Formin Mdia1, a Downstream Molecule of FMNL1, Regulates Proﬁlin1 for Actin Assembly and Spindle Organization During Mouse Oocyte Meiosis☆

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,FeiWanga,Ying-JieNiua,Hong-LinLiua,RongRuib, Xiang-Shun Cui c, Nam-Hyung Kim c, Shao-Chen Sun a,⁎ a College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China b College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China c Department of Animal Sciences, Chungbuk National University, Cheongju, Chungbuk, 361-763, Republic of Korea article info abstract

Article history: Mammalian diaphanous1 (mDia1) is a homologue of Drosophila diaphanous and belongs to the Formin- Received 26 August 2014 homology family of proteins that catalyze actin nucleation and polymerization. Although Formin family proteins, Received in revised form 6 October 2014 such as Drosophila diaphanous, have been shown to be essential for cytokinesis, whether and how mDia1 Accepted 4 November 2014 functions during meiosis remain uncertain. In this study, we explored possible roles and the signaling pathway Available online 15 November 2014 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 Keywords: fi mDia1 mouse oocytes and mDia1 knockdown resulted in reduced Pro lin1 expression. Depleting FMNL1, another Profilin1 Formin family member, resulted in reduced mDia1 expression, while RhoA inhibition did not alter mDia1 expres- Actin sion, which indicated that there was a FMNL1-mDia1-Profilin1 signaling pathway in mouse oocytes. Additionally, Spindle formation mDia1 knockdown resulted in disrupting oocyte spindle morphology, which was confirmed by aberrant p-MAPK Polar body extrusion localization. Thus, these results demonstrated indispensable roles for mDia1 in regulating mouse oocyte meiotic Oocyte maturation through its effects on actin assembly and spindle organization. © 2014 Elsevier B.V. All rights reserved.

1. Introduction germinal vesicle (GV) stage. During oocyte maturation, fully grown oocytes reinitiate meiosis, as indicated by germinal vesicle breakdown The meiosis and mitosis, both including DNA replication and (GVBD). Finally, an oocyte proceeds through the first meiosis, and cytokinesis events, are essential processes that emerge in mammal then transforms into a metaphase II (MII)-arrested oocyte with an development. The mitosis, a kind of continuous symmetric division, extruded small first polar body (pbI) until it is fertilized [1].Unlikethe encompasses a round of DNA replication and cytokinesis. Eventually, mitotic division that the actomyosin furrow ingression mainly depends the mitotic cell divides into two identical size daughter cells, which con- on the actomyosin ring contraction, interestingly, the polar body extru- tain the same genome as the parental cells. Unlike the mitosis, firstly, sion is a specialized cytokinesis, coordinated by the cortical membrane mammalian oocyte meiosis is characterized by a unique asymmetric di- protrusion and actomyosin ring contraction [2]. Additionally, in contrast vision, as the spindle migrates to the cortex and extrudes a small polar to the mitosis during which a centrally positioned spindle midzone body. Secondly, during meiosis, the mammalian oocyte undergoes two leads to the bilateral furrowing from the center of the somatic cells, consecutive rounds of asymmetric divisions while only one round of the meiotic spindle midzone-induced membrane furrow changes from DNA replication, generating a totipotent haploid egg. This brief period, the initial unilateral to the eventual bilateral during oocyte polar body so called ‘meiotic maturation’, is essential for maintaining female ge- extrusion [3]. nome integrity. Oocytes are arrested at the diplotene stage of the first Polar body extrusion is a highly coordinated process that depends on meiotic prophase within ovarian follicles, which is also defined as the the dynamic coordination of microtubules and actin filaments related events during divisions [4,5]. During meiosis I, microtubules form a

