Theriogenology xxx (2014) 1–8
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ROCK inhibitor Y-27632 prevents porcine oocyte maturation Yu Zhang a, b, Xing Duan a, Bo Xiong c, Xiang-Shun Cui d, Nam-Hyung Kim d, Rong Rui b, Shao-Chen Sun a, * a
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, China College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA d Department of Animal Sciences, Chungbuk National University, Cheongju, Chungbuk, Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 January 2014 Received in revised form 17 February 2014 Accepted 22 February 2014
The inhibitor Y-27632 is a specific selective inhibitor of Rho-associated protein kinases (ROCKs), which are downstream effectors of Rho guanosine triphosphatease (GTPases) and regulate Rho-associated cellular functions, including actin cytoskeletal organization. Little is known regarding the effects of Y-27632 on mammalian oocyte maturation. In the present study, we investigated the effects of Y-27632 on porcine oocyte meiosis and possible regulatory mechanisms of ROCK during porcine oocyte maturation. We found that ROCK accumulated not only at spindles, but also at the cortex in porcine oocytes. Y-27632 treatment reduced ROCK expression, and inhibited porcine oocyte meiotic maturation, which might be because of the impairment of actin expression and actin-related spindle positioning. Y-27632 treatment also disrupted the formation of actin cap and cortical granule-free domain, which further confirmed a spindle positioning failure. Thus, Y-27632 has significant effects on the meiotic competence of mammalian oocytes by reducing ROCK expression, and the regulation is related to its effects on actin-mediated spindle positioning. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Y-27632 Rho-associated protein kinase Actin Oocyte maturation Spindle migration
1. Introduction Mammalian oocyte meiosis involves a unique asymmetric division, which is responsible for successful fertilization. In mammalian ovaries, the fully grown oocytes are naturally arrested at prophase I before the first meiotic division, and then initiate meiotic maturation from germinal vesicle stage. All meiotic stages of porcine were completed within 42 to 48 hours, with germinal vesicle breakdown (GVBD) at 6 to 12 hours, metaphase I (MI) at 10 to 18 hours and anaphase/telophase I at 16 to 20 hours [1]. At the end of
* Corresponding author. Tel./fax: þ86 25 84399092. E-mail address: [email protected] (S.-C. Sun). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.02.020
meiosis, oocytes divide into highly polarized large metaphase II (MII)-arrested oocytes. Unlike common cells that induced polarity by ligand, the development of oocyte polarity and cortical reorganization is dependent on the intrinsic signal of oocytes instead of the involvement of any external ligand [2]. Furthermore, each meiotic division must ensure highly asymmetric separation of the cytoplasm and accurate symmetric segregation of the maternal genome. The formation of the first polar body is dependent on oocyte polarization, including spindle migration and positioning, cortical reorganization, and asymmetric division. The sequential events of these asymmetric divisions are regulated by microtubules and actin filaments [3,4]. After GVBD, the meiotic spindle forms and migrates from central areas to the periphery of an oocyte in an actin-dependent manner. In addition, the cortical
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granules (CGs) are redistributed to form a CG-free domain (CGFD) in the cortex area close to spindle where microvilli are lost, and actin becomes rich to form an actin cap [5,6]. When it becomes intense of cortical polarity, the oocyte extrudes a tiny polar body with the diameter approximately 20 mm of 100 mm mouse oocyte and 25 mm of 150 mm porcine oocyte, leaving a highly polarized MII egg. All these events are critical for the retention of maternal components for subsequent early embryo development in porcine oocytes. Until now, the molecular details of oocyte meiotic maturation have been poorly understood. Rho-associated serine-threonine protein kinase (Rhokinase/ROCK/ROK) belongs to the Protein Kinase A, G, C family