RESEARCH ARTICLE Molecular Reproduction & Development 81:725–734 (2014)
Involvement of Dynamin 2 in Actin-Based Polar-Body Extrusion During Porcine Oocyte Maturation YU ZHANG,1,2 QIAO-CHU WANG,1 JUN HAN,1 RUI CAO,1 XIANG-SHUN CUI,3 NAM-HYUNG KIM,3 RONG RUI2, 1 AND SHAO-CHEN SUN * 1
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, People’s Republic of, China College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, People’s Republic of, China 3 Department of Animal Sciences, Chungbuk National University, Cheongju, Chungbuk, Korea 2
SUMMARY Mammalian oocyte meiotic maturation involves a unique asymmetric division, but the regulatory mechanisms and signaling pathways involved are poorly understood. Dynamins are ubiquitous eukaryotic GTPases involved in membrane trafficking and actin dynamics, whose roles in mammalian oocyte maturation have not been determined. In this study, we used porcine oocytes to show that Dynamin 2 accumulated at the meiotic spindle and in the cortex of oocytes, with a distribution similar to that of actin. Inhibiting Dynamin 2 activity in porcine oocytes with the specific inhibitor dynasore resulted in failed polar-body extrusion. This phenotype may have been due to aberrant actin distribution and/or spindle positioning as inhibitor treatment disrupted the formation of the actin cap and cortical granule-free domain, which negatively impacted spindle positioning. Moreover, the distribution of ARP2, a key actin-nucleation factor, was severely reduced in the cortex after dynasore treatment. Thus, our results suggest that Dynamin 2 possibly regulates porcine oocyte maturation through its effects on actin-mediated spindle positioning and cytokinesis, and that this may depend on regulating ARP2 localization.
Mol. Reprod. Dev. 81: 725734, 2014. ß 2014 Wiley Periodicals, Inc. Received 15 April 2014; Accepted 8 May 2014
Corresponding author: College of Animal Sciences and Technology Nanjing Agricultural University Nanjing, People’s Republic of China. E-mail: [email protected]
Grant sponsor: National Basic Research Program of China; Grant number: 2014CB138503; Grant sponsor: Fundamental Research Funds for the Central Universities; Grant number: KJQN201402; Grant sponsor: Natural Science Foundation of Jiangsu Province, China; Grant number: BK20130671; Grant sponsor: Biogreen 21 Program; Grant numbers: PJ009594, PJ009080, PJ00909801; Grant sponsor: RDA, Republic of Korea
Published online 22 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22341
INTRODUCTION Mammalian oocytes remain arrested at the late diplotene stage of prophase I from birth until oocyte meiotic maturation, whose completion is responsible for successful fertilization. An oocyte initiates meiotic maturation from the germinal vesicle (GV) stage, passes through germinal vesicle breakdown (GVBD), completes a unique asymmet-
ß 2014 WILEY PERIODICALS, INC.
Abbreviations: ARP2/3 complex, actin-related protein 2/3 complex; CGFD, cortical-granule-free domain; COCs, cumulus oocyte complexes; DOs, denuded oocytes; GV[BD], germinal vesicle [breakdown]; MI/II, metaphase I/II.
Molecular Reproduction & Development
ric division involving the extrusion of a small, first polar body, and finally results in a highly polarized, metaphase II (MII) egg. These latter events are essential for retaining the most cytoplasm in the egg for the developing zygote. Polar-body formation is dependent on proper oocyte polarization. This process is controlled by the microtubule and microfilament cytoskeletons, resulting in spindle migration and positioning and in cortical reorganization (Brunet and Verlhac, 2011). After GVBD, the bipolar, meiosis-I spindle is assembled close to the center of the oocyte. During metaphase I (MI) stage, however, the spindle translocates to the equatorial plate and subsequently migrates to one side of the oocyte cortex, aligned along the spindle’s long axis. In addition to eccentric spindle positioning, another event pivotal for asymmetric division during oocyte maturation is the organization of cortical polarity: actin becomes enriched at an actin cap in the cortical area where cortical granules (CGs) and microvilli are also absent, resulting in a CG-free domain (CGFD) (Deng et al., 2003). After anaphase, cytokinesis segregates one set of homologous chromosomes to the first polar body. The oocyte then proceeds through meiosis I until MII stage, remaining arrested at this stage until fertilization. Involvement of the actin cytoskeleton in asymmetric spindle positioning and cortical polarization during oocyte meiotic division has been observed. Migration of the spindle (or chromosomes) is a dynamic, actin-dependent process. Previous work has implicated an actin feedback loop in the maintenance of meiotic spindle position during MII arrest (Yi et al., 2013a). The molecular mechanisms underlying spindle positioning emerged when it was reported that Formin-2 and ARP2/3 (actin-related protein 2/3 complex), which nucleate actin assembly, generate the force needed to propel chromosomes/the spindle towards the cortex (Yi et al., 2013b). Until now, the molecular details underlying these events of oocyte meiotic maturation were poorly understood. The Dynamin superfamily is comprised of Dynamin and several Dynamin-related proteins. Dynamins were originally identified as large GTPases homologous to the Drosophila Shibire gene product. There are three Dynamins in mammalian genomes (Cook et al., 1996), which share at least 70% homology. Dynamin 1 is expressed exclusively in the brain; Dynamin 2 is ubiquitously distributed; and Dynamin 3 is selectively expressed in the brain and testis (Cao et al., 1998); Dynamin 2 is the only isoform that functions in mammals (Thompson et al., 2002). Dynamins are involved in a variety of cell processes, including endocytosis, mitochondrial and chloroplast biogenesis, and cytokinesis (Praefcke and McMahon, 2004). Like other members of the GTPase superfamily, it appears that Dynamin itself does not mediate fission, instead acting as a molecular regulator during receptor-mediated endocytosis (Sever et al., 1999), possibly by regulating actin. In addition to its critical role in deforming membranes to create tubules or vesicles (Praefcke and McMahon, 2004), Dynamin particularly Dynamin 2 localizes to cellular membranes that are undergoing remodeling, specifically associating with actin filaments and thereby influencing
726
ZHANG
ET AL.
actin dynamics (Orth and McNiven, 2003). Consistent with this general function, Dynamin 2 is required for cytoskeletal reorganization, including actin-related podosome formation, membrane ruffling (Schafer, 2004), and actintail-based vesicle trafficking (Lee and De Camilli, 2002). It can nucleate actin at membranes, and regulates actincomet formation and movement in the rat and mouse (Orth et al., 2002). One function of Dynamin 2 appears to be based on its interactions with various cytoskeletal regulators during mitosis (Konopka et al., 2006). Cortactin is a Dynamin-2 binding partner. Actin filament nucleation by Dynamin 2 occurs through a Dynamin 2/Cortactin/ARP2/3 complex (Uruno et al., 2001; Schafer et al., 2002) that can bind filamentous actin (F-actin) in membrane ruffles, podosomes, actin comets, clathrin-coated pits, and the Golgi complex. It was recently found that another actin binding protein, ABP1, can directly bind to and co-localizes with Dynamin 2, linking the actin cytoskeleton to it (Kessels et al., 2001). Dynamin 2 can also regulate actin assembly by associating with three indirect actin-binding scaffold protein: Intersectin-1 (Hussain et al., 2001), Syndapin (Qualmann and Kelly, 2000), and Tuba (Salazar et al., 2003). Both Intersectin and Syndapin interact with N-WASP to regulate ARP2/3-dependent actin nucleation (Qualmann and Kelly, 2000). Profilin, the first protein identified from mouse brain that directly binds to Dynamin 2 (Witke et al., 1998), acts downstream of Dynamin 2 and is involved in actin polymerization (Pollard et al., 2000). In this study, we investigated the expression and functions of Dynamin 2 during porcine oocyte meiosis by using a specific inhibitor, dynasore. Our results showed that Dynamin 2 inhibition caused aberrant actin organization, failure of proper spindle positioning, and oocyte cytokinesis. We also found that ARP2 may act downstream of Dynamin 2 in the regulation of the actin cytoskeleton. Thus, our results demonstrated a functional mechanism for Dynamin 2 in porcine oocytes.
RESULTS Dynamin 2 Localization During Porcine Oocyte Meiotic Maturation We first examined the expression of the Dynamin 2 in cumulus oocyte complexes (COCs). Dynamin 2 was present in both cumulus cells and the oocytes (Fig. 1A). The subcellular localization of Dynamin 2 at different stages of meiotic maturation was also profiled using immunofluorescence staining (Fig. 1B): Dynamin 2 was observed in the cortex of GV oocytes. After GVBD, Dynamin 2 was found in the cortex while also accumulating at the spindle during all subsequent meiotic stages. To confirm this spindle localization, we co-stained for a-tubulin along with Dynamin 2, which revealed specific co-localization at this structure (Fig. 1C). Following dynasore inhibition of Dynamin, Dynamin 2 protein levels were significantly reduced compared to untreated porcine oocytes (Fig. 1B vs. 1D).
