Biochimica et Biophysica Acta 1853 (2015) 317–327

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Formin mDia1, a downstream molecule of FMNL1, regulates Profilin1 for actin assembly and spindle organization during mouse oocyte meiosis☆ Yu Zhang a, Fei Wang a, Ying-Jie Niu a, Hong-Lin Liu a, Rong Rui b, Xiang-Shun Cui c, Nam-Hyung Kim c, Shao-Chen Sun a,⁎ a b c

College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China Department of Animal Sciences, Chungbuk National University, Cheongju, Chungbuk, 361-763, Republic of Korea

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Article history: Received 26 August 2014 Received in revised form 6 October 2014 Accepted 4 November 2014 Available online 15 November 2014 Keywords: mDia1 Profilin1 Actin Spindle formation Polar body extrusion Oocyte

a b s t r a c t Mammalian diaphanous1 (mDia1) is a homologue of Drosophila diaphanous and belongs to the Forminhomology family of proteins that catalyze actin nucleation and polymerization. Although Formin family proteins, such as Drosophila diaphanous, have been shown to be essential for cytokinesis, whether and how mDia1 functions during meiosis remain uncertain. In this study, we explored possible roles and the signaling pathway involved for mDia1 using a mouse oocyte model. mDia1 depletion reduced polar body extrusion, which may have been due to reduced cortical actin assembly. mDia1 and Profilin1 had similar localization patterns in mouse oocytes and mDia1 knockdown resulted in reduced Profilin1 expression. Depleting FMNL1, another Formin family member, resulted in reduced mDia1 expression, while RhoA inhibition did not alter mDia1 expression, which indicated that there was a FMNL1-mDia1-Profilin1 signaling pathway in mouse oocytes. Additionally, mDia1 knockdown resulted in disrupting oocyte spindle morphology, which was confirmed by aberrant p-MAPK localization. Thus, these results demonstrated indispensable roles for mDia1 in regulating mouse oocyte meiotic maturation through its effects on actin assembly and spindle organization. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The meiosis and mitosis, both including DNA replication and cytokinesis events, are essential processes that emerge in mammal development. The mitosis, a kind of continuous symmetric division, encompasses a round of DNA replication and cytokinesis. Eventually, the mitotic cell divides into two identical size daughter cells, which contain the same genome as the parental cells. Unlike the mitosis, firstly, mammalian oocyte meiosis is characterized by a unique asymmetric division, as the spindle migrates to the cortex and extrudes a small polar body. Secondly, during meiosis, the mammalian oocyte undergoes two consecutive rounds of asymmetric divisions while only one round of DNA replication, generating a totipotent haploid egg. This brief period, so called ‘meiotic maturation’, is essential for maintaining female genome integrity. Oocytes are arrested at the diplotene stage of the first meiotic prophase within ovarian follicles, which is also defined as the ☆ Funding: This work was supported by the National Basic Research Program of China (2014CB138503), the Natural Science Foundation of Jiangsu Province (BK20140030), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20130097120055), and the Biogreen 21 Program (PJ009594, PJ009080 and PJ00909801), RDA, Republic of Korea. ⁎ Corresponding author at: College of Animal Sciences and Technology, Nanjing Agricultural University, Nanjing, China. Tel./fax: +86 25 84399092. E-mail address: [email protected] (S.-C. Sun).

http://dx.doi.org/10.1016/j.bbamcr.2014.11.005 0167-4889/© 2014 Elsevier B.V. All rights reserved.

germinal vesicle (GV) stage. During oocyte maturation, fully grown oocytes reinitiate meiosis, as indicated by germinal vesicle breakdown (GVBD). Finally, an oocyte proceeds through the first meiosis, and then transforms into a metaphase II (MII)-arrested oocyte with an extruded small first polar body (pbI) until it is fertilized [1]. Unlike the mitotic division that the actomyosin furrow ingression mainly depends on the actomyosin ring contraction, interestingly, the polar body extrusion is a specialized cytokinesis, coordinated by the cortical membrane protrusion and actomyosin ring contraction [2]. Additionally, in contrast to the mitosis during which a centrally positioned spindle midzone leads to the bilateral furrowing from the center of the somatic cells, the meiotic spindle midzone-induced membrane furrow changes from the initial unilateral to the eventual bilateral during oocyte polar body extrusion [3]. Polar body extrusion is a highly coordinated process that depends on the dynamic coordination of microtubules and actin filaments related events during divisions [4,5]. During meiosis I, microtubules form a specialized bipolar-shaped spindle at the metaphase I (MI) stage. Accurate spindle assembly is required for orderly meiosis during oocyte maturation [6]. In addition to the induction of actin organization by chromatin, spindle assembly is also attributed to this process. During cell cycle progression, the polymerized actin distributes parallel to the plasma membrane at the oocyte cortex, and plays a critical role in the establishment of cortical rigidity to act as a foundation for proper