of kinases that were first discovered to be downstream effectors of the small guanosine triphosphatease (GTPases) Rho. In mammals, Rho kinases have two isoforms: ROCK1/Rho-kinaseb/ROKb/p160ROCK [7] and ROCK2/Rhokinasea/ROKa [8]. The ROCK family has been implicated in a wide range of Rho-mediated fundamental cellular functions such as cell contraction, motility, morphology, polarity, cell division [9,10], and Rho-induced actin stress fiber and focal adhesion formation [7,11,12]. The roles of ROCKs are related to their phosphorylation of multiple proteins, including myosin light chain (MLC) phosphatase, lipase-modulator (LIM) kinases, adducin, and ezrin-radixin-moesin proteins. Rho-associated protein kinase phosphorylates MLC phosphatase and inhibits its activity [13]. Inhibiting ROCK results in increased MLC phosphorylation and actomyosin-based contractility, which contribute to smooth muscle contraction, stress fiber formation, and focal adhesions [14]. In addition, ROCK phosphorylates LIM kinase-1 (LIMK1) [15] and LIM kinase-2 (LIMK1) [16], which enhances their ability to phosphorylate Cofilin [17]. LIMK phosphorylation by ROCK and the subsequent increased phosphorylation of Coflin by LIMK contribute to Rho-mediated reorganization of the actin cytoskeleton [17]. Y-27632 is a well-known specific selective inhibitor of ROCKs in an ATP-competitive manner. Y-27632 contains a 4-aminopyridine ring, and affects ROCK to block phenylephrine-induced contraction of aortic strips in rabbits [18] and stress fiber formation in Swiss 3T3 cells [19]. Y-27632 has been widely used as a ROCK inhibitor to evaluate the roles of ROCK kinases in a variety of cell and animal models [19–22]. However, the roles of ROCK on the asymmetric division of mammalian oocytes are still unclear. The present study was aimed to investigate the function and regulatory mechanism of ROCK on the meiosis of porcine oocytes, extending the understanding regarding the regulators of oocyte asymmetric division, as to date only a few molecules have been identified as being involved in this process. Here, we examined the roles of ROCK using porcine oocytes as a model. Compared with mouse oocytes, firstly, the size of porcine oocytes is much larger, whereas the relative size of the first polar body is smaller, and the spindle is much smaller, which means that a longer distance for actinmediated spindle movement during porcine oocyte meiosis. Secondly, it takes 42 to 48 hours for porcine oocytes to complete the meiotic maturation, which are much longer than mouse oocytes. Accordingly, the window of spindle movement is correspondingly longer. Therefore, given the aspects of space and time, porcine oocyte is a good model for
studying the roles of actin on spindle migration during meiosis. Furthermore, data from porcine oocytes could provide the information about the conservative roles of ROCK among different species. Thus, in this study, using a special inhibitor of ROCK, we found that Y-27632 significantly affected porcine oocyte maturation. We also showed that ROCK inhibition caused aberrant actin expression and the failure of spindle positioning, which might have contributed to a failure of porcine oocyte polar body extrusion. Therefore, ROCK may regulate actin-mediated spindle positioning to participate in the porcine oocyte maturation. 2. Materials and methods 2.1. Antibodies and chemicals Basic maturation culture medium was TCM 199 (Sigma, St. Louis, MO, USA). Rabbit polyclonal anti-ROCK antibody was from Santa Cruz (Santa Cruz, CA, USA). PhalloidinTetramethylrhodamine, Phalloidin-fluorescein isothiocyanate (FITC), Lectin-FITC, and mouse monoclonal anti-a-tubulinFITC antibody were from Sigma. Alexa Fluor 488 and 568 antibodies were from Invitrogen (Carlsbad, CA, USA). Y-27632 was from Calbiochem (Darmstadt, Germany). All other chemicals and reagents were from Sigma-Aldrich unless otherwise stated (St. Louis, MO, USA). 