Mol. Reprod. Dev. 81:725–734 (2014)
DYNAMIN 2
IN
PORCINE OOCYTES
Figure 1. Dynamin 2 localization in porcine oocytes. A: Immunofluorescence staining for Dynamin 2 in COCs. Green, Dynamin 2; red, actin; blue, chromatin. Scale bar, 30 mm. B: Subcellular localization of Dynamin 2 during porcine oocyte meiotic maturation. Dynamin 2 accumulated at the cortex of GV-stage oocytes. From GVBD to MII, Dynamin 2 also accumulated at the spindle and at the oocytes cortex. Green, Dynamin 2; blue, chromatin. Scale bar, 20 mm. C: Immunofluorescence co-localization of a-tubulin and Dynamin 2 in oocytes. Red, Dynamin 2; green, a-tubulin; blue, chromatin. Scale bar, 20 mm. D: Immunofluorescence staining for Dynamin 2 in oocytes after dynasore exposure during porcine oocyte maturation. Dynamin 2 abundance was minimal after dynasore treatment. Green, Dynamin 2; blue, chromatin. Scale bar, 20 mm.
Dynamin 2 Inhibition Disrupts Porcine Oocyte Maturation In Vitro Based on the result that dynasore treatment suppresses Dynamin 2 abundance in oocytes, we examined if Dynamin 2 is involved in regulating porcine oocyte maturation. When COCs were treated with dynasore for 44 hr during maturation, cumulus expansion failed (Fig. 2A). Most COCs in the control group were surrounded by more than five layers of intact cumulus cells, whereas the cumulus layer became weakly diffused with 100 and 200 mM dynasore. A large proportion of inhibitor-treated oocytes also failed to extrude polar bodies in a dosedependent manner (Fig. 2B). After 44 hr of culture, most control oocytes extruded polar bodies and were arrested at the MII stage; the rate of first-polar-body extrusion for controls was 79.36 2.64% (n ¼ 179 COCs). The rate of polar-body extrusion was 70.99 3.13% (n ¼ 180 COCs) with 100 mM dynasore, and was significantly reduced to 36.98 7.71% (P < 0.05; n ¼ 182 COCs) with 200 mM
Mol. Reprod. Dev. 81:725–734 (2014)
treatment. A dynasore concentration of 200 mM was used for subsequent studies. To exclude the effects of cumulus cells on oocyte maturation, denuded oocytes (DOs) were also treated with dynasore (Fig. 2C). The rate of first-polar-body extrusion in the control group was 67.07 3.40% (n ¼ 88 DOs), which was significantly higher than the 200 mM treatment group (29.46 3.85%; n ¼ 75 DOs) (P < 0.05). Thus dynasore had direct effects on oocyte nuclear maturation. Considering the lower in vitro-maturation rates of DOs, however, COCs were adopted to investigate the mechanisms involving Dynamin 2 during porcine oocyte maturation.
Dynamin 2 Inhibition Affects Actin Assembly in Porcine Oocytes Previous studies established that Dynamin is a major actin nucleator. In this study, we investigated if Dynamin 2 affected actin filament organization in porcine oocytes.
727
Molecular Reproduction & Development
ZHANG
ET AL.
Figure 2. Effects of Dynasore treatment on porcine oocyte maturation. A: Representative image of porcine COCs maturation in the presence of dynasore, specifically showing cumulus expansion and first-polar-body (PB1) extrusion. In the control group, most COCs were surrounded by more than five layers of intact cumulus cells, whereas weak diffusion was observed after treatment with dynasore at 100 and 200 mM. Almost all control oocytes extruded polar bodies as compared with the two inhibitor-treated groups. Scale bar, 80 mm. B: Dynasore treatment resulted in a dosedependent, reduced first-polar-body extrusion rate. Compared to controls, the first-polar-body extrusion rate was significantly lower after 200 mM dynasore treatment (P < 0.05). C: Dynasore exposure resulted in a decreased first-polar-body extrusion rate for porcine DOs in a dose-dependent manner. The extrusion rate was significantly higher for controls than treated groups (P < 0.05).
Compared with control oocytes, actin abundance at the membrane and in the cytoplasm of oocytes decreased after dynasore treatment; actin fluorescence intensity was significantly higher in control than in treated oocytes, both at the membrane (91.43 8.94% vs. 52.00 6.43%; P < 0.01) and in the cytoplasm (39.40 3.92% vs. 14.93 1.20%) (P < 0.01) (Fig. 3A,B). These results demonstrated that Dynamin 2 is essential for actin organization in oocytes. To further assess if Dynamin 2 affected spindle positioning, we investigated actin cap and CGFD formation, which are features of successful spindle positioning. Actin caps formed in control oocytes, whereas they were undetectable after dynasore treatment (Fig. 3C). Similarly, CGs in control oocytes were absent at the region where chromosomes were close to the cortex at the MI stage, whereas CGs were uniformly distributed throughout the entire cortex after dynasore treatment (Fig. 3D). These results suggested that inhibiting Dynamin 2 disrupts the formation of an actin cap and CGFD, which are consistent with spindle positioning failure.
Dynamin 2 Inhibition Disrupts Spindle Positioning As dynasore-treated oocytes failed to undergo nuclear maturation, we first examined how spindle positioning was
728
affected at MI. After 26 hr of culture, when most oocytes had reached the late-MI stage, we stained oocytes with an anti-a-tubulin antibody and observed spindle location using confocal microscopy. Compared to control oocytes, which exhibited peripherally positioned spindles, a large proportion of 200 mM dynosore-treated oocytes had nearly centrally localized spindles (Fig. 4A,B). The frequency of oocytes with off-center spindles at the MI stage was significantly higher than that of control oocytes (56.48 6.56%, n ¼ 222 vs. 30.16 2.30%, n ¼ 240) (P < 0.05), whereas the proportions of oocytes with spindles at the cortex in the MI stage (37.11 9.47% vs. 26.23 8.34%) and in the anaphase-to-telophase (AT)/MII stage (32.73 7.50% vs. 17.29 5.48%) were reduced after dynasore treatment. Thus, inhibition of Dynamin 2 resulted in a failure of spindle migration and peripheral spindle positioning.
Dynamin 2 Inhibition Results in Disrupted ARP2 Distribution To investigate a possible signaling pathway for Dynamin 2 in porcine oocytes, we assessed changes in the expression of ARP2, an actin nucleator that belongs to the ARP2/3 complex, after dynasore treatment. Following treatment, the abundance of ARP2 protein decreased (Fig. 5A) and
Mol. Reprod. Dev. 81:725–734 (2014)
DYNAMIN 2
IN
PORCINE OOCYTES
Figure 3. Dynasore treatment affects actin assembly in porcine oocytes. A: Actin distribution in oocytes was significantly altered and abundance
lower after dynasore treatment at the membrane and in the cytoplasm. Red, actin. Scale bar, 20 mm. B: Average actin fluorescence intensity in porcine oocytes. Actin fluorescence intensity was significantly lower both at the membrane and in the cytoplasm after dynasore treatment (P < 0.01). C: Actin caps formation in oocytes when matured in the absence (top row) or presence (bottom row) of dynasore. Scale bar, 20 mm. D: CGFD formation during oocyte maturation in the absence (left) or presence (right) of dynasore. CGs were uniformly distributed throughout the entire cortex when dynasore was present during maturation. Scale bar, 20 mm.
its distribution was disrupted: in control oocytes, ARP2 resided at the cortex of porcine oocytes, but ARP2 levels at the cortex decreased in treated oocytes (Fig. 5B). ARP2 fluorescence intensity was significantly different between these two groups (59.47 4.45% vs. 25.61 1.41%) (P < 0.01) (Fig. 5C), demonstrating that Dynamin 2 inhibition might disrupt ARP2 distribution and therefore its function during meiotic maturation.
DISCUSSION In this study, we investigated the localization and possible mechanistic role of Dynamin 2 during porcine oocyte meiotic maturation. Based on our inhibitor results, we determined that Dynamin 2 contributes to oocyte maturation, spindle positioning, actin expression, and the formation of actin cap and CGFD. Additionally, our results suggest that Dynamin 2 might be an upstream regulator of ARP2/3 complex during porcine oocyte maturation.
Mol. Reprod. Dev. 81:725–734 (2014)
Dynamin 2 Localization Reflects Its Possible Effects on Porcine Oocyte Meiosis Cytokinesis produces two daughter cells, each of which contains a complete set of cytoplasmic organelles and chromosomes. Recent studies suggested that proteins, including members of the GTPase superfamily, vesiclecoat proteins, and syntaxins, contributed to the completion of cytokinesis (Finger and White, 2002). One study associated Dynamin 2 with the spindle midzone and identified that it is required for cytokinesis (Thompson et al., 2002). Dynamin homologs in Dictyostelium discoideum (Wienke et al., 1999), zebrafish (Feng et al., 2002), and plants (Lauber et al., 1997) have also been implicated in cell division. Based on our speculation that Dynamin 2 also functions during meiosis, we first determined the localization of Dynamin 2 in dividing porcine oocytes. We found that Dynamin 2 localized at the cortex and accumulated at spindles in porcine oocytes. These results were consistent with those of a previous study that reported a Dynamin
729
Molecular Reproduction & Development
ZHANG
ET AL.