☆ Funding: This work was supported by the National Basic Research Program of China specialized bipolar-shaped spindle at the metaphase I (MI) stage. (2014CB138503), the Natural Science Foundation of Jiangsu Province (BK20140030), the Accurate spindle assembly is required for orderly meiosis during oocyte Specialized Research Fund for the Doctoral Program of Higher Education of China maturation [6]. In addition to the induction of actin organization by (20130097120055), and the Biogreen 21 Program (PJ009594, PJ009080 and PJ00909801), chromatin, spindle assembly is also attributed to this process. During RDA, Republic of Korea. cell cycle progression, the polymerized actin distributes parallel to the ⁎ Corresponding author at: College of Animal Sciences and Technology, Nanjing Agricultural University, Nanjing, China. Tel./fax: +86 25 84399092. plasma membrane at the oocyte cortex, and plays a critical role in the E-mail address: [email protected] (S.-C. Sun). establishment of cortical rigidity to act as a foundation for proper

http://dx.doi.org/10.1016/j.bbamcr.2014.11.005 0167-4889/© 2014 Elsevier B.V. All rights reserved. 318 Y. Zhang et al. / Biochimica et Biophysica Acta 1853 (2015) 317–327 spindle assembly [7,8]. This regulation may be due to the physical inter- 2.2. Oocyte harvest and culture actions between the plus ends of astral microtubules with a rigid actin cortex that facilitate placing, shortening, and focusing of the nascent ICR mice were used for all experiments. Our experiments were ap- spindle [9,10]. Also, actin filaments ensure the targeting of microtubule proved by the Animal Care and Use Committee of Nanjing Agriculture spindle positioning at the periphery of an oocyte concomitant with University and were performed in accordance with Animal Research In- chromosome segregation [11,12]. Additionally, a fine network of micro- stitute Committee guidelines. Mice were housed in a temperature- filaments is found throughout the entire cortex and is responsible for controlled room with an appropriate light: dark cycle, fed a regular constructing the classical contractile ring, which constricts at the base diet, and maintained under the care of the Laboratory Animal Unit, of the protrusion for pinching off a polar body [2,13].Althoughseveral Nanjing Agricultural University. Young female mice (6–8 weeks) were molecules have been proposed to contribute to cytoskeletal regulation used for oocyte collection. For in vitro maturation, germinal vesicle- during oocyte meiosis in mammals, the molecular mechanisms that intact oocytes were harvested from female ovaries and cultured in M2 modulate the meiotic apparatus remain to be determined. medium under paraffin oil at 37 °C in a 5% CO2 atmosphere. Oocytes Formin family proteins are essential for diverse cellular processes, were removed from culture at different times for microinjection, real- including vesicle trafficking [14,15],cellmigration[16,17], and microtu- time RT-PCR, and immunofluorescent staining and Western blot bule stabilization [18,19]. Formins also have highly conserved, critical analysis. functions for cytokinesis [20–22]. Additionally, Formins are conserved fi actin nucleators that are involved in promoting the assembly of actin l- 2.3. Nocodazole treatment of oocytes aments [23]. Furthermore, a previous study has shown that a Formin protein, Formin2 is responsible for correct positioning of the metaphase For nocodazole treatment, 10 mg/ml nocodazole in DMSO stock was fi fi spindle and nal abscission of the rst polar body during meiosis [24]. diluted in M2 medium to give a final concentration of 20 μg/ml. After in- mDia proteins are the mammalian homologues of Drosophila diapha- cubating in M2 medium supplemented with nocodazole for 10 min, the nous, which is a member of the Formin-homology family of proteins oocytes were collected for immunofluorescence microscopy after 9 h and is a downstream effector of the small GTPase Rho. culture. In mammals, the mDia family comprises 3 isoforms, mDia1 (diapha- nous1), mDia2 (diaphanous2), and mDia3 (diaphanous3) [23,25], which vary in their subcellular localization, enzymatic activity, and 2.4. Real-time quantitative pcr analysis their targets. mDia1 and mDia2 are crucial for cell migration and cell adhesion [26]. In addition, mDia proteins are strongly implicated in cell di- Real-time quantitative PCR analysis was used to examine mDia1 vision. Several lines of evidence indicate that an activated mDia and mRNA expression in oocytes. Total RNA was extracted from 30 oocytes diaphanous are essential for catalyzing linear actin nucleation and poly- using a Dynabeads mRNA DIRECT kit (Invitrogen Dynal AS), and single- ™ merization during cytokinesis [27,28]. In addition to their functions for stranded cDNA was generated using a PrimeScript RT reagent kit – actin assembly, mDia proteins reportedly stabilize and align microtu- (Takara, Dalian, China) with Oligo (dT) 12 18 primers (Invitrogen). bules in mitotic cells [18,28,29]. Among these, mDia2 has been the The cDNA products were then diluted 6-fold with deionized water be- most extensively investigated. Studies have indicated that mDia2 was fore use as a template for real-time PCR. A cDNA mDia1 fragment was fi involved in actin assembly regulation during cytokinesis [30,31]. ampli ed using the following primers: forward, ACG CCA TCC TCT TCA mDia3, which is found at the kinetochores of condensed chromosomes AGC TA; reverse, TGG AGC CCG CAT TCA TAT AG. Each reaction mixture μ in HeLa cells, might be essential for microtubule-mediated chromosome included 10 l of FastStart Universal SYBR Green Master (Rox; Roche μ segregation during mitosis [29]. mDia1 is also crucial to a large variety of Applied Science, Mannheim, Germany), 0.6 l each of the forward and μ cellular and morphogenetic functions via its effects on actin assembly reverse primers, and 2 l of template cDNA. The total reaction volume μ [32,33]. Although mDia proteins have been implicated in multiple criti- was 20 l. Real-time quantitative PCR (qPCR) was done using a Step- cal biological processes during mitosis, their physiological functions and One plus Real-Time PCR System (Applied Biosystems, Life Technologies, fi the signaling cascade involved during oocyte meiosis have not been Carlsbad, CA, USA), and ampli cation was done using a 7500 Fast Dx investigated. Real-Time PCR instrument (Life Technologies, Carlsbad, CA, USA), ac- For this study, we proposed that mDia1 would have significant roles cording to the manufacturers' protocols. Cycling conditions were: initial during mouse oocyte maturation. To confirm our hypothesis, we used incubation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, fi RNA interference (RNAi) to deplete the mDia1 isoform in mouse and 60 °C for 31 s. A nal extension step at 60 °C for 1 min was added fi oocytes and assessed mDia1 effects with regard to cytoskeleton- at the end of the last cycle. Ampli cation for each sample was done in – mediated dynamic events during mouse oocyte meiotic maturation. triplicate. qRT PCR analyses were done 3 times with independent RNA samples. The fold-change in mRNA expression was determined Our results indicated a direct contribution of mDia1 in mouse oocyte −△△ meiosis in that mDia1-induced actin assembly and spindle organization with the reference gene GAPDH by using the 2 Ct method. were critical for polar body extrusion by dividing oocytes. 2.5. mDia1 siRNA, mDia1 antibody and FMNL1 morpholino (MO) injection