2.2. Oocyte isolation and IVC Animal care and use were in accordance with the Animal Research Institute Committee guidelines of Nanjing Agriculture University, China. Porcine ovaries were obtained from a local slaughterhouse, and transported to the laboratory within 3 hours after death in sterile saline (0.9% NaCl) containing 500 IU/mL of penicillin and 500 IU/mL of streptomycin at 30 C to 35 C. After washing twice in sterile PBS, cumulus oocyte complexes (COCs) were harvested from follicles (3–5 mm in diameter) by aspiration with a 20-ga needle. Oocytes were picked from the subnatant precipitate of follicular fluid, then washed twice with Dulbecco’ s PBS and modified medium 199 containing 0.1% (wt/vol) polyvinyl alcohol, 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 with intact cumulus cells and evenly granulated ooplasm were selected for further study. Cumulus oocyte complexes were cultured in 500 mL of a defined maturation medium consisting of 90% (vol/vol) modified M199, 10 ng/mL of EGF (mouse EGF; Sigma), 0.57 mM cysteine (Sigma), 10 IU/mL of hCG, 10 IU/mL of pregnant mare's serum gonadotropin, and 10% (vol/vol) pig follicular fluid. Oocytes were cultured for 26 or 44 hours in a four-well dish (NUNC, Roskilde, Denmark) at 38.5 C in a 5% CO2 atmosphere. 2.3. Y-27632 treatment For Y-27632 treatment, a stock solution of Y-27632 was diluted in DMSO to 5 mM and stored at 20 C. At the beginning of each culture, the stock solution was diluted with maturation medium 199 for final concentrations of 50
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Fig. 1. ROCK localization in porcine oocytes. (A) Subcellular ROCK localization during porcine oocyte meiotic maturation. Immunofluorescent staining was used to determine ROCK subcellular localization in porcine oocytes. In addition to spindle localization, ROCK also accumulated at the cortex and in the cytoplasm of oocytes from GVBD to the MII stage. Green, ROCK; blue, chromatin. Bar ¼ 20 mm. (B) Immunofluorescent staining for colocalization of a-tubulin with ROCK in porcine oocytes. This shows the colocalization of ROCK with spindles in a porcine oocyte at the MII stage. Red, ROCK; green, tubulin; blue, chromatin. Bar ¼ 20 mm. (C) Immunofluorescent staining for colocalization of actin with ROCK in oocytes. This shows a similar localization of ROCK with actin at the MI stage. Green, ROCK; red, actin; blue, chromatin. Bar ¼ 20 mm. (D) Immunofluorescent staining for ROCK in oocytes after Y-27632 treatment during porcine oocyte maturation. With Y-27632 treatment, ROCK was minimally expressed. Green, ROCK; blue, chromatin. Bar ¼ 20 mm. GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II; ROCK, Rho-associated protein kinase.
and 100 mM. These Y-27632 concentrations were determined from preliminary experiments (data not shown), and only those concentrations that had a significant effect on oocyte maturation were used. The COCs or denuded oocytes (DOs) were cultured with or without Y-27632 in vitro. After culture for 44 hours, the developmental competence of matured oocytes was evaluated by determining the rate of the first polar body extrusion during meiosis. Controls were cultured with pure DMSO at the same concentration under the same schedule. The COCs were denuded of their cumulus cells by gentle pipetting with 0.1% (wt/vol) hyaluronidase (Sigma). Oocytes with clearly extruded polar bodies were judged as matured oocytes. The occurrence of the first polar body extrusion in oocytes was examined after removing cumulus cells. 2.4. Confocal microscopy For immunofluorescent staining, oocytes were fixed with 4% paraformaldehyde (in PBS) for 30 minutes, and then treated with a membrane permeabilization solution (1% Triton X-100 in PBS) at room temperature for 8 to 12 hours. After 1 hour in blocking buffer (1% BSA in PBS), oocytes were then incubated overnight at 4 C or 5 hours at
room temperature with a rabbit polyclonal anti-ROCK antibody (1:50) or anti-a-tubulin-FITC (1:200) for 2 hours or with 10 mg/mL of phalloidin-Tetramethylrhodamine at room temperature for 1 hour. After three washes (2 minutes each) in wash buffer (0.1% Tween 20 and 0.01% Triton X-100 in PBS), oocytes were labeled with Alexa Fluor 488/568 goatantirabbit IgG (1:100) at room temperature for 1 hour (for ROCK). These samples were costained with Hoechst 33342 (Sigma, St. Louis, MO, USA) (10 mg/mL in PBS) for 10 minutes, mounted on glass slides, and then examined with a confocal laser-scanning microscope (Zeiss LSM 700 META, Germany).