Figure 4. Dynasore treatment affects spindle positioning. A: Population image of porcine oocyte spindle positioning after in vitro maturation. Almost all spindles migrated to the cortex of oocytes in control conditions; however, a majority of oocytes exhibited central spindle positioning when cultured in the presence of dynasore. Scale bar, 80 mm. B: Enlarged images of spindle positioning. In the control groups, spindles migrated to the periphery of oocytes whereas with dynasore treatment, spindles were arrested at central areas. Scale bar, 20 mm. C: Distribution of porcine spindle positions after 26 hr of maturation culture. The central localization of MI spindles is significantly higher in dynasore-treated populations than controls (P < 0.05).
homolog localizes to mitotic spindles in Caenorhabditis elegans (Clark et al., 1997). A similar observation in HIN3T3 cells identified Dynamin 2 co-associated with actin filaments and possibly involved with actin filament formation (Lim et al., 2012). These distribution patterns suggested that Dynamin 2 plays an essential role as an intermediator between actin and spindle-related processes during porcine oocyte maturation.
Dynamin 2 Influences Polar-Body Extrusion by Regulating Actin-Dependent Spindle Positioning Next, we investigated possible roles for Dynamin 2 during porcine oocyte maturation. Based on observations made among divergent models, including bacteria
730
(Dempwolff et al., 2012) and C. elegans (Thompson et al., 2002), we hypothesized that a conserved role for Dynamin 2 in cytokinesis may have been affected. In animal cells, the concerted efforts of microtubule and actin cytoskeletal elements are required to complete cytokinesis (Robinson and Spudich, 2000). Oocyte polar-body extrusion is also controlled by microtubule and microfilament cytoskeletons (Brunet and Verlhac, 2011). Our dynasoredependent inhibition data supported such a role for Dynamin 2 since overall polar-body extrusion was disrupted. During meiosis I, a spindle forms near the center of an oocyte and then migrates to the cortex in an actin-dependent manner (Verlhac et al., 2000; Sun and Kim, 2013). Inhibiting microtubule polymerization does not affect chromosome migration (Longo and Chen, 1985), whereas inhibiting actin
Mol. Reprod. Dev. 81:725–734 (2014)
DYNAMIN 2
IN
PORCINE OOCYTES
Figure 5. Expression and localization of ARP2 in porcine oocytes. A: Dynamin 2 protein abundance after
maturation; there was no difference between the control and treatment group. B: Localization of ARP2 at the cortex of porcine oocytes. ARP2 expression was significantly weaker after dynasore treatment. The average fluorescence intensity of ARP2 was significantly lower after dynasore treatment compared with the control group. Scale bar, 20 mm. C: Summary of Dynamin 2-regulated events in porcine oocyte maturation. Dynamin 2 may control ARP2/3 complex, which can influence actin assembly and thus further affect spindle positioning and polar-body extrusion.
filament polymerization or inducing actin polymerization and stabilization can prevent spindle migration, leaving a nearly centrally located set of chromosomes (Verlhac et al., 2000). Several factors that regulate actin assembly are involved in spindle positioning. Inhibiting the actin nucleator ARP2/3 blocks spindle migration and symmetric division (Sun et al., 2011). Other actin nucleators, such as Formin-2 and Spire 1/2, also participate in establishing spindle position in mouse oocytes (Leader et al., 2002; Dumont et al., 2007; Pfender et al., 2011). Moreover, mammalian Dynamins are integral regulators of the mitotic actin cytoskeleton during the final separation of cells (Thompson et al., 2002). Our results are consistent with the contribution of Dynamin 2 in meiotic spindle positioning as dynosore treatment reduced actin enrichment at both the oocyte membrane and cytoplasm while also preventing the complete translocation of the meiotic spindle. Therefore, we concluded that Dynamin 2 regulates spindle positioning through its effects on actin.
Mol. Reprod. Dev. 81:725–734 (2014)
Dynamin 2 Regulates Actin Assembly by Controlling the Distribution of ARP2 To determine how Dynamin 2 might influence actin assembly in porcine oocytes, we examined the effects of Dynamin 2 on the localization of ARP2. During mitosis, Dynamin 2 influences actin assembly together with ARP2/3 complex and Cortactin (Schafer et al., 2002). The ARP2/3 complex regulates asymmetric division and cytokinesis during the meiotic maturation of mouse oocytes (Sun et al., 2011). Our results showed that expression of ARP2 decreased after inhibition of Dynamin activity, suggesting that ARP2 might act downstream of Dynamin 2 during porcine oocyte meiosis and that its localization might require Dynamin 2 enrichment along the actin cytoskeletal network. Therefore, we propose that a conserved Dynamin 2/Arp2/3/actin signaling pathway is active in the porcine oocyte. In conclusion, our results indicated that Dynamin 2 regulates porcine oocyte maturation via regulation of the
731
Molecular Reproduction & Development
actin cytoskeleton. Both the cortical actin network and spindle positioning are affected in the absence of Dynamin 2 activity. These phenotypes could be a consequence of disrupted ARP2 distribution.
MATERIALS AND METHODS Antibodies and Chemicals Goat polyclonal anti-Dynamin 2 antibodies were purchased from Abcam (Cambridge, UK). Alexa Fluor-488 and -568 rabbit anti-goat antibodies were purchased from Invitrogen (Carlsbad, CA). Phalloidin-FITC and mouse monoclonal anti-a-tubulin antibody were purchased from Sigma (St Louis, MO). Rabbit polyclonal anti-ARP2 was purchased from Santa Cruz Biotechnology (Dallas, TX). Rabbit monoclonal anti-a-tubulin antibody and anti-rabbit horseradish peroxidase (HRP)-conjugated antibody was purchased from Cell Signaling Technology (Danvers, MA). Dynasore was purchased from Calbiochem (Darmstadt, Germany). Basic maturation culture medium M199 was from Gibco (Life Technologies, Carlsbad, CA). All other chemicals used in this study were from Sigma-Aldrich (St. Louis, MO), unless otherwise noted.
Oocytes Isolation and Culture Porcine ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to our laboratory in sterile saline (0.9% NaCl) containing 500 IU/ml of both penicillin and streptomycin at 30358C within 3 hr after slaughter. After washing twice in sterile phosphate-buffered saline (PBS), COCs were aspirated from antral follicles (25 mm in diameter) using a 20-gauge needle attached to a 5-ml disposable syringe. The COCs were separated from the cellular debris, and then washed four times with HEPESTLPPVA (tyrodelactatepyruvate polyvinylalcohol) and modified M199 containing 0.1% (w/v) polyvinyl alcohol (PVA), 32.5 mM sodium bicarbonate, 0.91 mM sodium pyruvate, 3.05 mM glucose, 75 mg/L of penicillin, and 50 mg/L of streptomycin. Only oocytes surrounded by three or more layers of intact cumulus cells and evenly granulated ooplasm were selected. COCs were cultured in a defined maturation medium consisting of 90% (v/v) modified M199, 10 ng/ml of epidermal growth factor (mouse EGF; Sigma), 0.57 mM cysteine (Sigma), 10 IU/ml hCG, 10 IU/ml PMSG, and 10% (v/v) pig follicular fluid. To prepare mature oocytes in vitro, a group of 70 COCs were transferred to 500 ml of maturation medium, then covered with 200 ml in a four-well dish (NUNC) of paraffin oil at 38.58C in a humidified, 5% CO2 atmosphere.
Dynasore Treatment During Porcine Oocyte Maturation For dynasore treatment, stock dynasore (50 mM in dimethylsulfoxide [DMSO]) was diluted in M199 to final concentrations of 100 and 200 mM. A control group was cultured in DMSO at the same relative concentration of
732
ZHANG
ET AL.
solvent. COCs or DOs were cultured with dynasore to evaluate its effects on oocyte maturation and Dynamin 2 expression. COCs were denuded of their cumulus cells by gentle pipetting with 0.1% (w/v) hyaluronidase (Sigma). Oocytes with clearly extruded polar bodies were considered to be matured. The occurrence of first-polar-body extrusion in oocytes was examined after removing cumulus cells.
Confocal Microscopy Oocytes were fixed for immunostaining in 4% paraformaldehyde in PBS for 30 min at room temperature. Oocytes were then transferred to a membrane permeabilization solution (1% Triton X-100 in PBS) for 812 hr at room temperature. After 1 hr in blocking buffer (1% bovine serum albumin [BSA] in PBS), oocytes were subsequently incubated overnight at 48C or 4 hr at room temperature with a goat anti-Dynamin 2 primary antibody (1:25), or anti-atubulin-FITC (1:200) for 2 hr, or 10 mg/ml of phalloidinTRITC for 1 hr at room temperature. After three washes (2 min each) in wash buffer (0.1% Tween 20 and 0.01% Triton X-100 in PBS), oocytes were labeled with Alexa Fluor-488 or -568 rabbit-anti-goat IgG (1:100) for 1 hr at room temperature (for Dynamin 2). These samples were counterstained with Hoechst 33342 (10 mg/ml in PBS) for 10 min, mounted on glass slides, and then examined with a confocal laser-scanning microscope (Zeiss LSM 700 META, Oberkochen, Germany). Each experiment was repeated three times; at least 30 oocytes were examined per experimental condition.