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spindle assembly [7,8]. This regulation may be due to the physical interactions between the plus ends of astral microtubules with a rigid actin cortex that facilitate placing, shortening, and focusing of the nascent spindle [9,10]. Also, actin filaments ensure the targeting of microtubule spindle positioning at the periphery of an oocyte concomitant with chromosome segregation [11,12]. Additionally, a fine network of microfilaments is found throughout the entire cortex and is responsible for constructing the classical contractile ring, which constricts at the base of the protrusion for pinching off a polar body [2,13]. Although several molecules have been proposed to contribute to cytoskeletal regulation during oocyte meiosis in mammals, the molecular mechanisms that modulate the meiotic apparatus remain to be determined. Formin family proteins are essential for diverse cellular processes, including vesicle trafficking [14,15], cell migration [16,17], and microtubule stabilization [18,19]. Formins also have highly conserved, critical functions for cytokinesis [20–22]. Additionally, Formins are conserved actin nucleators that are involved in promoting the assembly of actin filaments [23]. Furthermore, a previous study has shown that a Formin protein, Formin2 is responsible for correct positioning of the metaphase spindle and final abscission of the first polar body during meiosis [24]. mDia proteins are the mammalian homologues of Drosophila diaphanous, which is a member of the Formin-homology family of proteins and is a downstream effector of the small GTPase Rho. In mammals, the mDia family comprises 3 isoforms, mDia1 (diaphanous1), mDia2 (diaphanous2), and mDia3 (diaphanous3) [23,25], which vary in their subcellular localization, enzymatic activity, and their targets. mDia1 and mDia2 are crucial for cell migration and cell adhesion [26]. In addition, mDia proteins are strongly implicated in cell division. Several lines of evidence indicate that an activated mDia and diaphanous are essential for catalyzing linear actin nucleation and polymerization during cytokinesis [27,28]. In addition to their functions for actin assembly, mDia proteins reportedly stabilize and align microtubules in mitotic cells [18,28,29]. Among these, mDia2 has been the most extensively investigated. Studies have indicated that mDia2 was involved in actin assembly regulation during cytokinesis [30,31]. mDia3, which is found at the kinetochores of condensed chromosomes in HeLa cells, might be essential for microtubule-mediated chromosome segregation during mitosis [29]. mDia1 is also crucial to a large variety of cellular and morphogenetic functions via its effects on actin assembly [32,33]. Although mDia proteins have been implicated in multiple critical biological processes during mitosis, their physiological functions and the signaling cascade involved during oocyte meiosis have not been investigated. For this study, we proposed that mDia1 would have significant roles during mouse oocyte maturation. To confirm our hypothesis, we used RNA interference (RNAi) to deplete the mDia1 isoform in mouse oocytes and assessed mDia1 effects with regard to cytoskeletonmediated dynamic events during mouse oocyte meiotic maturation. Our results indicated a direct contribution of mDia1 in mouse oocyte meiosis in that mDia1-induced actin assembly and spindle organization were critical for polar body extrusion by dividing oocytes.

2.2. Oocyte harvest and culture ICR mice were used for all experiments. Our experiments were approved by the Animal Care and Use Committee of Nanjing Agriculture University and were performed in accordance with Animal Research Institute Committee guidelines. Mice were housed in a temperaturecontrolled room with an appropriate light: dark cycle, fed a regular diet, and maintained under the care of the Laboratory Animal Unit, Nanjing Agricultural University. Young female mice (6–8 weeks) were used for oocyte collection. For in vitro maturation, germinal vesicleintact oocytes were harvested from female ovaries and cultured in M2 medium under paraffin oil at 37 °C in a 5% CO2 atmosphere. Oocytes were removed from culture at different times for microinjection, realtime RT-PCR, and immunofluorescent staining and Western blot analysis. 2.3. Nocodazole treatment of oocytes For nocodazole treatment, 10 mg/ml nocodazole in DMSO stock was diluted in M2 medium to give a final concentration of 20 μg/ml. After incubating in M2 medium supplemented with nocodazole for 10 min, the oocytes were collected for immunofluorescence microscopy after 9 h culture. 2.4. Real-time quantitative pcr analysis Real-time quantitative PCR analysis was used to examine mDia1 mRNA expression in oocytes. Total RNA was extracted from 30 oocytes using a Dynabeads mRNA DIRECT kit (Invitrogen Dynal AS), and singlestranded cDNA was generated using a PrimeScript™ RT reagent kit (Takara, Dalian, China) with Oligo (dT) 12–18 primers (Invitrogen). The cDNA products were then diluted 6-fold with deionized water before use as a template for real-time PCR. A cDNA mDia1 fragment was amplified using the following primers: forward, ACG CCA TCC TCT TCA AGC TA; reverse, TGG AGC CCG CAT TCA TAT AG. Each reaction mixture included 10 μl of FastStart Universal SYBR Green Master (Rox; Roche Applied Science, Mannheim, Germany), 0.6 μl each of the forward and reverse primers, and 2 μl of template cDNA. The total reaction volume was 20 μl. Real-time quantitative PCR (qPCR) was done using a StepOne plus Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA, USA), and amplification was done using a 7500 Fast Dx Real-Time PCR instrument (Life Technologies, Carlsbad, CA, USA), according to the manufacturers' protocols. Cycling conditions were: initial incubation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 31 s. A final extension step at 60 °C for 1 min was added at the end of the last cycle. Amplification for each sample was done in triplicate. qRT–PCR analyses were done 3 times with independent RNA samples. The fold-change in mRNA expression was determined with the reference gene GAPDH by using the 2−△△Ct method. 2.5. mDia1 siRNA, mDia1 antibody and FMNL1 morpholino (MO) injection

2. Materials and methods 2.1. Antibodies and chemicals Rabbit monoclonal anti-mDia1, mouse monoclonal anti-Profilin1, and rabbit polyclonal anti-FMNL1 antibodies were from Abcam (Cambridge, MA, USA). Phalloidin-TRITC and mouse monoclonal anti-α-tubulin-FITC antibodies were from Sigma-Aldrich Corp. (St. Louis, MO, USA). FITC-conjugated and TRITC-conjugated goat anti-rabbit IgG and TRITC-conjugated goat anti-mouse IgG were from Zhongshan Golden Bridge Biotechnology, Co., Ltd. (Beijing). All other chemicals and reagents were from Sigma-Aldrich Corp., unless otherwise stated.