2. Materials and methods Three target-speciﬁc short interfering double-stranded RNA oligo- mers (siRNAs) (sc-35191; Santa Cruz) were used for mDia 1 RNAi. 2.1. Antibodies and chemicals Stealth RNAi negative control duplexes were used as a control. For mDia1 knockdown (KD) experiments, mDia1 siRNA was diluted with Rabbit monoclonal anti-mDia1, mouse monoclonal anti-Proﬁlin1, RNAase free water to give a 50 nM stock solution, and ≈5–10 pl of and rabbit polyclonal anti-FMNL1 antibodies were from Abcam mDia1 siRNA solution was microinjected into the cytoplasm of a fully (Cambridge, MA, USA). Phalloidin-TRITC and mouse monoclonal grown GV oocyte using an Eppendorf FemtoJet (Eppendorf AG) with a anti-α-tubulin-FITC antibodies were from Sigma-Aldrich Corp. Nikon Diaphot ECLIPSE TE300 inverted microscope (Nikon U.K. Ltd) (St. Louis, MO, USA). FITC-conjugated and TRITC-conjugated goat equipped with a Narishige MM0-202N hydraulic three-dimensional mi- anti-rabbit IgG and TRITC-conjugated goat anti-mouse IgG were from cromanipulator (Narishige Inc. Tokyo, Japan). Negative control siRNA Zhongshan Golden Bridge Biotechnology, Co., Ltd. (Beijing). All other (5–10 pl) was microinjected into oocytes as a control. For antibody in- chemicals and reagents were from Sigma-Aldrich Corp., unless other- jection, 5–10 pl of an mDia1 antibody was microinjected and the same wise stated. volume of water was injected as a control. Y. Zhang et al. / Biochimica et Biophysica Acta 1853 (2015) 317–327 319