Fluorescence Intensity Analysis To analyze actin fluorescence intensity, samples of control and treated oocytes were mounted on the same glass slide. Image J software was used to define a region of interest (ROI), and the average fluorescence intensity per unit area within the ROI was determined. Independent measurements using identically sized ROIs were taken for the cell membrane and cytoplasm. The average values of all measurements were used to compare the final average intensities between control and treatment groups.
Protein Extraction and Western Blot Analysis A total of 100 porcine oocytes at the MI stage were collected, lysed in Laemmli sample buffer (SDS sample buffer with 2-mercaptoethanol), and boiled at 1008C for 5 min. Proteins subjected to SDSpolyacrylamide gel electrophoresis (PAGE) using 8% gels. After electrophoretic separation, proteins were transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA). To avoid nonspecific binding, membranes were blocked with Trisbuffered saline (TBS) containing 0.1% (w/w) Tween 20 (TBST) and 5% (w/v) nonfat dry milk powder for 2 hr at room temperature. After a short wash in TBST, the membrane was incubated with rabbit polyclonal anti-ARP2 (1:200) primary antibody at 48C, overnight. After washing
Mol. Reprod. Dev. 81:725–734 (2014)
DYNAMIN 2
three times in TBST (10 min each), membranes were incubated for 2 hr with secondary anti-rabbit HRP-conjugated antibodies (1:2,000) in 5% nonfat dry milk in TBST. The membrane was then exposed to enhanced chemiluminescence reagent (EMD Millipore, Billerica, MA). Detection of a-tubulin, with rabbit monoclonal anti-a-tubulin antibody (1:2,000) and secondary anti-rabbit antibody was used as a loading control.
Statistical Analysis For each treatment, at least three replicates were performed. Results were expressed as means standard errors of the mean. Statistical comparisons were made by ANOVA, followed by Duncan’s multiple comparisons tests. P < 0.05 was considered significant.
ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (2014CB138503), the Fundamental Research Funds for the Central Universities (KJQN201402), the Natural Science Foundation of Jiangsu Province (BK20130671), China; and the Biogreen 21 Program (PJ009594, PJ009080, and PJ00909801), RDA, Republic of Korea.
REFERENCES Brunet S, Verlhac MH. 2011. Positioning to get out of meiosis: The asymmetry of division. Hum Reprod Update 17:6875. Cao H, Garcia F, McNiven MA. 1998. Differential distribution of dynamin isoforms in mammalian cells. Mol Biol Cell 9: 25952609. Clark SG, Shurland D-L, Meyerowitz EM, Bargmann CI, Van Der Bliek AM. 1997. A dynamin GTPase mutation causes a rapid and reversible temperature-inducible locomotion defect in C. elegans. Proc Natl Acad Sci 94:1043810443. Cook T, Mesa K, Urrutia R. 1996. Three dynamin-encoding genes are differentially expressed in developing rat brain. J Neurochem 67:927931. Dempwolff F, Wischhusen HM, Specht M, Graumann PL. 2012. The deletion of bacterial dynamin and flotillin genes results in pleiotrophic effects on cell division, cell growth and in cell shape maintenance. BMC Microbiol 12:298. Deng M, Kishikawa H, Yanagimachi R, Kopf GS, Schultz RM, Williams CJ. 2003. Chromatin-mediated cortical granule redistribution is responsible for the formation of the cortical granulefree domain in mouse eggs. Dev Biol 257:166176. Dumont J, Million K, Sunderland K, Rassinier P, Lim H, Leader B, Verlhac MH. 2007. Formin-2 is required for spindle migration and for the late steps of cytokinesis in mouse oocytes. Dev Biol 301:254265.
Mol. Reprod. Dev. 81:725–734 (2014)
IN
PORCINE OOCYTES
Feng B, Schwarz H, Jesuthasan S. 2002. Furrow-specific endocytosis during cytokinesis of zebrafish blastomeres. Exp Cell Res 279:1420. Finger FP, White JG. 2002. Fusion and fission: Membrane trafficking in animal cytokinesis. Cell 108:727730. Hussain NK, Jenna S, Glogauer M, Quinn CC, Wasiak S, Guipponi M, Antonarakis SE, Kay BK, Stossel TP, Lamarche-Vane N. 2001. Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP. Nat Cell Biol 3:927932. Kessels MM, Engqvist-Goldstein éE, Drubin DG, Qualmann B. 2001. Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin. J Cell Biol 153:351366. Konopka CA, Schleede JB, Skop AR, Bednarek SY. 2006. Dynamin and cytokinesis. Traffic 7:239247. Lauber MH, Waizenegger I, Steinmann T, Schwarz H, Mayer U, Hwang I, Lukowitz W, Jürgens G. 1997. The Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin. J Cell Biol 139:14851493. Leader B, Lim H, Carabatsos MJ, Harrington A, Ecsedy J, Pellman D, Maas R, Leder P. 2002. Formin-2, polyploidy, hypofertility and positioning of the meiotic spindle in mouse oocytes. Nat Cell Biol 4:921928. Lee E, De Camilli P. 2002. Dynamin at actin tails. Proc Natl Acad Sci 99:161166. Lim DS, Kim SR, Choi BD, Jeong SJ, Wang G, Kim HK, Kim BH, Goo JH, Jeong MJ. 2012. Dynamin II involves in cell migration and actin formation of NIH3T3 cells. J Nanosci Nanotechnol 12:690695. Longo FJ, Chen DY. 1985. Development of cortical polarity in mouse eggs: Involvement of the meiotic apparatus. Dev Biol 107:382394. Orth JD, Krueger E, Cao H, McNiven MA. 2002. The large GTPase dynamin regulates actin comet formation and movement in living cells. Proc Natl Acad Sci 99:167172. Orth JD, McNiven MA. 2003. Dynamin at the actinmembrane interface. Current Opin Cell Biol 15:3139. Pfender S, Kuznetsov V, Pleiser S, Kerkhoff E, Schuh M. 2011. Spire-type actin nucleators cooperate with Formin-2 to drive asymmetric oocyte division. Curr Biol 21:955960. Pollard TD, Blanchoin L, Mullins RD. 2000. Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu Rev Biophys Biomol Structure 29:545576. Praefcke GJ, McMahon HT. 2004. The dynamin superfamily: Universal membrane tubulation and fission molecules? Nat Rev Mol Cell Biol 5:133147. Qualmann B, Kelly RB. 2000. Syndapin isoforms participate in receptor-mediated endocytosis and actin organization. J Cell Biol 148:10471062.
733
Molecular Reproduction & Development
ZHANG
ET AL.
Robinson DN, Spudich JA. 2000. Towards a molecular understanding of cytokinesis. Trends Cell Biol 10:228237.
midzone and is required for cytokinesis. Curr Biol 12: 21112117.
Salazar MA, Kwiatkowski AV, Pellegrini L, Cestra G, Butler MH, Rossman KL, Serna DM, Sondek J, Gertler FB, De Camilli P. 2003. Tuba, a novel protein containing bin/amphiphysin/Rvs and Dbl homology domains, links dynamin to regulation of the actin cytoskeleton. J Biol Chem 278:4903149043.
Uruno T, Liu J, Zhang P, Fan Yx, Egile C, Li R, Mueller SC, Zhan X. 2001. Activation of Arp2/3 complex-mediated actin polymerization by cortactin. Nat Cell Biol 3:259266.
Schafer DA. 2004. Regulating actin dynamics at membranes: A focus on dynamin. Traffic 5:463469. Schafer DA, Weed SA, Binns D, Karginov AV, Parsons JT, Cooper JA. 2002. Dynamin2 and cortactin regulate actin assembly and filament organization. Curr Biol 12:18521857. Sever S, Muhlberg AB, Schmid SL. 1999. Impairment of dynamin’s GAP domain stimulates receptor-mediated endocytosis. Nature 398:481486. Sun SC, Wang ZB, Xu YN, Lee SE, Cui XS, Kim NH. 2011. Arp2/3 complex regulates asymmetric division and cytokinesis in mouse oocytes. PLoS ONE 6:e18392. Sun SC, Kim NH. 2013. Molecular mechanisms of asymmetric division in oocytes. Microsc Microanal 19:883897. Thompson HM, Skop AR, Euteneuer U, Meyer BJ, McNiven MA. 2002. The large GTPase dynamin associates with the spindle
734
Verlhac M-H, Lefebvre C, Guillaud P, Rassinier P, Maro B. 2000. Asymmetric division in mouse oocytes: With or without Mos. Curr Biol 10:13031306. Wienke DC, Knetsch ML, Neuhaus EM, Reedy MC, Manstein DJ. 1999. Disruption of a dynamin homologue affects endocytosis, organelle morphology, and cytokinesis in Dictyostelium discoideum. Mol Biol Cell 10:225243. Witke W, Podtelejnikov AV, Di Nardo A, Sutherland JD, Gurniak CB, Dotti C, Mann M. 1998. In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly. EMBO J 17:967976. Yi K, Rubinstein B, Li R. 2013a. Symmetry breaking and polarity establishment during mouse oocyte maturation. Philos Trans R Soc Lond B Biol Sci 368:20130002. Yi K, Rubinstein B, Unruh JR, Guo F, Slaughter BD, Li R. 2013b. Sequential actin-based pushing forces drive meiosis I chromosome migration and symmetry breaking in oocytes. J Cell Biol 200:567576.