2.5. Fluorescence intensity analysis To analyze actin fluorescence intensity, samples of control oocytes and treated oocytes were mounted on the same glass slide. ImageJ software (v1.47) 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 made for the cell membrane and cytoplasm. The average values of all measurements were used to compare the final average intensities between control and treated oocytes.
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Fig. 2. Y-27632 effects on porcine oocyte meiotic competence. (A) Judgment standard for porcine oocyte maturation that involves the diffusion of COCs and polar body extrusion. For controls, most COCs were surrounded by more than five layers of intact cumulus cells, whereas weak diffusion was observed after treatment with Y-27632 at 50 and 100 mM. (B) Y-27632 treatment resulted in reduced meiotic competence for porcine COCs in a dose-dependent manner. Compared with controls, the first polar body extrusion rate was significantly lower with Y-27632 treatment at 50 and 100 mM (P < 0.01). Meiotic competence with 50 mM was significantly better than with 100 mM (P < 0.05). (C) Y-27632 treatment results in a decreased first polar body extrusion rate for porcine DOs in a dose-dependent manner. The first polar body extrusion rate of controls was significantly higher as compared with Y-27632 treatment (P < 0.05). COC, cumulus oocyte complex; DO, denuded oocyte.
2.6. Statistical analysis
3.2. Effects of Y-27632 on porcine oocyte maturation
Each experiment was repeated three times. A total of 60 oocytes were cultured with or without Y-27632. At least 30 oocytes were examined for each experimental condition, and the results were expressed as means standard errors of the mean. Statistical comparisons were made by ANOVA followed by Duncan multiple comparisons tests. A value of Pless than 0.05 was considered significant.
The COCs were treated with Y-27632 for 44 hours to inhibit endogenous ROCK expression during maturation. As shown in Figure 2A, compared with controls that most COCs were surrounded by more than five layers of intact cumulus cells, the expansion of COCs was weaker after treatment with 50 mM Y-27632. Similar effects were observed after incubation with 100 mM Y-27632. Furthermore, the first polar body extrusion of COCs decreased in a dose-dependent manner (Fig. 2B). For controls, most oocytes had extruded polar bodies, and were arresting at the MII stage after culture for 44 hours in vitro; the rate of the first polar body extrusion was 73.59 3.00% (n ¼ 132). However, the rate decreased to 50.54 1.27% (n ¼ 112; P < 0.01) when treated with 50 mM Y-27632, and the vast majority of oocytes failed to extrude polar bodies after 100 mM Y-27632 treatment, with a significantly reduced rate of 32.26 1.90% (n ¼ 138; P < 0.01). These results show that porcine oocyte maturation is affected by Y-27632 treatment. A concentration of 100 mM Y-27632 was adopted for subsequent experiments. Denuded oocytes were studied with Y-27632 to exclude the effects of cumulus cells on the oocyte maturation (Fig. 2C). For controls, the rate of the first polar body extrusion was 56.52 4.14% (n ¼ 75 DOs), which was significantly higher than that of 100 mM Y-27632 treatment groups (24.22 1.14%; n ¼ 81 DOs; P < 0.05). This indicates that Y-27632 has a direct effect on porcine oocyte nuclear maturation. Thus, COCs were used to investigate the
3. Results 3.1. Intracellular localization of ROCK during porcine oocyte meiosis We examined subcellular localization of ROCK at different developmental stages of porcine meiotic maturation. Rho-associated protein kinase was observed at spindles from GVBD to MII stage (Fig. 1A). In addition, ROCK also accumulated in the cortex and the cytoplasm of porcine oocytes during GVBD to the MII stage. To confirm this localization pattern, we performed double staining for ROCK and a-tubulin or actin. As shown in Figure 1B, ROCK colocalized with spindle at MII stage. Also, ROCK colocalized with actin at the cortex (Fig. 1C). Furthermore, we tested ROCK localization in Y-27632-treated oocytes. Compared with control oocytes, the expression of ROCK was significantly reduced at the spindles after treatment (Fig. 1D).