Three target-specific short interfering double-stranded RNA oligomers (siRNAs) (sc-35191; Santa Cruz) were used for mDia 1 RNAi. Stealth RNAi negative control duplexes were used as a control. For mDia1 knockdown (KD) experiments, mDia1 siRNA was diluted with RNAase free water to give a 50 nM stock solution, and ≈ 5–10 pl of mDia1 siRNA solution was microinjected into the cytoplasm of a fully grown GV oocyte using an Eppendorf FemtoJet (Eppendorf AG) with a Nikon Diaphot ECLIPSE TE300 inverted microscope (Nikon U.K. Ltd) equipped with a Narishige MM0-202N hydraulic three-dimensional micromanipulator (Narishige Inc. Tokyo, Japan). Negative control siRNA (5–10 pl) was microinjected into oocytes as a control. For antibody injection, 5–10 pl of an mDia1 antibody was microinjected and the same volume of water was injected as a control.

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Formin-like 1 (FMNL1) MO microinjection was used to deplete FMNL1 in mouse oocytes. FMNL1-MO 5′-CTCCAGCCTCGGAGATCCAG TTTTC-3′ (Gene Tools, Philomath, OR, USA) that targeted translation initiation was diluted with water to give a 300 nM stock solution, and 5–10 pl of MO solution was injected into oocytes. An MO standard control (5–10 pl) was injected as a control. After injection, oocytes arrested at the GV stage were cultured in M2 medium supplemented with 5 μM milrinone for 24 h to facilitate mDia1 mRNA translation KD. After washing 3 times (2 min each) in milrinone-free fresh M2 medium, oocytes were transferred to fresh M2 medium and cultured for 14 h to determine their maturation status (Pb1 extrusion) at 37 °C in a 5% CO2 atmosphere. Spindle and chromosome morphology, actin expression, and chromosome localization were examined. 2.6. RhoA inhibitor (Rhosin) treatment A specific inhibitor of RhoA, Rhosin (Calbiochem, Darmstadt, Germany), was used to inhibit intracellular RhoA activity during meiosis of oocytes. A Rhosin solution prepared in dimethyl sulfoxide (DMSO; 50 mM) was diluted in M2 medium to a concentration of 200 μM. GV oocytes were cultured in this medium for 9 h and used for Western blot analysis. Controls were cultured in fresh M2 medium using the same concentration of DMSO. 2.7. Confocal microscopy For single mDia1, actin, α-tubulin, or Profilin1 staining, oocytes were fixed in 4% paraformaldehyde (in PBS) at room temperature for 30 min and then permeabilized with 0.5% Triton X-100 in PBS for 20 min. To reduce non-specific IgG binding, oocytes were blocked in blocking buffer (1% BSA-supplemented PBS) at room temperature for 1 h. For mDia1 or Profilin1 staining, oocytes were incubated with a rabbit monoclonal anti-mDia1 antibody (1:100) or a mouse monoclonal anti-Profilin1 antibody (1:100) at 4 °C overnight or at room temperature for 4 h. After 3 washes (2 min each) with wash buffer (0.1% Tween 20 and 0.01% Triton X-100 in PBS), oocytes were labeled with an appropriate secondary antibody coupled to Alexa Fluor 488/568 goat-anti-rabbit IgG (1:100; for mDia1 staining) or goat-anti-mouse IgG (1:100; for Profilin1 staining) at room temperature for 1 h. For α-tubulin-FITC staining, oocytes were incubated with an anti-α-tubulin-FITC antibody (1:400) at room temperature for 2 h. For actin staining, oocytes were incubated with Phalloidin-TRITC (5 μg/ml in PBS) at room temperature for 1 h. After 3 washes in wash buffer, oocytes were co-stained with Hoechst 33342 to examine chromosomes. After staining, samples were mounted on glass slides and observed with a confocal laser-scanning microscope (Zeiss LSM 700 META, Germany). 2.8. Western blot analysis A total of 100 mouse oocytes were lysed in 15 μl of Laemmli sample buffer (SDS sample buffer and 2-mercaptoethanol) and boiled at 100 °C for 10 min. After cooling on ice and centrifugation at 12,000 ×g for 4 min, samples were frozen at − 20 °C until used. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) at 100 V for 100 min. Membranes were then blocked with TBST [TBS with 0.1% (w/w) Tween 20] that contained 5% nonfat dry milk powder at room temperature for 3 h. The membrane was then labeled with a rabbit monoclonal anti-mDia1 (1:2000) or a mouse monoclonal antiProfilin1 (1:1000) primary antibody diluted in blocking buffer. After overnight incubation at 4 °C, membranes were washed 3 times (5 min each) with TBST, and then incubated with HRP conjugated Pierce antirabbit IgG (for mDia1; 1:5000 dilution in blocking buffer) or antimouse IgG (for Profilin1, 1:5000; Cell Signaling Technology, Danvers,