Mol. Reprod. Dev. 81:725–734 (2014)
Involvement of Dynamin 2 in Actin-Based Polar-Body Extrusion During Porcine Oocyte Maturation YU ZHANG,1,2 QIAO-CHU WANG,1 JUN HAN,1 RUI CAO,1 XIANG-SHUN CUI,3 NAM-HYUNG KIM,3 RONG RUI2, 1 AND SHAO-CHEN SUN * 1
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, People’s Republic of, China College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, People’s Republic of, China 3 Department of Animal Sciences, Chungbuk National University, Cheongju, Chungbuk, Korea 2
SUMMARY Mammalian oocyte meiotic maturation involves a unique asymmetric division, but the regulatory mechanisms and signaling pathways involved are poorly understood. Dynamins are ubiquitous eukaryotic GTPases involved in membrane trafficking and actin dynamics, whose roles in mammalian oocyte maturation have not been determined. In this study, we used porcine oocytes to show that Dynamin 2 accumulated at the meiotic spindle and in the cortex of oocytes, with a distribution similar to that of actin. Inhibiting Dynamin 2 activity in porcine oocytes with the specific inhibitor dynasore resulted in failed polar-body extrusion. This phenotype may have been due to aberrant actin distribution and/or spindle positioning as inhibitor treatment disrupted the formation of the actin cap and cortical granule-free domain, which negatively impacted spindle positioning. Moreover, the distribution of ARP2, a key actin-nucleation factor, was severely reduced in the cortex after dynasore treatment. Thus, our results suggest that Dynamin 2 possibly regulates porcine oocyte maturation through its effects on actin-mediated spindle positioning and cytokinesis, and that this may depend on regulating ARP2 localization.
Mol. Reprod. Dev. 81: 725734, 2014. ß 2014 Wiley Periodicals, Inc. Received 15 April 2014; Accepted 8 May 2014
Corresponding author: College of Animal Sciences and Technology Nanjing Agricultural University Nanjing, People’s Republic of China. E-mail: [email protected]
Grant sponsor: National Basic Research Program of China; Grant number: 2014CB138503; Grant sponsor: Fundamental Research Funds for the Central Universities; Grant number: KJQN201402; Grant sponsor: Natural Science Foundation of Jiangsu Province, China; Grant number: BK20130671; Grant sponsor: Biogreen 21 Program; Grant numbers: PJ009594, PJ009080, PJ00909801; Grant sponsor: RDA, Republic of Korea
Published online 22 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22341
INTRODUCTION Mammalian oocytes remain arrested at the late diplotene stage of prophase I from birth until oocyte meiotic maturation, whose completion is responsible for successful fertilization. An oocyte initiates meiotic maturation from the germinal vesicle (GV) stage, passes through germinal vesicle breakdown (GVBD), completes a unique asymmet-
ß 2014 WILEY PERIODICALS, INC.
Abbreviations: ARP2/3 complex, actin-related protein 2/3 complex; CGFD, cortical-granule-free domain; COCs, cumulus oocyte complexes; DOs, denuded oocytes; GV[BD], germinal vesicle [breakdown]; MI/II, metaphase I/II.
Molecular Reproduction & Development
ric division involving the extrusion of a small, first polar body, and finally results in a highly polarized, metaphase II (MII) egg. These latter events are essential for retaining the most cytoplasm in the egg for the developing zygote. Polar-body formation is dependent on proper oocyte polarization. This process is controlled by the microtubule and microfilament cytoskeletons, resulting in spindle migration and positioning and in cortical reorganization (Brunet and Verlhac, 2011). After GVBD, the bipolar, meiosis-I spindle is assembled close to the center of the oocyte. During metaphase I (MI) stage, however, the spindle translocates to the equatorial plate and subsequently migrates to one side of the oocyte cortex, aligned along the spindle’s long axis. In addition to eccentric spindle positioning, another event pivotal for asymmetric division during oocyte maturation is the organization of cortical polarity: actin becomes enriched at an actin cap in the cortical area where cortical granules (CGs) and microvilli are also absent, resulting in a CG-free domain (CGFD) (Deng et al., 2003). After anaphase, cytokinesis segregates one set of homologous chromosomes to the first polar body. The oocyte then proceeds through meiosis I until MII stage, remaining arrested at this stage until fertilization. Involvement of the actin cytoskeleton in asymmetric spindle positioning and cortical polarization during oocyte meiotic division has been observed. Migration of the spindle (or chromosomes) is a dynamic, actin-dependent process. Previous work has implicated an actin feedback loop in the maintenance of meiotic spindle position during MII arrest (Yi et al., 2013a). The molecular mechanisms underlying spindle positioning emerged when it was reported that Formin-2 and ARP2/3 (actin-related protein 2/3 complex), which nucleate actin assembly, generate the force needed to propel chromosomes/the spindle towards the cortex (Yi et al., 2013b). Until now, the molecular details underlying these events of oocyte meiotic maturation were poorly understood. The Dynamin superfamily is comprised of Dynamin and several Dynamin-related proteins. Dynamins were originally identified as large GTPases homologous to the Drosophila Shibire gene product. There are three Dynamins in mammalian genomes (Cook et al., 1996), which share at least 70% homology. Dynamin 1 is expressed exclusively in the brain; Dynamin 2 is ubiquitously distributed; and Dynamin 3 is selectively expressed in the brain and testis (Cao et al., 1998); Dynamin 2 is the only isoform that functions in mammals (Thompson et al., 2002). Dynamins are involved in a variety of cell processes, including endocytosis, mitochondrial and chloroplast biogenesis, and cytokinesis (Praefcke and McMahon, 2004). Like other members of the GTPase superfamily, it appears that Dynamin itself does not mediate fission, instead acting as a molecular regulator during receptor-mediated endocytosis (Sever et al., 1999), possibly by regulating actin. In addition to its critical role in deforming membranes to create tubules or vesicles (Praefcke and McMahon, 2004), Dynamin particularly Dynamin 2 localizes to cellular membranes that are undergoing remodeling, specifically associating with actin filaments and thereby influencing
726
ZHANG
ET AL.
actin dynamics (Orth and McNiven, 2003). Consistent with this general function, Dynamin 2 is required for cytoskeletal reorganization, including actin-related podosome formation, membrane ruffling (Schafer, 2004), and actintail-based vesicle trafficking (Lee and De Camilli, 2002). It can nucleate actin at membranes, and regulates actincomet formation and movement in the rat and mouse (Orth et al., 2002). One function of Dynamin 2 appears to be based on its interactions with various cytoskeletal regulators during mitosis (Konopka et al., 2006). Cortactin is a Dynamin-2 binding partner. Actin filament nucleation by Dynamin 2 occurs through a Dynamin 2/Cortactin/ARP2/3 complex (Uruno et al., 2001; Schafer et al., 2002) that can bind filamentous actin (F-actin) in membrane ruffles, podosomes, actin comets, clathrin-coated pits, and the Golgi complex. It was recently found that another actin binding protein, ABP1, can directly bind to and co-localizes with Dynamin 2, linking the actin cytoskeleton to it (Kessels et al., 2001). Dynamin 2 can also regulate actin assembly by associating with three indirect actin-binding scaffold protein: Intersectin-1 (Hussain et al., 2001), Syndapin (Qualmann and Kelly, 2000), and Tuba (Salazar et al., 2003). Both Intersectin and Syndapin interact with N-WASP to regulate ARP2/3-dependent actin nucleation (Qualmann and Kelly, 2000). Profilin, the first protein identified from mouse brain that directly binds to Dynamin 2 (Witke et al., 1998), acts downstream of Dynamin 2 and is involved in actin polymerization (Pollard et al., 2000). In this study, we investigated the expression and functions of Dynamin 2 during porcine oocyte meiosis by using a specific inhibitor, dynasore. Our results showed that Dynamin 2 inhibition caused aberrant actin organization, failure of proper spindle positioning, and oocyte cytokinesis. We also found that ARP2 may act downstream of Dynamin 2 in the regulation of the actin cytoskeleton. Thus, our results demonstrated a functional mechanism for Dynamin 2 in porcine oocytes.
RESULTS Dynamin 2 Localization During Porcine Oocyte Meiotic Maturation We first examined the expression of the Dynamin 2 in cumulus oocyte complexes (COCs). Dynamin 2 was present in both cumulus cells and the oocytes (Fig. 1A). The subcellular localization of Dynamin 2 at different stages of meiotic maturation was also profiled using immunofluorescence staining (Fig. 1B): Dynamin 2 was observed in the cortex of GV oocytes. After GVBD, Dynamin 2 was found in the cortex while also accumulating at the spindle during all subsequent meiotic stages. To confirm this spindle localization, we co-stained for a-tubulin along with Dynamin 2, which revealed specific co-localization at this structure (Fig. 1C). Following dynasore inhibition of Dynamin, Dynamin 2 protein levels were significantly reduced compared to untreated porcine oocytes (Fig. 1B vs. 1D).