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Fig. 3. Y-27632 effects on actin assembly in porcine oocytes. (A) Actin expression in oocytes was examined by immunofluorescent staining. Actin expression was significantly lower after Y-27632 treatment at the membrane and in the cytoplasm. Red, actin. (B) Average actin fluorescence intensity in porcine oocytes. Actin fluorescence intensity was significantly lower in Y-27632-treated oocytes both at the membrane (P < 0.01) and in the cytoplasm (P < 0.05). (C) Actin cap formation after Y-27632 treatment. Compared with controls, actin caps disappeared after Y-27632 treatment.
preliminary mechanisms of ROCK during porcine oocyte meiotic maturation.
P < 0.01). These results demonstrate that ROCK inhibition by Y-27632 results in a reduced actin expression.
3.3. Effects of Y-27632 on actin assembly in porcine oocytes
3.4. Effects of Y-27632 on spindle positioning in porcine oocytes
Next, we investigated the effects of Y-27632 on actin filament expression in porcine oocytes. As compared with control oocytes, actin expression decreased both at the membrane and in the cytoplasm of oocytes after Y-27632 treatment (Fig. 3A). In addition, using ImageJ software, we confirmed the change of actin expression by quantitative analysis of actin fluorescence intensity levels in oocytes. As shown in Figure 3B, for control oocytes, the actin fluorescence intensity (80.10 5.79% vs. 48.73 4.59%; P < 0.01) was significantly higher than that of treated oocytes at the membrane. Similar results were found in the cytoplasm that actin fluorescence signals were significantly reduced after Y-27632 treatment (25.59 2.19% vs. 16.85 1.76%;
As noted above, Y-27632-treated porcine oocytes failed to undergo nuclear maturation. Thus, we focused on the roles of Y-27632 on spindle positioning at MI stage. We treated oocytes with Y-27632 for 26 hours when most oocytes had reached the late MI stage. Using confocal microscopy, immunofluorescent images of spindles labeled with an anti-a-tubulin antibody were acquired to assess the spindle locations. As shown in Figure 4A, almost all of the spindles arrested at the central area of cytoplasm after treatment with 100 mM Y-27632 as compared with control oocytes that exhibited peripherally located spindles. Enlarged images also confirmed this (Fig. 4B).
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Fig. 4. Y-27632 effects on spindle positioning. (A) Porcine oocyte spindle positioning. Almost all spindles migrated to the cortex of oocytes; however, most oocytes exhibited central spindle positioning. (B) Enlarged images of spindle positioning. For controls, spindles migrated to the periphery of oocytes, whereas with Y-27632 treatment, spindles were arrested at central areas. (C) Distributions of porcine spindle positions after 26 hours of maturation culture. Compared with controls, there were significantly more MI oocytes with centrally located spindles among Y-27632-treated oocytes (P < 0.05). (D) Cortical granule-free domain (CGFD) formation during oocyte maturation after Y-27632 treatment. Y-27632 treatment disrupted CGFDs and cortical granules were uniformly distributed throughout the entire cortex. AT, anaphase and telophase; CG, cortical granule; DIC, differential interference contrast; MI, metaphase I; MII, metaphase II.