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MA, USA) at room temperature for 1.5 h. Finally, membranes were processed using an enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA). Equal protein loading was confirmed by the levels of α-tubulin (rabbit monoclonal anti-α-tubulin antibody; 1:5000; Cell Signaling Technology, Danvers, MA, USA). This experiment was repeated at least 3 times using independent samples. To quantify Western blot results, band intensity values were determined using ImageJ software. Control band intensity (mDia1: α-Tubulin or Profilin1: α-Tubulin) was set to 1. Three replicates were used for this analysis. 2.9. Fluorescence intensity analysis Actin staining intensity was quantified using ImageJ software. Control and treatment group samples were mounted on the same glass slide. After immunofluorescent staining, the average fluorescence intensity per unit area within a region of interest (ROI) of immunofluorescence images was determined. Independent measurements using identically sized ROIs were made for the cell membrane. The average values of all measurements were used to compare the final average intensities between control and mDia1 KD groups. 2.10. Statistical analysis At least 3 replicates were used for each treatment and at least 30 oocytes were examined each time. Results were given as means ± SEM's. Statistical comparisons were made by analysis of variance (ANOVA), followed by Duncan's multiple comparisons test. A P-value of b 0.05 was considered significant. 3. Results 3.1. Subcellular distribution of mDia1 during mouse oocyte meiosis To acquire insights into mDia1 mechanisms of action during meiosis, we first examined the subcellular distribution of endogenous mDia1 at different developmental stages during mouse oocyte meiosis using immunofluorescence microscopy. Oocytes were cultured for 3, 9, and 14 h, which corresponded to the times taken to reach the GVBD, MI, and MII stages, respectively. Immunofluorescent staining clearly showed that mDia1 was expressed in mouse oocytes. mDia1 was primarily localized around chromosomes at the GVBD stage. Interestingly, when a spindle formed in the central cytoplasm at the MI stage, mDia1 accumulated around the meiotic spindle region. During anaphase I (AI) and MII, mDia1 continued to be associated with the meiotic spindle region (Fig. 1A). As shown in Fig. 2B, we also used double staining for mDia1 and spindles to assess their spatial interrelationship. Metaphase oocytes that were labeled with both mDia1 and α-tubulin antibodies clearly showed overlapping immunofluorescent signals (yellow). Nocodazole treatment abolished spindle localization, although mDia1 remained around chromosomes (Fig. 1B, white arrow), which supported mDia1 localization around the meiotic spindle region. Its distribution pattern suggested that mDia1 may function to participate in meiotic spindlerelated progression. 3.2. mDia1 depletion adversely affects mouse oocyte meiotic progression To investigate possible functions for mDia1 during meiosis, fully grown oocytes that had been microinjected with mDia1 siRNA for mDia1 knockdown (KD) were arrested at the GV stage and placed in medium supplemented with milrinone for 24 h. After washing in milrinone-free medium, oocytes were then analyzed for meiotic progression by pbI extrusion. After mDia1 siRNA injection, mDia1 mRNA expression was significantly reduced (0.18 ± 0.04 vs. 1 control; P b 0.01; Fig. 2A). Additionally, reduced mDia1 protein expression due to

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Fig. 1. Confocal image analysis of mDia1 subcellular localization during mouse oocyte meiotic maturation. (A) Subcellular localization of mDia1 in mouse oocytes. Oocytes were immunolabeled with an mDia1 antibody. During the germinal vesicle breakdown (GVBD) stage, mDia1 was primarily found around chromosomes. During the MI, AI, and MII stages, mDia1 accumulated where meiotic spindles were distributed. Green, mDia1; blue, chromatin. Bar = 30 μm. (B) Immunofluorescent staining for mDia1 and α-tubulin co-localization in oocytes. Oocytes were treated with 10 mg/ml of nocodazole for 2 h to depolymerize spindles. Yellow fluorescence is an overlay of mDia1 (red) and spindles (green). Nocodazole treatment abolished spindles without affecting mDia1 expression in oocytes (white arrow). Double labeling confirmed mDia1 localization similar to that of meiotic spindles. Green, α-tubulin; red, mDia1; blue, chromatin. Bar = 30 μm.

mDia1 RNAi was verified by Western blot analysis. Immunoblotting confirmed that the mDia1 protein level was reduced as compared with control levels (relative mDia1/α-tubulin intensity of 0.21 vs. 1 control; P b 0.01; Fig. 2B). After mDia1 KD, a large proportion of oocytes exhibited abnormal polar body extrusion as compared with the controls. In control oocytes, polar body extrusion was successful. However, after mDia1 depletion, meiosis progression was not maintained properly, including an oocyte phenotype that failed to emit a polar body or underwent symmetric division (Fig. 2C). After culture for 14 h, only 62.22 ± 4.55% (n = 563) of mDia1 KD oocytes extruded a pbI, which was significantly reduced compared to control siRNA-injected oocytes (84.85 ± 2.57%; n = 275; P b 0.01; Fig. 2D). In a small number of cases for which a meiotic division appeared to have been completed, a symmetrical “2-cell-like” egg was observed (Fig. 2C, white arrowheads), a phenotype that was 9 times more common than in control oocytes (11.05 ± 1.68% vs. 1.18 ± 0.71% control; P b 0.05; Fig. 2E). These pbI extrusion defects were also apparent when antibody injection was used. The rate of polar body extrusion after antibody injection was significantly reduced to 43.22 ± 5.96% (n = 135) as compared with that of controls (74.61 ± 3.55%; n = 135; P b 0.01; Fig. 2F). Taken together, these results suggested that mDia1 was fundamentally important for polar body extrusion during meiotic divisions. 3.3. mDia1 depletion alters cortical actin assembly but not spindle positioning in mouse oocytes To investigate how mDia1 depletion resulted in polar body extrusion failure, actin expression was monitored using intact MI oocytes. As shown in Fig. 3A, F-actin staining with phalloidin reflected that mDia1