Mol. Reprod. Dev. 81:725–734 (2014)
DYNAMIN 2
IN
PORCINE OOCYTES
Figure 1. Dynamin 2 localization in porcine oocytes. A: Immunofluorescence staining for Dynamin 2 in COCs. Green, Dynamin 2; red, actin; blue, chromatin. Scale bar, 30 mm. B: Subcellular localization of Dynamin 2 during porcine oocyte meiotic maturation. Dynamin 2 accumulated at the cortex of GV-stage oocytes. From GVBD to MII, Dynamin 2 also accumulated at the spindle and at the oocytes cortex. Green, Dynamin 2; blue, chromatin. Scale bar, 20 mm. C: Immunofluorescence co-localization of a-tubulin and Dynamin 2 in oocytes. Red, Dynamin 2; green, a-tubulin; blue, chromatin. Scale bar, 20 mm. D: Immunofluorescence staining for Dynamin 2 in oocytes after dynasore exposure during porcine oocyte maturation. Dynamin 2 abundance was minimal after dynasore treatment. Green, Dynamin 2; blue, chromatin. Scale bar, 20 mm.
Dynamin 2 Inhibition Disrupts Porcine Oocyte Maturation In Vitro Based on the result that dynasore treatment suppresses Dynamin 2 abundance in oocytes, we examined if Dynamin 2 is involved in regulating porcine oocyte maturation. When COCs were treated with dynasore for 44 hr during maturation, cumulus expansion failed (Fig. 2A). Most COCs in the control group were surrounded by more than five layers of intact cumulus cells, whereas the cumulus layer became weakly diffused with 100 and 200 mM dynasore. A large proportion of inhibitor-treated oocytes also failed to extrude polar bodies in a dosedependent manner (Fig. 2B). After 44 hr of culture, most control oocytes extruded polar bodies and were arrested at the MII stage; the rate of first-polar-body extrusion for controls was 79.36 2.64% (n ¼ 179 COCs). The rate of polar-body extrusion was 70.99 3.13% (n ¼ 180 COCs) with 100 mM dynasore, and was significantly reduced to 36.98 7.71% (P < 0.05; n ¼ 182 COCs) with 200 mM
Mol. Reprod. Dev. 81:725–734 (2014)
treatment. A dynasore concentration of 200 mM was used for subsequent studies. To exclude the effects of cumulus cells on oocyte maturation, denuded oocytes (DOs) were also treated with dynasore (Fig. 2C). The rate of first-polar-body extrusion in the control group was 67.07 3.40% (n ¼ 88 DOs), which was significantly higher than the 200 mM treatment group (29.46 3.85%; n ¼ 75 DOs) (P < 0.05). Thus dynasore had direct effects on oocyte nuclear maturation. Considering the lower in vitro-maturation rates of DOs, however, COCs were adopted to investigate the mechanisms involving Dynamin 2 during porcine oocyte maturation.
Dynamin 2 Inhibition Affects Actin Assembly in Porcine Oocytes Previous studies established that Dynamin is a major actin nucleator. In this study, we investigated if Dynamin 2 affected actin filament organization in porcine oocytes.
727
Molecular Reproduction & Development
ZHANG
ET AL.
Figure 2. Effects of Dynasore treatment on porcine oocyte maturation. A: Representative image of porcine COCs maturation in the presence of dynasore, specifically showing cumulus expansion and first-polar-body (PB1) extrusion. In the control group, most COCs were surrounded by more than five layers of intact cumulus cells, whereas weak diffusion was observed after treatment with dynasore at 100 and 200 mM. Almost all control oocytes extruded polar bodies as compared with the two inhibitor-treated groups. Scale bar, 80 mm. B: Dynasore treatment resulted in a dosedependent, reduced first-polar-body extrusion rate. Compared to controls, the first-polar-body extrusion rate was significantly lower after 200 mM dynasore treatment (P < 0.05). C: Dynasore exposure resulted in a decreased first-polar-body extrusion rate for porcine DOs in a dose-dependent manner. The extrusion rate was significantly higher for controls than treated groups (P < 0.05).
Compared with control oocytes, actin abundance at the membrane and in the cytoplasm of oocytes decreased after dynasore treatment; actin fluorescence intensity was significantly higher in control than in treated oocytes, both at the membrane (91.43 8.94% vs. 52.00 6.43%; P < 0.01) and in the cytoplasm (39.40 3.92% vs. 14.93 1.20%) (P < 0.01) (Fig. 3A,B). These results demonstrated that Dynamin 2 is essential for actin organization in oocytes. To further assess if Dynamin 2 affected spindle positioning, we investigated actin cap and CGFD formation, which are features of successful spindle positioning. Actin caps formed in control oocytes, whereas they were undetectable after dynasore treatment (Fig. 3C). Similarly, CGs in control oocytes were absent at the region where chromosomes were close to the cortex at the MI stage, whereas CGs were uniformly distributed throughout the entire cortex after dynasore treatment (Fig. 3D). These results suggested that inhibiting Dynamin 2 disrupts the formation of an actin cap and CGFD, which are consistent with spindle positioning failure.
Dynamin 2 Inhibition Disrupts Spindle Positioning As dynasore-treated oocytes failed to undergo nuclear maturation, we first examined how spindle positioning was
728
affected at MI. After 26 hr of culture, when most oocytes had reached the late-MI stage, we stained oocytes with an anti-a-tubulin antibody and observed spindle location using confocal microscopy. Compared to control oocytes, which exhibited peripherally positioned spindles, a large proportion of 200 mM dynosore-treated oocytes had nearly centrally localized spindles (Fig. 4A,B). The frequency of oocytes with off-center spindles at the MI stage was significantly higher than that of control oocytes (56.48 6.56%, n ¼ 222 vs. 30.16 2.30%, n ¼ 240) (P < 0.05), whereas the proportions of oocytes with spindles at the cortex in the MI stage (37.11 9.47% vs. 26.23 8.34%) and in the anaphase-to-telophase (AT)/MII stage (32.73 7.50% vs. 17.29 5.48%) were reduced after dynasore treatment. Thus, inhibition of Dynamin 2 resulted in a failure of spindle migration and peripheral spindle positioning.
Dynamin 2 Inhibition Results in Disrupted ARP2 Distribution To investigate a possible signaling pathway for Dynamin 2 in porcine oocytes, we assessed changes in the expression of ARP2, an actin nucleator that belongs to the ARP2/3 complex, after dynasore treatment. Following treatment, the abundance of ARP2 protein decreased (Fig. 5A) and
Mol. Reprod. Dev. 81:725–734 (2014)
DYNAMIN 2
IN
PORCINE OOCYTES
Figure 3. Dynasore treatment affects actin assembly in porcine oocytes. A: Actin distribution in oocytes was significantly altered and abundance
lower after dynasore treatment at the membrane and in the cytoplasm. Red, actin. Scale bar, 20 mm. B: Average actin fluorescence intensity in porcine oocytes. Actin fluorescence intensity was significantly lower both at the membrane and in the cytoplasm after dynasore treatment (P < 0.01). C: Actin caps formation in oocytes when matured in the absence (top row) or presence (bottom row) of dynasore. Scale bar, 20 mm. D: CGFD formation during oocyte maturation in the absence (left) or presence (right) of dynasore. CGs were uniformly distributed throughout the entire cortex when dynasore was present during maturation. Scale bar, 20 mm.
its distribution was disrupted: in control oocytes, ARP2 resided at the cortex of porcine oocytes, but ARP2 levels at the cortex decreased in treated oocytes (Fig. 5B). ARP2 fluorescence intensity was significantly different between these two groups (59.47 4.45% vs. 25.61 1.41%) (P < 0.01) (Fig. 5C), demonstrating that Dynamin 2 inhibition might disrupt ARP2 distribution and therefore its function during meiotic maturation.
DISCUSSION In this study, we investigated the localization and possible mechanistic role of Dynamin 2 during porcine oocyte meiotic maturation. Based on our inhibitor results, we determined that Dynamin 2 contributes to oocyte maturation, spindle positioning, actin expression, and the formation of actin cap and CGFD. Additionally, our results suggest that Dynamin 2 might be an upstream regulator of ARP2/3 complex during porcine oocyte maturation.
Mol. Reprod. Dev. 81:725–734 (2014)
Dynamin 2 Localization Reflects Its Possible Effects on Porcine Oocyte Meiosis Cytokinesis produces two daughter cells, each of which contains a complete set of cytoplasmic organelles and chromosomes. Recent studies suggested that proteins, including members of the GTPase superfamily, vesiclecoat proteins, and syntaxins, contributed to the completion of cytokinesis (Finger and White, 2002). One study associated Dynamin 2 with the spindle midzone and identified that it is required for cytokinesis (Thompson et al., 2002). Dynamin homologs in Dictyostelium discoideum (Wienke et al., 1999), zebrafish (Feng et al., 2002), and plants (Lauber et al., 1997) have also been implicated in cell division. Based on our speculation that Dynamin 2 also functions during meiosis, we first determined the localization of Dynamin 2 in dividing porcine oocytes. We found that Dynamin 2 localized at the cortex and accumulated at spindles in porcine oocytes. These results were consistent with those of a previous study that reported a Dynamin
729
Molecular Reproduction & Development
ZHANG
ET AL.