Formin-like 1 (FMNL1) MO microinjection was used to deplete MA, USA) at room temperature for 1.5 h. Finally, membranes were FMNL1 in mouse oocytes. FMNL1-MO 5′-CTCCAGCCTCGGAGATCCAG processed using an enhanced chemiluminescence reagent (Millipore, TTTTC-3′ (Gene Tools, Philomath, OR, USA) that targeted translation Billerica, MA, USA). Equal protein loading was conﬁrmed by the levels initiation was diluted with water to give a 300 nM stock solution, and of α-tubulin (rabbit monoclonal anti-α-tubulin antibody; 1:5000; Cell 5–10 pl of MO solution was injected into oocytes. An MO standard con- Signaling Technology, Danvers, MA, USA). This experiment was repeat- trol (5–10 pl) was injected as a control. ed at least 3 times using independent samples. After injection, oocytes arrested at the GV stage were cultured in To quantify Western blot results, band intensity values were M2 medium supplemented with 5 μM milrinone for 24 h to facilitate determined using ImageJ software. Control band intensity (mDia1: mDia1 mRNA translation KD. After washing 3 times (2 min each) in α-Tubulin or Proﬁlin1: α-Tubulin) was set to 1. Three replicates milrinone-free fresh M2 medium, oocytes were transferred to fresh were used for this analysis. M2 medium and cultured for 14 h to determine their maturation status