The rate of control oocytes with centrally located spindles in the MI stage was significantly lower than that of Y-27632-treated oocytes (33.15 6.78%, n ¼ 213 vs. 61.93 5.66%, n ¼ 213; P < 0.05), whereas the control oocytes that exhibited peripherally located spindles in the MI stage (35.69 4.85% vs. 24.92 5.99%) and in the anaphase and telophase/MII stage (31.16 7.57% vs. 13.15 2.90%) were significantly higher than treatment groups (Fig. 4C). Thus, ROCK inhibition by Y-27632 interferes with spindle migration, which results in centrally located spindle positioning. To further confirm the spindle positioning affected by ROCK, we investigated two characteristics of successful spindle positioning, actin cap formation, and CGFD. Microfilaments were enriched to form an actin cap in control oocytes, whereas a large proportion of oocytes had no actin caps formed after Y-27632 treatment (Fig. 4D). We also examined the formation of CGFD. In control oocytes, CGs were absent in the region close to chromosomes at the MI stage, whereas it was uniformly distributed throughout the entire cortex after Y-27632 treatment (Fig. 4D). These results suggest that ROCK inhibition disrupts the formation of actin caps and CGFD, which indicates spindle-positioning failure. 4. Discussion In the present study, using the special ROCK inhibitor Y-27632, we investigated the roles and preliminary mechanisms of ROCK on porcine oocyte meiotic maturation. The
results showed that Y-27632 significantly affected porcine oocyte maturation. In addition, ROCK inhibition by Y-27632 resulted in a failure of spindle positioning and actin expression. Therefore, ROCK might play an essential role in porcine oocyte maturation. We first examined whether ROCK was expressed in porcine oocytes, and we found that ROCK had a specific localization during porcine oocyte maturation. Our results showed that ROCK colocalized with spindles in the cytoplasm and also colocalized with actin at the membranes, which were consistent with the previous studies that ROCK was primarily found in the cytoplasm and membranes in Cos-7 and National Institutes of Health 3T3 cells and confluent Madin-Darby canine kidney cells [8,23,24]. Because ROCK plays a crucial role in cytoskeletal dynamics [25] and actin is involved in the spindle migration during meiosis [26], the localization pattern of ROCK in porcine oocyte might suggest a possibility that ROCK is related to actin dynamics in porcine oocyte, and plays a significant role during porcine oocyte meiotic maturation. To verify our hypothesis, we disrupted ROCK activity by Y-27632 treatment. We found that the polar body extrusion of porcine oocytes failed after ROCK inhibition, indicating a failure of porcine oocyte maturation. A number of studies have shown that the small GTPase Rho participates in cytokinesis [27–29]. Rho-associated protein kinase is one of the effectors for Rho, and is also considered to be involved in the progression of cytokinesis in several models [30]. Another study also indicates that ROCK2 is essential for
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cytokinesis in Xenopus embryos and mammalian cells by regulating glial filament segregation [31]. Our results are consistent with the previous studies. Therefore, ROCK plays a significant role during porcine oocyte meiotic maturation. To find the possible reason of ROCK inhibition on porcine oocyte maturation defect, we first examined the actin expression, because the cytokinesis in animal cells involves the concerted efforts of the actin cytoskeleton [32,33]. The results showed that the inhibition of ROCK activity caused the degradation of actin expression and the barrier of clear actin cap formation. These results were similar to previous studies that ROCK is involved in cytoskeletal rearrangements and the formation of stress fibers [34,35]. Our results may suggest that ROCK regulates the actin assembly. Similar