Figure 4. Dynasore treatment affects spindle positioning. A: Population image of porcine oocyte spindle positioning after in vitro maturation. Almost all spindles migrated to the cortex of oocytes in control conditions; however, a majority of oocytes exhibited central spindle positioning when cultured in the presence of dynasore. Scale bar, 80 mm. B: Enlarged images of spindle positioning. In the control groups, spindles migrated to the periphery of oocytes whereas with dynasore treatment, spindles were arrested at central areas. Scale bar, 20 mm. C: Distribution of porcine spindle positions after 26 hr of maturation culture. The central localization of MI spindles is significantly higher in dynasore-treated populations than controls (P < 0.05).
homolog localizes to mitotic spindles in Caenorhabditis elegans (Clark et al., 1997). A similar observation in HIN3T3 cells identified Dynamin 2 co-associated with actin filaments and possibly involved with actin filament formation (Lim et al., 2012). These distribution patterns suggested that Dynamin 2 plays an essential role as an intermediator between actin and spindle-related processes during porcine oocyte maturation.
Dynamin 2 Influences Polar-Body Extrusion by Regulating Actin-Dependent Spindle Positioning Next, we investigated possible roles for Dynamin 2 during porcine oocyte maturation. Based on observations made among divergent models, including bacteria
730
(Dempwolff et al., 2012) and C. elegans (Thompson et al., 2002), we hypothesized that a conserved role for Dynamin 2 in cytokinesis may have been affected. In animal cells, the concerted efforts of microtubule and actin cytoskeletal elements are required to complete cytokinesis (Robinson and Spudich, 2000). Oocyte polar-body extrusion is also controlled by microtubule and microfilament cytoskeletons (Brunet and Verlhac, 2011). Our dynasoredependent inhibition data supported such a role for Dynamin 2 since overall polar-body extrusion was disrupted. During meiosis I, a spindle forms near the center of an oocyte and then migrates to the cortex in an actin-dependent manner (Verlhac et al., 2000; Sun and Kim, 2013). Inhibiting microtubule polymerization does not affect chromosome migration (Longo and Chen, 1985), whereas inhibiting actin
Mol. Reprod. Dev. 81:725–734 (2014)
DYNAMIN 2
IN
PORCINE OOCYTES
Figure 5. Expression and localization of ARP2 in porcine oocytes. A: Dynamin 2 protein abundance after
maturation; there was no difference between the control and treatment group. B: Localization of ARP2 at the cortex of porcine oocytes. ARP2 expression was significantly weaker after dynasore treatment. The average fluorescence intensity of ARP2 was significantly lower after dynasore treatment compared with the control group. Scale bar, 20 mm. C: Summary of Dynamin 2-regulated events in porcine oocyte maturation. Dynamin 2 may control ARP2/3 complex, which can influence actin assembly and thus further affect spindle positioning and polar-body extrusion.
filament polymerization or inducing actin polymerization and stabilization can prevent spindle migration, leaving a nearly centrally located set of chromosomes (Verlhac et al., 2000). Several factors that regulate actin assembly are involved in spindle positioning. Inhibiting the actin nucleator ARP2/3 blocks spindle migration and symmetric division (Sun et al., 2011). Other actin nucleators, such as Formin-2 and Spire 1/2, also participate in establishing spindle position in mouse oocytes (Leader et al., 2002; Dumont et al., 2007; Pfender et al., 2011). Moreover, mammalian Dynamins are integral regulators of the mitotic actin cytoskeleton during the final separation of cells (Thompson et al., 2002). Our results are consistent with the contribution of Dynamin 2 in meiotic spindle positioning as dynosore treatment reduced actin enrichment at both the oocyte membrane and cytoplasm while also preventing the complete translocation of the meiotic spindle. Therefore, we concluded that Dynamin 2 regulates spindle positioning through its effects on actin.
Mol. Reprod. Dev. 81:725–734 (2014)
Dynamin 2 Regulates Actin Assembly by Controlling the Distribution of ARP2 To determine how Dynamin 2 might influence actin assembly in porcine oocytes, we examined the effects of Dynamin 2 on the localization of ARP2. During mitosis, Dynamin 2 influences actin assembly together with ARP2/3 complex and Cortactin (Schafer et al., 2002). The ARP2/3 complex regulates asymmetric division and cytokinesis during the meiotic maturation of mouse oocytes (Sun et al., 2011). Our results showed that expression of ARP2 decreased after inhibition of Dynamin activity, suggesting that ARP2 might act downstream of Dynamin 2 during porcine oocyte meiosis and that its localization might require Dynamin 2 enrichment along the actin cytoskeletal network. Therefore, we propose that a conserved Dynamin 2/Arp2/3/actin signaling pathway is active in the porcine oocyte. In conclusion, our results indicated that Dynamin 2 regulates porcine oocyte maturation via regulation of the
731
Molecular Reproduction & Development
actin cytoskeleton. Both the cortical actin network and spindle positioning are affected in the absence of Dynamin 2 activity. These phenotypes could be a consequence of disrupted ARP2 distribution.
MATERIALS AND METHODS Antibodies and Chemicals Goat polyclonal anti-Dynamin 2 antibodies were purchased from Abcam (Cambridge, UK). Alexa Fluor-488 and -568 rabbit anti-goat antibodies were purchased from Invitrogen (Carlsbad, CA). Phalloidin-FITC and mouse monoclonal anti-a-tubulin antibody were purchased from Sigma (St Louis, MO). Rabbit polyclonal anti-ARP2 was purchased from Santa Cruz Biotechnology (Dallas, TX). Rabbit monoclonal anti-a-tubulin antibody and anti-rabbit horseradish peroxidase (HRP)-conjugated antibody was purchased from Cell Signaling Technology (Danvers, MA). Dynasore was purchased from Calbiochem (Darmstadt, Germany). Basic maturation culture medium M199 was from Gibco (Life Technologies, Carlsbad, CA). All other chemicals used in this study were from Sigma-Aldrich (St. Louis, MO), unless otherwise noted.
Oocytes Isolation and Culture Porcine ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to our laboratory in sterile saline (0.9% NaCl) containing 500 IU/ml of both penicillin and streptomycin at 30358C within 3 hr after slaughter. After washing twice in sterile phosphate-buffered saline (PBS), COCs were aspirated from antral follicles (25 mm in diameter) using a 20-gauge needle attached to a 5-ml disposable syringe. The COCs were separated from the cellular debris, and then washed four times with HEPESTLPPVA (tyrodelactatepyruvate polyvinylalcohol) and modified M199 containing 0.1% (w/v) polyvinyl alcohol (PVA), 32.5 mM sodium bicarbonate, 0.91 mM sodium pyruvate, 3.05 mM glucose, 75 mg/L of penicillin, and 50 mg/L of streptomycin. Only oocytes surrounded by three or more layers of intact cumulus cells and evenly granulated ooplasm were selected. COCs were cultured in a defined maturation medium consisting of 90% (v/v) modified M199, 10 ng/ml of epidermal growth factor (mouse EGF; Sigma), 0.57 mM cysteine (Sigma), 10 IU/ml hCG, 10 IU/ml PMSG, and 10% (v/v) pig follicular fluid. To prepare mature oocytes in vitro, a group of 70 COCs were transferred to 500 ml of maturation medium, then covered with 200 ml in a four-well dish (NUNC) of paraffin oil at 38.58C in a humidified, 5% CO2 atmosphere.
Dynasore Treatment During Porcine Oocyte Maturation For dynasore treatment, stock dynasore (50 mM in dimethylsulfoxide [DMSO]) was diluted in M199 to final concentrations of 100 and 200 mM. A control group was cultured in DMSO at the same relative concentration of
732
ZHANG
ET AL.
solvent. COCs or DOs were cultured with dynasore to evaluate its effects on oocyte maturation and Dynamin 2 expression. COCs were denuded of their cumulus cells by gentle pipetting with 0.1% (w/v) hyaluronidase (Sigma). Oocytes with clearly extruded polar bodies were considered to be matured. The occurrence of first-polar-body extrusion in oocytes was examined after removing cumulus cells.
Confocal Microscopy Oocytes were fixed for immunostaining in 4% paraformaldehyde in PBS for 30 min at room temperature. Oocytes were then transferred to a membrane permeabilization solution (1% Triton X-100 in PBS) for 812 hr at room temperature. After 1 hr in blocking buffer (1% bovine serum albumin [BSA] in PBS), oocytes were subsequently incubated overnight at 48C or 4 hr at room temperature with a goat anti-Dynamin 2 primary antibody (1:25), or anti-atubulin-FITC (1:200) for 2 hr, or 10 mg/ml of phalloidinTRITC for 1 hr at room temperature. After three washes (2 min each) in wash buffer (0.1% Tween 20 and 0.01% Triton X-100 in PBS), oocytes were labeled with Alexa Fluor-488 or -568 rabbit-anti-goat IgG (1:100) for 1 hr at room temperature (for Dynamin 2). These samples were counterstained with Hoechst 33342 (10 mg/ml in PBS) for 10 min, mounted on glass slides, and then examined with a confocal laser-scanning microscope (Zeiss LSM 700 META, Oberkochen, Germany). Each experiment was repeated three times; at least 30 oocytes were examined per experimental condition.
Fluorescence Intensity Analysis To analyze actin fluorescence intensity, samples of control and treated oocytes were mounted on the same glass slide. Image J software was used to define a region of interest (ROI), and the average fluorescence intensity per unit area within the ROI was determined. Independent measurements using identically sized ROIs were taken for the cell membrane and cytoplasm. The average values of all measurements were used to compare the final average intensities between control and treatment groups.