(Pb1 extrusion) at 37 °C in a 5% CO2 atmosphere. Spindle and chromo- 2.9. Fluorescence intensity analysis some morphology, actin expression, and chromosome localization were examined. Actin staining intensity was quantified using ImageJ software. Control and treatment group samples were mounted on the same 2.6. RhoA inhibitor (Rhosin) treatment glass slide. After immunofluorescent staining, the average fluorescence intensity per unit area within a region of interest (ROI) of immunofluo- Aspecific inhibitor of RhoA, Rhosin (Calbiochem, Darmstadt, rescence images was determined. Independent measurements using Germany), was used to inhibit intracellular RhoA activity during meiosis identically sized ROIs were made for the cell membrane. The average of oocytes. A Rhosin solution prepared in dimethyl sulfoxide (DMSO; values of all measurements were used to compare the final average in- 50 mM) was diluted in M2 medium to a concentration of 200 μM. GV tensities between control and mDia1 KD groups. oocytes were cultured in this medium for 9 h and used for Western blot analysis. Controls were cultured in fresh M2 medium using the 2.10. Statistical analysis same concentration of DMSO. At least 3 replicates were used for each treatment and at least 30 oo- 2.7. Confocal microscopy cytes were examined each time. Results were given as means ± SEM's. Statistical comparisons were made by analysis of variance (ANOVA), For single mDia1, actin, α-tubulin, or Profilin1 staining, oocytes were followed by Duncan's multiple comparisons test. A P-value of b0.05 fixed in 4% paraformaldehyde (in PBS) at room temperature for 30 min was considered significant. and then permeabilized with 0.5% Triton X-100 in PBS for 20 min. To re- duce non-specific IgG binding, oocytes were blocked in blocking buffer 3. Results (1% BSA-supplemented PBS) at room temperature for 1 h. For mDia1 or Profilin1 staining, oocytes were incubated with a rabbit monoclonal 3.1. Subcellular distribution of mDia1 during mouse oocyte meiosis 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 To acquire insights into mDia1 mechanisms of action during meiosis, washes (2 min each) with wash buffer (0.1% Tween 20 and 0.01% Triton we first examined the subcellular distribution of endogenous mDia1 at X-100 in PBS), oocytes were labeled with an appropriate secondary an- different developmental stages during mouse oocyte meiosis using im- tibody coupled to Alexa Fluor 488/568 goat-anti-rabbit IgG (1:100; for munofluorescence microscopy. Oocytes were cultured for 3, 9, and 14 h, mDia1 staining) or goat-anti-mouse IgG (1:100; for Profilin1 staining) which corresponded to the times taken to reach the GVBD, MI, and MII at room temperature for 1 h. For α-tubulin-FITC staining, oocytes stages, respectively. Immunofluorescent staining clearly showed that were incubated with an anti-α-tubulin-FITC antibody (1:400) at room mDia1 was expressed in mouse oocytes. mDia1 was primarily localized temperature for 2 h. For actin staining, oocytes were incubated with around chromosomes at the GVBD stage. Interestingly, when a spindle Phalloidin-TRITC (5 μg/ml in PBS) at room temperature for 1 h. After 3 formed in the central cytoplasm at the MI stage, mDia1 accumulated washes in wash buffer, oocytes were co-stained with Hoechst 33342 around the meiotic spindle region. During anaphase I (AI) and MII, to examine chromosomes. After staining, samples were mounted on mDia1 continued to be associated with the meiotic spindle region glass slides and observed with a confocal laser-scanning microscope (Fig. 1A). (Zeiss LSM 700 META, Germany). As shown in Fig. 2B, we also used double staining for mDia1 and spindles to assess their spatial interrelationship. Metaphase oocytes 2.8. Western blot analysis that were labeled with both mDia1 and α-tubulin antibodies clearly showed overlapping immunofluorescent signals (yellow). Nocodazole A total of 100 mouse oocytes were lysed in 15 μl of Laemmli sample treatment abolished spindle localization, although mDia1 remained buffer (SDS sample buffer and 2-mercaptoethanol) and boiled at 100 °C around chromosomes (Fig. 1B, white arrow), which supported mDia1 for 10 min. After cooling on ice and centrifugation at 12,000 ×g for localization around the meiotic spindle region. Its distribution pattern 4 min, samples were frozen at −20 °C until used. Proteins were suggested that mDia1 may function to participate in meiotic spindle- separated by sodium dodecyl sulfate-polyacrylamide gel electrophore- related progression. sis (SDS-PAGE) and electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) at 100 V for 3.2. mDia1 depletion adversely affects mouse oocyte meiotic progression 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 To investigate possible functions for mDia1 during meiosis, fully temperature for 3 h. The membrane was then labeled with a rabbit grown oocytes that had been microinjected with mDia1 siRNA for monoclonal anti-mDia1 (1:2000) or a mouse monoclonal anti- mDia1 knockdown (KD) were arrested at the GV stage and placed in Profilin1 (1:1000) primary antibody diluted in blocking buffer. After medium supplemented with milrinone for 24 h. After washing in overnight incubation at 4 °C, membranes were washed 3 times (5 min milrinone-free medium, oocytes were then analyzed for meiotic pro- each) with TBST, and then incubated with HRP conjugated Pierce anti- gression by pbI extrusion. After mDia1 siRNA injection, mDia1 mRNA rabbit IgG (for mDia1; 1:5000 dilution in blocking buffer) or anti- expression was significantly reduced (0.18 ± 0.04 vs. 1 control; P b mouse IgG (for Profilin1, 1:5000; Cell Signaling Technology, Danvers, 0.01; Fig. 2A). Additionally, reduced mDia1 protein expression due to 320 Y. Zhang et al. / Biochimica et Biophysica Acta 1853 (2015) 317–327