KD caused an apparent alteration in the arrangement of the actin cytoskeleton. Immunofluorescence analysis showed that the accumulation of actin signals at the cortical region appeared weaker in mDia1-RNAi oocytes as compared with control-RNAi oocytes. Furthermore, a decrease in actin expression in mDia1 KD oocytes was verified by a statistical analysis of fluorescence intensity levels in oocytes. Fig. 3B shows that there was a significant reduction in actin fluorescence intensity in the cortex after mDia1 KD. Actin fluorescence intensity in the control group was set to 1, whereas in mDia1 KD oocytes it was 0.50 ± 0.026 (n = 61; P b 0.01), Thus, mDia1 appeared to be required for regulating actin assembly during mouse oocyte meiosis. As noted above, mDia1 KD oocytes exhibited polar body extrusion failure. We next examined spindle positioning that, in addition to the actin cytoskeleton, is also important for polar body extrusion. Spindle positioning in MI oocytes was assessed using immunofluorescent images of spindles that were labeled with an anti-α-tubulin antibody. As shown in Fig. 3C, after culture for 9 h, spindle positioning in MI oocytes could be categorized into 3 phenotypes: (1) spindles that migrated to the cortex; (2) spindles that localized between the cortex and the center of an oocyte; and (3) spindles that localized in the center of cytoplasm. After statistically analyzing spindle positioning, no significant differences in the proportions of spindle positioning during each stage were found. In control oocytes, the proportions of spindle positioning were: GVBD: 7.44 ± 2.03%; MI-1: 34.73 ± 6.27%; MI-2: 20.78 ± 2.44%; MI-3: 24.07 ± 5.35%; and ATI/MII: 12.99 ± 7.25% (n = 151). By comparison, in mDia1 KD oocytes, these phenotypes were: GVBD: 5.87 ± 2.76%; MI-1: 45.16 ± 5.47%; MI-2: 17.60 ± 3.42%; MI-3: 21.91 ± 5.98%; and ATI/MII: 9.45 ± 3.11% (n = 164). These results indicated that mDia1 KD did not affect spindle positioning during polar body extrusion.

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Fig. 2. mDia1 KD effects on mouse oocyte meiotic maturation. (A) mDia1 mRNA levels were significantly reduced after siRNA injection. (B) Knockdown (KD) of endogenous mDia1 protein expression after mDia1-siRNA injection was verified by Western blot analysis. mDia1 protein expression was significantly reduced after siRNA injection. (C) Phase-contrast images of control negative siRNA-injected and mDia1-KD oocytes; Bar = 100 μm. For a control, an oocyte in the polar body extrusion stage (black arrow) is shown. For mDia1 KD oocytes, oocytes at the MI stage (black arrowheads) or with a 2-cell-like morphology (white arrowheads) are shown. Bar = 30 μm. (D) Polar body extrusion rates by control and mDia1 KD oocytes. (E) Symmetrical division rates for control and mDia1 KD oocytes. (F) Polar body extrusion rates for control and mDia1 antibody injected oocytes. *Significantly different vs. control (P b 0.05).

3.4. mDia1 is involved in spindle formation and chromosome alignment The specific localization of mDia1 at the spindle region prompted us to hypothesize that mDia1 played a regulatory role in the meiotic apparatus. Thus, we assessed the consequences of mDia1 depletion in oocytes. Among controls, most oocytes had typical barrel-shaped spindles and well-aligned chromosomes on the metaphase plate (Fig. 4Aa). However, mDia1-depleted oocytes exhibited different types of malformed spindles (Fig. 4Ab, c; red arrowheads) and multipolar spindles (Fig. 4Ad, red arrowheads) with displacement of one or several chromosomes from the equator (Fig. 4A, white arrowheads). Notably, another severe phenotype of mDia1 KD oocytes was that even the basic spindle structure did not form and with irregularly appearing chromatin (Fig. 4Ae, white arrowheads). Only 9.97 ± 2.62% (n = 199) of control oocytes exhibited abnormal spindles and chromosomes (Fig. 4B). In striking contrast, there was a high frequency of spindle defects and chromosome disorganization in mDia1 KD oocytes (26.33 ± 2.32%, n = 203, P b 0.01; Fig. 3B). To determine a possible function for mDia1 in meiotic spindle assembly, the localization of p-MAPK, a well-known microtubule regulator, was determined. Fig. 4C showed the fluorescence staining pattern for p-MAPK that was specifically concentrated at the spindle poles in control MI stage oocytes (white arrows). In contrast, its specific

localization was lost in mDia1 KD oocytes (white arrowhead). These results suggested that mDia1 KD oocytes could not properly organize their meiotic spindles or align their meiotic chromosomes. 3.5. mDia1 depletion interferes with Profilin1 expression in mouse oocytes The effects of mDia1 KD on polar body extrusion prompted us to consider the possible target in oocytes that might mediate this process. To date, mDia1 has been shown to play essential roles in actin assembly. Also worth noting, as an ATP nucleotide exchange factor, Profilin1 is critically involved in actin polymerization in mitotic cells. However, its function during meiosis remains unknown. To test whether the effect of mDia1 depletion on actin assembly may have been mediated by Profilin1, we first explored the localization/expression of endogenous Profilin1 by immunofluorescent staining. As shown in Fig. 5A, Profilin1 exhibited a specific subcellular localization at the spindle region, which was consistent with the mDia1 distribution in MI oocytes. Metaphase oocytes co-stained with mDia1 and Profilin1 antibodies clearly showed overlapping fluorescent signals (yellow). Next, we investigated whether Profilin1 cooperated with mDia1 in a functional unit. The immunofluorescence results shown in Fig. 5B indicated reduced Profilin1 immunofluorescent signals in mDia1-siRNA injected oocytes (white arrow). As expected, immunoblot analysis