research has shown that the actin nucleation activity of formin-2 is responsible for actin filament assembly and actin-driven chromosomal motility, which lead to asymmetric division in meiotic oocytes [36]. Therefore, our results might reflect that ROCK regulates porcine oocyte meiosis by its effect on actin assembly. Recently, in mammalian oocyte, the progressive formation of an actin filament meshwork has been confirmed to regulate the spindle movement for polar body extrusion during asymmetric division [4]. Previous studies have established that several factors regulate actin assembly and polymerization, which are involved in spindle positioning. Formin-2 is responsible for actin filament assembly, and is critical for driving spindle migration in mouse oocytes [37]. The actin nucleator Arp2/3 complex promotes actin polymerization, and contributes to spindle migration and oocyte asymmetric division [38,39]. Moreover, Rho GTPase subfamily molecules are critical for regulating the actin cytoskeleton. For example, the small GTPase Cdc42 has been recently found to participate in spindle migration and asymmetric division [40,41]. In addition, another small GTPase, Rac1, is confirmed to play a similar role in spindle positioning [42,43]. Therefore, we next examined the spindle position after ROCK inhibition. Our results show that Y-27632-treated porcine oocytes exhibit spindle positioning defects. Rho-associated protein kinase inhibition results in centrally arrested spindles, and the disruption of actin cap and CGFD formation, which are in agreement with the previous studies. Therefore, ROCK regulates porcine oocyte polar body extrusion by affecting meiotic spindle positioning. 4.1. Conclusions In summary, we found that ROCK is an actin assembly regulator of meiotic spindle positioning in porcine oocytes. Our results also establish a possible underlying mechanism of ROCK for the polar body extrusion during porcine oocyte meiotic maturation. Acknowledgments This work was supported by the National Basic Research Program of China (2014CB138503), the Natural Science Foundation of Jiangsu Province (BK20130671), China; and the Biogreen 21 Program (PJ009594 & PJ00909801), RDA, Republic of Korea.
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[34] Katoh K, Kano Y, Amano M, Onishi H, Kaibuchi K, Fujiwara K. Rhokinase–mediated contraction of isolated stress fibers. J Cell Biol 2001;153:569–84. [35] Chen Xq, Tan I, Ng CH, Hall C, Lim L, Leung T. Characterization of RhoA-binding kinase ROKalpha implication of the pleckstrin homology domain in ROKa function using region-specific antibodies. J Biol Chem 2002;277:12680–8. [36] Li H, Guo F, Rubinstein B, Li R. Actin-driven chromosomal motility leads to symmetry breaking in mammalian meiotic oocytes. Nat Cell Biol 2008;10:1301–8. [37] Leader B, Lim H, Carabatsos MJ, Harrington A, Ecsedy J, Pellman D, et al. Formin-2, polyploidy, hypofertility and positioning of the meiotic spindle in mouse oocytes. Nat Cell Biol 2002;4:921–8. [38] Sun SC, Wang ZB, Xu YN, Lee SE, Cui XS, Kim NH. Arp2/3 complex regulates asymmetric division and cytokinesis in mouse oocytes. PLoS One 2011;6:e18392. [39] Yi K, Unruh JR, Deng M, Slaughter BD, Rubinstein B, Li R. Dynamic maintenance of asymmetric meiotic spindle position through Arp2/ 3-complex-driven cytoplasmic streaming in mouse oocytes. Nat Cell Biol 2011;13:1252–8. [40] Na J, Zernicka-Goetz M. Asymmetric positioning and organization of the meiotic spindle of mouse oocytes requires CDC42 function. Curr Biol 2006;16:1249–54. [41] Dehapiot B, Carrière V, Carrollc J, Halet G. Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes. Dev Biol 2013;377:201–12. [42] Halet G, Carroll J. Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes. Dev Cell 2007;12:309–17. [43] Dehapiot B, Halet G. Ran GTPase promotes oocyte polarization by regulating ERM (Ezrin/Radixin/Moesin) activation. Cell Cycle 2013; 12:1672–8.