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 KD caused an apparent alteration in the arrangement of the actin cyto- confirmed that the mDia1 protein level was reduced as compared skeleton. Immunofluorescence analysis showed that the accumulation with control levels (relative mDia1/α-tubulin intensity of 0.21 vs. 1 con- of actin signals at the cortical region appeared weaker in mDia1-RNAi trol; P b 0.01; Fig. 2B). oocytes as compared with control-RNAi oocytes. Furthermore, a de- After mDia1 KD, a large proportion of oocytes exhibited abnormal crease in actin expression in mDia1 KD oocytes was verified by a statis- polar body extrusion as compared with the controls. In control oocytes, tical analysis of fluorescence intensity levels in oocytes. Fig. 3B shows polar body extrusion was successful. However, after mDia1 depletion, that there was a significant reduction in actin fluorescence intensity in meiosis progression was not maintained properly, including an oocyte the cortex after mDia1 KD. Actin fluorescence intensity in the control phenotype that failed to emit a polar body or underwent symmetric di- group was set to 1, whereas in mDia1 KD oocytes it was 0.50 ± 0.026 vision (Fig. 2C). After culture for 14 h, only 62.22 ± 4.55% (n = 563) of (n = 61; P b 0.01), Thus, mDia1 appeared to be required for regulating mDia1 KD oocytes extruded a pbI, which was significantly reduced com- actin assembly during mouse oocyte meiosis. pared to control siRNA-injected oocytes (84.85 ± 2.57%; n = 275; P b As noted above, mDia1 KD oocytes exhibited polar body extrusion 0.01; Fig. 2D). In a small number of cases for which a meiotic division failure. We next examined spindle positioning that, in addition to the appeared to have been completed, a symmetrical “2-cell-like” egg was actin cytoskeleton, is also important for polar body extrusion. Spindle observed (Fig. 2C, white arrowheads), a phenotype that was 9 times positioning in MI oocytes was assessed using immunofluorescent im- more common than in control oocytes (11.05 ± 1.68% vs. 1.18 ± ages of spindles that were labeled with an anti-α-tubulin antibody. As 0.71% control; P b 0.05; Fig. 2E). These pbI extrusion defects were also shown in Fig. 3C, after culture for 9 h, spindle positioning in MI oocytes apparent when antibody injection was used. The rate of polar body ex- could be categorized into 3 phenotypes: (1) spindles that migrated to trusion after antibody injection was significantly reduced to 43.22 ± the cortex; (2) spindles that localized between the cortex and the center 5.96% (n = 135) as compared with that of controls (74.61 ± 3.55%; of an oocyte; and (3) spindles that localized in the center of cytoplasm. n = 135; P b 0.01; Fig. 2F). Taken together, these results suggested After statistically analyzing spindle positioning, no significant differ- that mDia1 was fundamentally important for polar body extrusion dur- ences in the proportions of spindle positioning during each stage were ing meiotic divisions. 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: 3.3. mDia1 depletion alters cortical actin assembly but not spindle position- 24.07 ± 5.35%; and ATI/MII: 12.99 ± 7.25% (n = 151). By comparison, ing in mouse oocytes 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%; To investigate how mDia1 depletion resulted in polar body extrusion and ATI/MII: 9.45 ± 3.11% (n = 164). These results indicated that failure, actin expression was monitored using intact MI oocytes. As mDia1 KD did not affect spindle positioning during polar body shown in Fig. 3A, F-actin staining with phalloidin reflected that mDia1 extrusion. Y. Zhang et al. / Biochimica et Biophysica Acta 1853 (2015) 317–327 321

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 localization was lost in mDia1 KD oocytes (white arrowhead). These results suggested that mDia1 KD oocytes could not properly organize their The specific localization of mDia1 at the spindle region prompted us meiotic spindles or align their meiotic chromosomes. to hypothesize that mDia1 played a regulatory role in the meiotic apparatus. Thus, we assessed the consequences of mDia1 depletion in 3.5. mDia1 depletion interferes with Profilin1 expression in mouse oocytes oocytes. Among controls, most oocytes had typical barrel-shaped spindles and well-aligned chromosomes on the metaphase plate (Fig. 4Aa). The effects of mDia1 KD on polar body extrusion prompted us to However, mDia1-depleted oocytes exhibited different types of mal- consider the possible target in oocytes that might mediate this process. formed spindles (Fig. 4Ab, c; red arrowheads) and multipolar spindles To date, mDia1 has been shown to play essential roles in actin assembly. (Fig. 4Ad, red arrowheads) with displacement of one or several chromo- Also worth noting, as an ATP nucleotide exchange factor, Profilin1 is somes from the equator (Fig. 4A, white arrowheads). Notably, another critically involved in actin polymerization in mitotic cells. However, its severe phenotype of mDia1 KD oocytes was that even the basic spindle function during meiosis remains unknown. To test whether the effect structure did not form and with irregularly appearing chromatin of mDia1 depletion on actin assembly may have been mediated by (Fig. 4Ae, white arrowheads). Only 9.97 ± 2.62% (n = 199) of control Profilin1, we first explored the localization/expression of endogenous oocytes exhibited abnormal spindles and chromosomes (Fig. 4B). In Profilin1 by immunofluorescent staining. As shown in Fig. 5A, Profilin1 striking contrast, there was a high frequency of spindle defects and exhibited a specific subcellular localization at the spindle region, chromosome disorganization in mDia1 KD oocytes (26.33 ± 2.32%, which was consistent with the mDia1 distribution in MI oocytes. Meta- n=203,Pb 0.01; Fig. 3B). phase oocytes co-stained with mDia1 and Profilin1 antibodies clearly To determine a possible function for mDia1 in meiotic spindle as- showed overlapping fluorescent signals (yellow). sembly, the localization of p-MAPK, a well-known microtubule regula- Next, we investigated whether Profilin1 cooperated with mDia1 in a tor, was determined. Fig. 4Cshowedthefluorescence staining pattern functional unit. The immunofluorescence results shown in Fig. 5Bindi- for p-MAPK that was specifically concentrated at the spindle poles in cated reduced Profilin1 immunofluorescent signals in mDia1-siRNA control MI stage oocytes (white arrows). In contrast, its specific injected oocytes (white arrow). As expected, immunoblot analysis 322 Y. Zhang et al. / Biochimica et Biophysica Acta 1853 (2015) 317–327