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Fig. 3. mDia1 KD results in reduced cortical actin expression but has no effects on spindle positioning. (A) Immunofluorescence microscopy for actin expression in oocyte membranes. Actin expression was reduced after mDia1 KD. Red, actin. Bar = 30 μm (40×). (B) Average actin fluorescence intensities in membranes. Compared with controls, actin fluorescence intensity at the membrane was significantly reduced after mDia1 KD. (C) Three typical images for spindle positioning in MI oocytes. Green, α-tubulin. Bar = 30 μm. Histogram shows the proportions of oocytes at different cell cycle stages after 9 h of culture for control and mDia1 KD oocytes. There were no significant differences at each stage. *Significantly different vs. control (P b 0.05).

showed a drastic reduction in Profilin1 protein expression in mDia1 deficient oocytes. The relative Profilin1/α-tubulin intensity of 1 in control oocytes vs. 0.20 in mDia1 KD oocytes also confirmed this (P b 0.01; Fig. 5C). Taken together, these results suggested that mDia1 was likely essential for Profilin1 expression during mouse oocyte meiotic maturation.

3.6. FMNL1 depletion but not RhoA inhibition alters mDia1 expression in oocytes We then searched for a signaling pathway that might account for the mDia1 requirement for maintaining actin assembly in oocytes. It is well documented that FMNL1 is a major component of the Formin family of actin nucleators and has been implicated in regulating actin assembly in mitotic cells. Thus, we hypothesized that there was a relationship between FMNL1 and mDia1. To test this, we first assessed the effects of FMNL1 depletion on mDia1 expression levels in oocytes by Western blot analysis. This showed that mDia1 protein expression in FMNL1-MO oocytes was significantly reduced as compared to that in control oocytes (Fig. 6A). Band intensities determined with ImageJ software also confirmed this (mDia1: α-tubulin, 1.0 vs. 0.63; P b 0.05;

Fig. 6A). This indicated that mDia1 expression/levels during oocyte meiosis depended on FMNL1. Additionally, it is also thought that Rho GTPases, such as RhoA, act on downstream effectors, such as ROCK and mDia1 [34]. Thus, we investigated whether RhoA inhibition affected mDia1 during meiosis. However, Western blot and quantitative analysis results showed that RhoA activity inhibition in oocytes had no significant effects on mDia1 expression as compared to that in controls (1.07 vs. 1 control, P N 0.05; Fig. 6B). This suggested that RhoA might not have effects on mDia1 expression in oocytes, and that FMNL1 may act as the upstream molecule for mDia1Profilin in mouse oocytes (Fig. 6C).

4. Discussion The experiments discussed here were designed to explore possible mDia1 functions during mouse oocyte meiosis. We found that mDia1 was localized specifically at the spindle region for regulating polar body formation. This may have been due to the substantially reduced expression of actin accumulation at the membrane and malformed and/or disorganized meiotic spindles in the absence of mDia1. Our results also suggested the importance of the FMNL1-mDia1-Profilin1

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Fig. 4. mDia1 knockdown results in spindle defects and chromosome misalignments during oocyte meiosis. (A-a) Control MI oocytes exhibit typical barrel-shaped spindles and wellaligned chromosomes on the metaphase plate. (b–e) Spindle defects (red arrowheads) and chromosome misalignments (white arrowheads) were frequently observed in mDia1-KD MI oocytes. Green, tubulin; blue, chromatin. Bar = 30 μm. (B) Rates of control and mDia1 KD oocytes with spindle defects or chromosome misalignments among MI oocytes. The percentage of mDia1 KD oocytes with abnormalities was significantly higher than that of controls. (C) p-MAPK localization after mDia1-KD. p-MAPK accumulated at the spindle poles in control oocytes. This specific p-MAPK localization was disrupted after mDia1-KD. Blue, chromatin. Red, p-MAPK. Bar = 30 μm. *Significantly different vs. control (P b 0.05).

signaling pathway for regulating actin assembly during polar body extrusion.

4.1. mDia1 plays pivotal roles in polar body extrusion Polar body extrusion is frequently described as asymmetric division. Although it has been confirmed that mDia1 is involved in regulating cell division by somatic cells, its function and mechanisms of action during mouse oocyte meiosis have remained uncertain. In this study, we first sought to determine whether mDia1 was expressed in mouse oocytes. Our results indicated that mDia1 was localized in the meiotic spindle region of oocytes, which was similar to previous results that mDia1 was detected along the midbodies of dividing cells [32]. This localization pattern was also consistent with those in a previous report that mDia1 was localized to the mitotic spindles in HeLa cells [35]. Furthermore, it was also shown that mDia1 was a component of meiotic spindles in mouse

oocytes [36]. Thus, our results suggest that mDia1 may have various functions during meiosis. Because mDia1 was localized at the meiotic spindle region in dividing oocytes, we further explored its role in meiosis. Most mDia1 siRNA-injected oocytes failed to emit a polar body as compared to control oocytes. Microinjection of mDia1 antibodies confirmed this. A number of studies have confirmed the involvement of Formins in cytokinesis in many organisms, including plants, budding and fission yeasts, Drosophila, Caenorhabditis elegans, and vertebrates. Diaphanous was required for normal cytokinesis in Drosophila [20]. In addition, diaphanous related Formin (DRF) proteins were also essential for cell division [37]. Other studies have shown that an mDia member, mDia2, was essential during mammalian cell division, as its degradation blocked cytokinesis [30,31]. Based on these findings, our findings were somewhat expected because microinjecting an anti-mDia1 antibody interfered with cytokinesis, which resulted in binucleate cells [32]. Additionally, the Formin2 protein is considered as a pivotal regulator

