after WDR62 knockdown, its localization would be affected.

405

However, this hypothesis required further verification.

406

In conclusion, our results demonstrated a novel function of WDR62 in spindle

407

migration and asymmetric cytokinesis during mouse oocyte maturation. However, it

408

remains unclear what role WDR62 has in the transmission of chromatin signal during

409

polar body extrusion, and warranted further investigations.

410

Author Contributions

411

Y.S.W. and L.J.H. conceived and designed experiments; Y.S.W and X.F.J.

412

performed experiments; F.C, D.W, Z.M.D. and Y.L.M provided new tools and

413

reagents; Y.S.W. and L.J.H. wrote the manuscript; L.J.H. made manuscript revisions.

414

Funding

415

This work was supported by the National Key Research and Development Program

416

(2017YFD0501701), the Natural Science Foundation of Hubei Province (Grant#

417

2018CFA015) and the Fundamental Research Funds for the Central Universities

418

(Program No. 2662018PY037).

419

Conflicts of interest Statement

19

420

The authors declare that the research was conducted in the absence of any commercial

421

or financial relationships that could be construed as a potential conflict of interest.

422

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591 592 593 594 595

27

Figure legends

596 597

Figure 1

Subcellular localization and expression pattern of WDR62 during

598

mouse oocyte maturation. (A) Subcellular localization of WDR62 detected by

599

immunofluorescent staining. Oocytes at indicated stages were immunostained for

600

WDR62 (red), microtubule (α-tubulin; green) and DNA (blue). Scale bar, 20 µm. (B)

601

Expression pattern of WDR62 during mouse oocyte meiotic maturation. Oocytes were

602

collected after 0, 2, 8 or 14 h in culture, corresponding to GV, GVBD, MI and MII

603

stage, respectively. The molecular weight of WDR62 and β -actin was 167 kD and 43

604

kD respectively. Normalized signal intensity of WDR62 was presented. Data were

605

presented as mean percentage (mean ± SEM) of at least three independent

606

experiments. **P < 0.01 and ***P < 0.001.

607

Figure 2: Knockdown of WDR62 affects the first polar body extrusion and

608

asymmetric division. Live GV-stage oocytes microinjected with control or Wdr62

609

siRNA were incubated in M2 medium containing 50 µM IBMX for 24 h and then

610

released into IBMX-free M16 medium for in vitro maturation. (A) Knockdown of

611

endogenous WDR62 expression after Wdr62-siRNA injection was verified by Western

612

blot analysis with α-tubulin as a loading control. Band intensity was measured by

613

Image J software. (B) Rate of GVBD in control and RNAi oocytes subsequently

614

continuously observed at 2 h. (C) Representative images of polar body in control and

615

RNAi oocytes. (D) Rate of polar body extrusion (PBE) in control and RNAi oocytes

616

after 14 culture. (E) Rate of the large polar body extrusion in control and RNAi

617

oocytes after 14 culture. A total of 146 oocytes in control group and 144 oocytes in

28

618

RNAi group were analyzed. Data were presented as mean percentage (mean ± SEM)

619

of at least three independent experiments. NS, not significant, *P < 0.05, **P < 0.01

620

and ***P < 0.001.

621

Figure 3: Effects of WDR62 knockdown on spindle organization and

622

chromosome alignment in oocyte. (A) Oocytes at metaphase I stage were stained

623

with anti-tubulin antibody to visualize spindle (green) and counterstained with DAPI

624

to visualize chromosomes (blue). Representative confocal images from control and

625

RNAi oocytes were shown. The control oocyte exhibited typical barrel-shaped spindle

626

and well-aligned chromosomes on the metaphase plate. Spindle defects and

627

chromosomes misalignment were frequently observed in RNAi oocytes. Scale bar, 20

628

µm. (B and C) Rate of aberrant spindle and misaligned chromosome in control (n =

629

80) and RNAi (n = 82) oocytes. Data were presented as mean ± SEM. **P < 0.01 and

630

***P < 0.001.

631

Figure 4: Knockdown of WDR62 affects spindle migration in oocyte meiosis. (A)

632

After culturing for 9 h, oocytes were stained with anti-tubulin antibody to visualize

633

spindle (green) and counterstained with DAPI to visualize chromosomes (blue). The

634

spindle formed in the central cytoplasm and migrated to oocyte cortex in control

635

oocytes. However, the spindle was still in the central cytoplasm in RNAi oocytes.

636

Scale bar, 20 µm. (B) Quantitative analysis of spindle position (center and cortex) in

637

control (n = 86) and RNAi (n = 78) oocytes. Data were presented as mean ± SEM.

638

**P < 0.01.

639

Figure 5: Knockdown of WDR62 disturbs the formation of actin cap during

29

640

oocyte maturation. MI, AI and MII oocytes were labeled with phalloidin to visualize

641

actin (green) and were counterstained with DAPI for chromosomes (blue). (A)

642

Representative confocal images showed actin distribution in control and RNAi

643

oocytes. Arrows indicated the position of actin cap in control oocytes. Scale bar, 20

644

µm. (B) Quantitative analysis of the proportion of actin cap formation in control (n =

645

83) and RNAi (n = 77) oocytes at anaphase I stage. Data were presented as mean ±

646

SEM. *P < 0.05.

647

Figure 6: Effects of WDR62 knockdown on the localization and expression of

648

Arp3 in oocyte. (A) Representative images showed the distribution of Arp3 in control

649

and RNAi oocytes. Oocytes cultured for 9 hours were immunostained for Arp3 (red),

650

actin (green) and DNA (blue). The arrow indicated the polarized distribution and the

651

asterisk indicated the position of actin cap in control oocytes. Scale bar, 20 µm. (B)

652

Quantitative analysis of control (n = 51) and RNAi (n = 46) oocytes with polarized

653

distribution of Arp3. Data were presented as mean ± SEM. **P < 0.01. (C) The Arp3

654

expression was increased in RNAi oocytes. Band intensity analysis also showed that

655

Arp3 expression was increased compared with that of control. *P < 0.05.

656

Figure 7: Effects of WDR62 knockdown on p-MAPK in oocyte.

657

Representative images showed the distribution of p-MAPK in control and RNAi

658

oocytes. Oocytes at post-MI stage were immunostained for p-MAPK (red), actin

659

(green) and DNA (blue). Scale bar, 20 µm. (B) Expression of p-MAPK in control and

660

RNAi oocytes.

661

Figure 8: A diagram of WDR62 functions in spindle migration during mouse

30

(A)

662

oocyte maturation. The Arp2/3 complex which localizes to the cortical cap in a

663

RanGTP gradient - dependent manner regulates meiotic spindle migration by generating

664

an actin polymerization-driven cytoplamic streaming during meiotic asymmetric

665

division [1], and WDR62 may participate in chromatin signal transmission for Arp2/3

666

activation or localization by cooperating with RCC1 to regulate the RanGTP gradient.

667 668 669 670 671

31

Highlights •

WDR62 depletion compromised the first polar body extrusion and asymmetric division in mouse oocytes.

•

WDR62 knockdown disrupted spindle organization and chromosome alignment in mouse oocytes.

•

WDR62 participated in regulating meiotic spindle migration in mouse oocytes.

•

WDR62 participated in regulating the distribution of cortical actin and Arp2/3 complex in mouse oocytes.

Author Contributions Y.S.W. and L.J.H. conceived and designed experiments; Y.S.W and X.F.J. performed experiments; F.C, D.W, Z.M.D. and Y.L.M provided new tools and reagents; Y.S.W. and L.J.H. wrote the manuscript; L.J.H. made manuscript revisions.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.