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 de- Fig. 6A). This indicated that mDia1 expression/levels during oocyte ficient oocytes. The relative Profilin1/α-tubulin intensity of 1 in control meiosis depended on FMNL1. oocytes vs. 0.20 in mDia1 KD oocytes also confirmed this (P b 0.01; Additionally, it is also thought that Rho GTPases, such as RhoA, act on Fig. 5C). Taken together, these results suggested that mDia1 was likely downstream effectors, such as ROCK and mDia1 [34].Thus,weinvesti- essential for Profilin1 expression during mouse oocyte meiotic gated whether RhoA inhibition affected mDia1 during meiosis. Howev- maturation. er, 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). 3.6. FMNL1 depletion but not RhoA inhibition alters mDia1 expression in This suggested that RhoA might not have effects on mDia1 expression in oocytes oocytes, and that FMNL1 may act as the upstream molecule for mDia1- Profilin in mouse oocytes (Fig. 6C). 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 4. Discussion family of actin nucleators and has been implicated in regulating actin assembly in mitotic cells. Thus, we hypothesized that there was a re- The experiments discussed here were designed to explore possible lationship between FMNL1 and mDia1. To test this, we first assessed mDia1 functions during mouse oocyte meiosis. We found that mDia1 the effects of FMNL1 depletion on mDia1 expression levels in oocytes was localized specifically at the spindle region for regulating polar by Western blot analysis. This showed that mDia1 protein expression in body formation. This may have been due to the substantially reduced FMNL1-MO oocytes was significantly reduced as compared to that in expression of actin accumulation at the membrane and malformed control oocytes (Fig. 6A). Band intensities determined with ImageJ soft- and/or disorganized meiotic spindles in the absence of mDia1. Our re- ware also confirmed this (mDia1: α-tubulin, 1.0 vs. 0.63; P b 0.05; sults also suggested the importance of the FMNL1-mDia1-Profilin1 Y. Zhang et al. / Biochimica et Biophysica Acta 1853 (2015) 317–327 323

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 well- aligned 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 oocytes [36]. Thus, our results suggest that mDia1 may have various extrusion. functions during meiosis. Because mDia1 was localized at the meiotic spindle region in dividing oocytes, we further explored its role in meiosis. Most mDia1 4.1. mDia1 plays pivotal roles in polar body extrusion siRNA-injected oocytes failed to emit a polar body as compared to control oocytes. Microinjection of mDia1 antibodies confirmed this. A num- Polar body extrusion is frequently described as asymmetric division. ber of studies have confirmed the involvement of Formins in cytokinesis Although it has been confirmed that mDia1 is involved in regulating cell in many organisms, including plants, budding and fission yeasts, Dro- division by somatic cells, its function and mechanisms of action during sophila, Caenorhabditis elegans, and vertebrates. Diaphanous was re- mouse oocyte meiosis have remained uncertain. In this study, we first quired for normal cytokinesis in Drosophila [20].Inaddition, sought to determine whether mDia1 was expressed in mouse oocytes. diaphanous related Formin (DRF) proteins were also essential for cell Our results indicated that mDia1 was localized in the meiotic spindle re- division [37]. Other studies have shown that an mDia member, mDia2, gion of oocytes, which was similar to previous results that mDia1 was was essential during mammalian cell division, as its degradation detected along the midbodies of dividing cells [32]. This localization pat- blocked cytokinesis [30,31]. Based on these findings, our findings were tern was also consistent with those in a previous report that mDia1 was somewhat expected because microinjecting an anti-mDia1 antibody in- localized to the mitotic spindles in HeLa cells [35]. Furthermore, it was terfered with cytokinesis, which resulted in binucleate cells [32].Addi- also shown that mDia1 was a component of meiotic spindles in mouse tionally, the Formin2 protein is considered as a pivotal regulator 324 Y. Zhang et al. / Biochimica et Biophysica Acta 1853 (2015) 317–327