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Fig. 5. mDia1 depletion reduces Profilin1 expression in oocytes. (A) Subcellular localization of Profilin1 in metaphase mouse oocytes. Oocytes were co-labeled with mDia1 and Profilin1 antibodies at the MI stage. Profilin1 was localized at the spindle region, which was consistent with the mDia1 distribution. Green, mDia1; red, Profilin1; blue, chromatin. (B) The immunofluorescence results indicated reduced Profilin1 immunofluorescent signals (white arrow) in mDia1-KD oocytes compared with the controls. Green, α-tubulin; red, Profilin1; blue, chromatin. Bar = 30 μM. (C) Western blot analysis showed reduced Profilin1 protein expression in oocytes with endogenous mDia1 KD as compared to controls. α-Tubulin was used as a loading control. Band intensity was determined using ImageJ software, after which Profilin1/α-tubulin expression ratios were determined. *Significantly different vs. control (P b 0.05).

during mouse meiosis [24]. Our results also showed that there was a higher percentage of oocytes that underwent symmetric division after mDia1 KD. Thus, we suggest that mDia1 is involved for appropriate pbI extrusion in mouse oocytes. 4.2. mDia1 regulates actin assembly and spindle organization during mouse oocyte meiosis How mDia1 is involved in mouse oocyte regulation in time and space remains elusive. Formin proteins are well-established regulators that are involved in a considerable range of actin-based processes during mammalian cell cytokinesis. Among the better-characterized Formins, mDia1 is essential for assembling actin filaments [38,39]. Previous studies suggested the involvement of mDia1 in actin reorganization, such as stress fiber formation [32,40]. mDia1 or mDia2 RNAi inhibited nuclear actin assembly in NIH3TS cells [41]. Another study showed that diaphanous was involved in regulating actin organization during Drosophila cytokinesis [42]. mDia2 also contributed to actin assembly that was necessary for regulating cytokinesis in NIH3T3 cells [31]. Recent studies showed that Formin2 played a critical role in actin polymerization for mouse oocyte meiosis [11,12,43]. In our study, we examined actin assembly during meiosis. We found that cortical actin cytoskeleton assembly was significantly blocked in the absence of mDia1. During mammalian oocyte polar body extrusion, the meiotic spindle midzone induces the formation of membrane furrows; meanwhile, the special actin-based cortical bulges also

give rise to the cytokinetic furrows, and eventually, constriction of the actomyosin contractile ring forms to pinch off the polar body. Previous work indicated that actin assembly is crucial for the targeting and dynamic maintenance of asymmetric meiotic spindle position during mammalian oocytes meiosis, and a positive effect of actin feedback loop on maintaining the meiotic spindle at oocyte cell periphery during MII arrest [11,44]; additionally, actin acts as the requirement for cortical reorganization that is pivotal for cortical polarization and polar body extrusion [45, 46]. Furthermore, the generation of actin-based force is involved in cortical membrane protrusion that is the critical process of physical cleavage at the end of each oocyte division [2]. Thus, our results suggest that mDia1 drives polar body extrusion by promoting cortical actin assembly. In mammalian cells, spindle microtubules play a pivotal role in promoting cytokinesis [47]. Evidence also indicates that Formins might aid in the assembly or function of mitotic spindles [35]. A possible function of mDia1 that is localized to mitotic spindles is that mDia1 is involved in regulating meiotic spindles during polar body extrusion. Our results indicated that mDia1 was not involved in spindle positioning but was critical for proper spindle formation and chromosome alignment. In support of this, similar findings with mitotic cells showed that a Drosophila diaphanous mutation affected the organization of central spindles during cytokinesis [48]. The Rho-mDia pathway has also been suggested to have a novel role for microtubule stabilization and early mitotic spindle organization [18,49,50]. A recent study showed that overexpression of an active mDia1 mutant rescued the parallel

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Fig. 6. FMNL1 depletion reduces mDia1 expression in oocytes. (A) Western blot results showed dramatically reduced mDia1 protein expression in oocytes after FMNL1-MO injection as compared with that in control oocytes. α-Tubulin was used as a loading control. Relative mDia1 (mDia1/α-tubulin) intensity confirmed this. *Significantly different vs. control (P b 0.05). (B) mDia1 protein expression was unaffected by RhoA activity inhibition as shown by Western blot. Relative mDia1 (mDia1/α-tubulin) intensity confirmed this. *Significantly different vs. control (P b 0.05). (C) Summary of mDia1-related events during mouse oocyte maturation. mDia1 can control polar body extrusion by organizing microtubules and catalyzing actin assembly that is regulated via the FMNL1-mDia1-Profilin1 signaling pathway.

arrangement of microtubules in HeLa cells [28]. Indeed, mDia2 has been implicated in microtubule growth from MTOC's in mouse oocytes [51]. Another Formin, mDia3, is considered to play a role in the appropriate assembly of metaphase spindles [29]. Furthermore, interfering with mDia1 by anti-mDia1 antibody injection resulted in both disorganized

mitotic spindles and mitotic arrest in prophase Rat-2 cells [52]. Thus, our results suggest that mDia1 may act on spindle organization for regulating oocyte maturation. Taken together, the Formin mDia1 may be responsible for coordinating the mechanisms that control both the actin and microtubule dynamics that are required for oocyte meiosis.