Protein Extraction and Western Blot Analysis A total of 100 porcine oocytes at the MI stage were collected, lysed in Laemmli sample buffer (SDS sample buffer with 2-mercaptoethanol), and boiled at 1008C for 5 min. Proteins subjected to SDSpolyacrylamide gel electrophoresis (PAGE) using 8% gels. After electrophoretic separation, proteins were transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA). To avoid nonspecific binding, membranes were blocked with Trisbuffered saline (TBS) containing 0.1% (w/w) Tween 20 (TBST) and 5% (w/v) nonfat dry milk powder for 2 hr at room temperature. After a short wash in TBST, the membrane was incubated with rabbit polyclonal anti-ARP2 (1:200) primary antibody at 48C, overnight. After washing
Mol. Reprod. Dev. 81:725–734 (2014)
DYNAMIN 2
three times in TBST (10 min each), membranes were incubated for 2 hr with secondary anti-rabbit HRP-conjugated antibodies (1:2,000) in 5% nonfat dry milk in TBST. The membrane was then exposed to enhanced chemiluminescence reagent (EMD Millipore, Billerica, MA). Detection of a-tubulin, with rabbit monoclonal anti-a-tubulin antibody (1:2,000) and secondary anti-rabbit antibody was used as a loading control.
Statistical Analysis For each treatment, at least three replicates were performed. Results were expressed as means standard errors of the mean. Statistical comparisons were made by ANOVA, followed by Duncan’s multiple comparisons tests. P < 0.05 was considered significant.
ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (2014CB138503), the Fundamental Research Funds for the Central Universities (KJQN201402), the Natural Science Foundation of Jiangsu Province (BK20130671), China; and the Biogreen 21 Program (PJ009594, PJ009080, and PJ00909801), RDA, Republic of Korea.
REFERENCES Brunet S, Verlhac MH. 2011. Positioning to get out of meiosis: The asymmetry of division. Hum Reprod Update 17:6875. Cao H, Garcia F, McNiven MA. 1998. Differential distribution of dynamin isoforms in mammalian cells. Mol Biol Cell 9: 25952609. Clark SG, Shurland D-L, Meyerowitz EM, Bargmann CI, Van Der Bliek AM. 1997. A dynamin GTPase mutation causes a rapid and reversible temperature-inducible locomotion defect in C. elegans. Proc Natl Acad Sci 94:1043810443. Cook T, Mesa K, Urrutia R. 1996. Three dynamin-encoding genes are differentially expressed in developing rat brain. J Neurochem 67:927931. Dempwolff F, Wischhusen HM, Specht M, Graumann PL. 2012. The deletion of bacterial dynamin and flotillin genes results in pleiotrophic effects on cell division, cell growth and in cell shape maintenance. BMC Microbiol 12:298. Deng M, Kishikawa H, Yanagimachi R, Kopf GS, Schultz RM, Williams CJ. 2003. Chromatin-mediated cortical granule redistribution is responsible for the formation of the cortical granulefree domain in mouse eggs. Dev Biol 257:166176. Dumont J, Million K, Sunderland K, Rassinier P, Lim H, Leader B, Verlhac MH. 2007. Formin-2 is required for spindle migration and for the late steps of cytokinesis in mouse oocytes. Dev Biol 301:254265.
Mol. Reprod. Dev. 81:725–734 (2014)
IN
PORCINE OOCYTES
Feng B, Schwarz H, Jesuthasan S. 2002. Furrow-specific endocytosis during cytokinesis of zebrafish blastomeres. Exp Cell Res 279:1420. Finger FP, White JG. 2002. Fusion and fission: Membrane trafficking in animal cytokinesis. Cell 108:727730. Hussain NK, Jenna S, Glogauer M, Quinn CC, Wasiak S, Guipponi M, Antonarakis SE, Kay BK, Stossel TP, Lamarche-Vane N. 2001. Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP. Nat Cell Biol 3:927932. Kessels MM, Engqvist-Goldstein éE, Drubin DG, Qualmann B. 2001. Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin. J Cell Biol 153:351366. Konopka CA, Schleede JB, Skop AR, Bednarek SY. 2006. Dynamin and cytokinesis. Traffic 7:239247. Lauber MH, Waizenegger I, Steinmann T, Schwarz H, Mayer U, Hwang I, Lukowitz W, Jürgens G. 1997. The Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin. J Cell Biol 139:14851493. Leader B, Lim H, Carabatsos MJ, Harrington A, Ecsedy J, Pellman D, Maas R, Leder P. 2002. Formin-2, polyploidy, hypofertility and positioning of the meiotic spindle in mouse oocytes. Nat Cell Biol 4:921928. Lee E, De Camilli P. 2002. Dynamin at actin tails. Proc Natl Acad Sci 99:161166. Lim DS, Kim SR, Choi BD, Jeong SJ, Wang G, Kim HK, Kim BH, Goo JH, Jeong MJ. 2012. Dynamin II involves in cell migration and actin formation of NIH3T3 cells. J Nanosci Nanotechnol 12:690695. Longo FJ, Chen DY. 1985. Development of cortical polarity in mouse eggs: Involvement of the meiotic apparatus. Dev Biol 107:382394. Orth JD, Krueger E, Cao H, McNiven MA. 2002. The large GTPase dynamin regulates actin comet formation and movement in living cells. Proc Natl Acad Sci 99:167172. Orth JD, McNiven MA. 2003. Dynamin at the actinmembrane interface. Current Opin Cell Biol 15:3139. Pfender S, Kuznetsov V, Pleiser S, Kerkhoff E, Schuh M. 2011. Spire-type actin nucleators cooperate with Formin-2 to drive asymmetric oocyte division. Curr Biol 21:955960. Pollard TD, Blanchoin L, Mullins RD. 2000. Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu Rev Biophys Biomol Structure 29:545576. Praefcke GJ, McMahon HT. 2004. The dynamin superfamily: Universal membrane tubulation and fission molecules? Nat Rev Mol Cell Biol 5:133147. Qualmann B, Kelly RB. 2000. Syndapin isoforms participate in receptor-mediated endocytosis and actin organization. J Cell Biol 148:10471062.
733
Molecular Reproduction & Development
ZHANG
ET AL.
Robinson DN, Spudich JA. 2000. Towards a molecular understanding of cytokinesis. Trends Cell Biol 10:228237.
midzone and is required for cytokinesis. Curr Biol 12: 21112117.
Salazar MA, Kwiatkowski AV, Pellegrini L, Cestra G, Butler MH, Rossman KL, Serna DM, Sondek J, Gertler FB, De Camilli P. 2003. Tuba, a novel protein containing bin/amphiphysin/Rvs and Dbl homology domains, links dynamin to regulation of the actin cytoskeleton. J Biol Chem 278:4903149043.
Uruno T, Liu J, Zhang P, Fan Yx, Egile C, Li R, Mueller SC, Zhan X. 2001. Activation of Arp2/3 complex-mediated actin polymerization by cortactin. Nat Cell Biol 3:259266.
Schafer DA. 2004. Regulating actin dynamics at membranes: A focus on dynamin. Traffic 5:463469. Schafer DA, Weed SA, Binns D, Karginov AV, Parsons JT, Cooper JA. 2002. Dynamin2 and cortactin regulate actin assembly and filament organization. Curr Biol 12:18521857. Sever S, Muhlberg AB, Schmid SL. 1999. Impairment of dynamin’s GAP domain stimulates receptor-mediated endocytosis. Nature 398:481486. Sun SC, Wang ZB, Xu YN, Lee SE, Cui XS, Kim NH. 2011. Arp2/3 complex regulates asymmetric division and cytokinesis in mouse oocytes. PLoS ONE 6:e18392. Sun SC, Kim NH. 2013. Molecular mechanisms of asymmetric division in oocytes. Microsc Microanal 19:883897. Thompson HM, Skop AR, Euteneuer U, Meyer BJ, McNiven MA. 2002. The large GTPase dynamin associates with the spindle
734
Verlhac M-H, Lefebvre C, Guillaud P, Rassinier P, Maro B. 2000. Asymmetric division in mouse oocytes: With or without Mos. Curr Biol 10:13031306. Wienke DC, Knetsch ML, Neuhaus EM, Reedy MC, Manstein DJ. 1999. Disruption of a dynamin homologue affects endocytosis, organelle morphology, and cytokinesis in Dictyostelium discoideum. Mol Biol Cell 10:225243. Witke W, Podtelejnikov AV, Di Nardo A, Sutherland JD, Gurniak CB, Dotti C, Mann M. 1998. In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly. EMBO J 17:967976. Yi K, Rubinstein B, Li R. 2013a. Symmetry breaking and polarity establishment during mouse oocyte maturation. Philos Trans R Soc Lond B Biol Sci 368:20130002. Yi K, Rubinstein B, Unruh JR, Guo F, Slaughter BD, Li R. 2013b. Sequential actin-based pushing forces drive meiosis I chromosome migration and symmetry breaking in oocytes. J Cell Biol 200:567576.
Mol. Reprod. Dev. 81:725–734 (2014)