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 give rise to the cytokinetic furrows, and eventually, constriction of the higher percentage of oocytes that underwent symmetric division after actomyosin contractile ring forms to pinch off the polar body. Previous mDia1 KD. Thus, we suggest that mDia1 is involved for appropriate work indicated that actin assembly is crucial for the targeting and dynam- pbI extrusion in mouse oocytes. ic maintenance of asymmetric meiotic spindle position during mammalian oocytes meiosis, and a positive effect of actin feedback loop on 4.2. mDia1 regulates actin assembly and spindle organization during mouse maintaining the meiotic spindle at oocyte cell periphery during MII arrest oocyte meiosis [11,44]; additionally, actin acts as the requirement for cortical reorganization that is pivotal for cortical polarization and polar body extrusion [45, How mDia1 is involved in mouse oocyte regulation in time and space 46]. Furthermore, the generation of actin-based force is involved in corti- remains elusive. Formin proteins are well-established regulators that are cal membrane protrusion that is the critical process of physical cleavage involved in a considerable range of actin-based processes during mam- at the end of each oocyte division [2]. Thus, our results suggest that malian cell cytokinesis. Among the better-characterized Formins, mDia1 mDia1 drives polar body extrusion by promoting cortical actin assembly. is essential for assembling actin filaments [38,39]. Previous studies sug- In mammalian cells, spindle microtubules play a pivotal role in pro- gested the involvement of mDia1 in actin reorganization, such as stress moting cytokinesis [47]. Evidence also indicates that Formins might aid fiber formation [32,40]. mDia1 or mDia2 RNAi inhibited nuclear actin as- in the assembly or function of mitotic spindles [35]. A possible function sembly in NIH3TS cells [41]. Another study showed that diaphanous was of mDia1 that is localized to mitotic spindles is that mDia1 is involved in involved in regulating actin organization during Drosophila cytokinesis regulating meiotic spindles during polar body extrusion. Our results [42]. mDia2 also contributed to actin assembly that was necessary for indicated that mDia1 was not involved in spindle positioning but was regulating cytokinesis in NIH3T3 cells [31]. Recent studies showed that critical for proper spindle formation and chromosome alignment. In Formin2 played a critical role in actin polymerization for mouse oocyte support of this, similar findings with mitotic cells showed that a meiosis [11,12,43]. In our study, we examined actin assembly during mei- Drosophila diaphanous mutation affected the organization of central osis. We found that cortical actin cytoskeleton assembly was significantly spindles during cytokinesis [48]. The Rho-mDia pathway has also been blocked in the absence of mDia1. During mammalian oocyte polar body suggested to have a novel role for microtubule stabilization and early extrusion, the meiotic spindle midzone induces the formation of mem- mitotic spindle organization [18,49,50]. A recent study showed that brane furrows; meanwhile, the special actin-based cortical bulges also overexpression of an active mDia1 mutant rescued the parallel Y. Zhang et al. / Biochimica et Biophysica Acta 1853 (2015) 317–327 325

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 mitotic spindles and mitotic arrest in prophase Rat-2 cells [52]. Thus, implicated in microtubule growth from MTOC's in mouse oocytes [51]. our results suggest that mDia1 may act on spindle organization for reg- Another Formin, mDia3, is considered to play a role in the appropriate ulating oocyte maturation. Taken together, the Formin mDia1 may be assembly of metaphase spindles [29]. Furthermore, interfering with responsible for coordinating the mechanisms that control both the mDia1 by anti-mDia1 antibody injection resulted in both disorganized actin and microtubule dynamics that are required for oocyte meiosis. 326 Y. Zhang et al. / Biochimica et Biophysica Acta 1853 (2015) 317–327

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