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4.3. mDia1 cooperates with FMNL1 and Profilin1 in mouse oocytes To better understand the mDia1 mechanism of action for polar body extrusion, we used Western blotting to investigate possible relationships between mDia1 and FMNL1 and Profilin1, which have been shown to regulate actin dynamics during polar body extrusion. FMNL1 is a member of Formin-homology proteins that are related to diaphanous1 and diaphanous2 and might be involved in polarity control, migration, invasion, and metastasis through its regulatory roles on the Rho-related signaling pathway [53]. It has been shown that FMNL1 and mDia2 were involved in the formation of actin filament networks [54,55]. FMNL1 or mDia1 was considered to be a novel regulator for actin-related filopodia formation [56]. In the present study, as shown by FMNL1 KD and Western blot results, FMNL1 was involved in controlling mDia1 expression levels. In contrast, RhoA activity inhibition did not alter mDia1 expression. Thus, mDia1 might be a downstream effector of FMNL1 for regulating actin assembly during meiosis. Profilin was originally thought to have a promoting effect on actin polymerization [57]. Several studies reported that Formins catalyzed actin nucleation and polymerization and accelerated actin elongation by binding to the actin monomer-binding protein Profilin [58,59]. Furthermore, mDia1 is a downstream target for RhoA regulated actin polymerization by Profilin [27]. In the present study, abolishing endogenous mDia1 resulted in significantly reduced Profilin1 expression. Thus, mDia1 might regulate actin assembly via Profilin1 during meiosis. Taken together, our findings support that mDia1 contributes to actin assembly through the FMNL1-mDia1-Profilin1 signaling pathway in oocytes. In summary, our results suggest important functions for mDia1 during mouse oocyte meiosis. First, mDia1 appeared to be involved in organizing spindle. Second, we provided novel evidence showing that mDia1 could catalyze cortical actin assembly in oocytes via an FMNL1mDia1-Profilin1 signaling pathway. References [1] J.J. Eppig, M. O'Brien, K. Wigglesworth, Mammalian oocyte growth and development in vitro, Mol. Reprod. Dev. 44 (1996) 260–273. [2] K. Yi, R. Li, Actin cytoskeleton in cell polarity and asymmetric division during mouse oocyte maturation, Cytoskeleton 69 (2012) 727–737. [3] Q. Wang, C. Racowsky, M. Deng, Mechanism of the chromosome-induced polar body extrusion in mouse eggs, Cell Div. 6 (2011) 17. [4] M.H. Verlhac, J. Dumont, Interactions between chromosomes, microfilaments and microtubules revealed by the study of small GTPases in a big cell, the vertebrate oocyte, Mol. Cell. Endocrinol. 282 (2008) 12–17. [5] S.C. Sun, N.H. Kim, Molecular mechanisms of asymmetric division in oocytes, Microsc. Microanal. 19 (2013) 883–897. [6] L. Zhang, X. Hou, R. Ma, K. Moley, T. Schedl, Q. Wang, Sirt2 functions in spindle organization and chromosome alignment in mouse oocyte meiosis, FASEB J. 28 (2014) 1435–1445. [7] P. Kunda, A.E. Pelling, T. Liu, B. Baum, Moesin controls cortical rigidity, cell rounding, and spindle morphogenesis during mitosis, Curr. Biol. 18 (2008) 91–101. [8] J. Rosenblatt, L.P. Cramer, B. Baum, K.M. McGee, Myosin II-dependent cortical movement is required for centrosome separation and positioning during mitotic spindle assembly, Cell 117 (2004) 361–372. [9] G. Lansbergen, A. Akhmanova, Microtubule plus end: a hub of cellular activities, Traffic 7 (2006) 499–507. [10] N.M. Mahoney, G. Goshima, A.D. Douglass, R.D. Vale, Making microtubules and mitotic spindles in cells without functional centrosomes, Curr. Biol. 16 (2006) 564–569. [11] H. Li, F. Guo, B. Rubinstein, R. Li, Actin-driven chromosomal motility leads to symmetry breaking in mammalian meiotic oocytes, Nat. Cell Biol. 10 (2008) 1301–1308. [12] M. Schuh, J. Ellenberg, A new model for asymmetric spindle positioning in mouse oocytes, Curr. Biol. 18 (2008) 1986–1992. [13] J. Leblanc, X. Zhang, D. McKee, Z.B. Wang, R. Li, C. Ma, Q.Y. Sun, X.J. Liu, The small GTPase Cdc42 promotes membrane protrusion during polar body emission via ARP2-nucleated actin polymerization, Mol. Hum. Reprod. 17 (2011) 305–316. [14] B.J. Wallar, A.D. Deward, J.H. Resau, A.S. Alberts, RhoB and the mammalian Diaphanous-related formin mDia2 in endosome trafficking, Exp. Cell Res. 313 (2007) 560–571. [15] M. Fernandez-Borja, L. Janssen, D. Verwoerd, P. Hordijk, J. Neefjes, RhoB regulates endosome transport by promoting actin assembly on endosomal membranes through Dia1, J. Cell Sci. 118 (2005) 2661–2670. [16] K.M. Eisenmann, R.A. West, D. Hildebrand, S.M. Kitchen, J. Peng, R. Sigler, J. Zhang, K.A. Siminovitch, A.S. Alberts, T cell responses in mammalian diaphanous-related formin mDia1 knock-out mice, J. Biol. Chem. 282 (2007) 25152–25158.

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