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ROCK inhibitor Y-27632 prevents porcine oocyte maturation Yu Zhang a, b, Xing Duan a, Bo Xiong c, Xiang-Shun Cui d, Nam-Hyung Kim d, Rong Rui b, Shao-Chen Sun a, * a
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, China College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA d Department of Animal Sciences, Chungbuk National University, Cheongju, Chungbuk, Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 January 2014 Received in revised form 17 February 2014 Accepted 22 February 2014
The inhibitor Y-27632 is a specific selective inhibitor of Rho-associated protein kinases (ROCKs), which are downstream effectors of Rho guanosine triphosphatease (GTPases) and regulate Rho-associated cellular functions, including actin cytoskeletal organization. Little is known regarding the effects of Y-27632 on mammalian oocyte maturation. In the present study, we investigated the effects of Y-27632 on porcine oocyte meiosis and possible regulatory mechanisms of ROCK during porcine oocyte maturation. We found that ROCK accumulated not only at spindles, but also at the cortex in porcine oocytes. Y-27632 treatment reduced ROCK expression, and inhibited porcine oocyte meiotic maturation, which might be because of the impairment of actin expression and actin-related spindle positioning. Y-27632 treatment also disrupted the formation of actin cap and cortical granule-free domain, which further confirmed a spindle positioning failure. Thus, Y-27632 has significant effects on the meiotic competence of mammalian oocytes by reducing ROCK expression, and the regulation is related to its effects on actin-mediated spindle positioning. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Y-27632 Rho-associated protein kinase Actin Oocyte maturation Spindle migration
1. Introduction Mammalian oocyte meiosis involves a unique asymmetric division, which is responsible for successful fertilization. In mammalian ovaries, the fully grown oocytes are naturally arrested at prophase I before the first meiotic division, and then initiate meiotic maturation from germinal vesicle stage. All meiotic stages of porcine were completed within 42 to 48 hours, with germinal vesicle breakdown (GVBD) at 6 to 12 hours, metaphase I (MI) at 10 to 18 hours and anaphase/telophase I at 16 to 20 hours [1]. At the end of
* Corresponding author. Tel./fax: þ86 25 84399092. E-mail address: [email protected] (S.-C. Sun). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.02.020
meiosis, oocytes divide into highly polarized large metaphase II (MII)-arrested oocytes. Unlike common cells that induced polarity by ligand, the development of oocyte polarity and cortical reorganization is dependent on the intrinsic signal of oocytes instead of the involvement of any external ligand [2]. Furthermore, each meiotic division must ensure highly asymmetric separation of the cytoplasm and accurate symmetric segregation of the maternal genome. The formation of the first polar body is dependent on oocyte polarization, including spindle migration and positioning, cortical reorganization, and asymmetric division. The sequential events of these asymmetric divisions are regulated by microtubules and actin filaments [3,4]. After GVBD, the meiotic spindle forms and migrates from central areas to the periphery of an oocyte in an actin-dependent manner. In addition, the cortical
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granules (CGs) are redistributed to form a CG-free domain (CGFD) in the cortex area close to spindle where microvilli are lost, and actin becomes rich to form an actin cap [5,6]. When it becomes intense of cortical polarity, the oocyte extrudes a tiny polar body with the diameter approximately 20 mm of 100 mm mouse oocyte and 25 mm of 150 mm porcine oocyte, leaving a highly polarized MII egg. All these events are critical for the retention of maternal components for subsequent early embryo development in porcine oocytes. Until now, the molecular details of oocyte meiotic maturation have been poorly understood. Rho-associated serine-threonine protein kinase (Rhokinase/ROCK/ROK) belongs to the Protein Kinase A, G, C family of kinases that were first discovered to be downstream effectors of the small guanosine triphosphatease (GTPases) Rho. In mammals, Rho kinases have two isoforms: ROCK1/Rho-kinaseb/ROKb/p160ROCK [7] and ROCK2/Rhokinasea/ROKa [8]. The ROCK family has been implicated in a wide range of Rho-mediated fundamental cellular functions such as cell contraction, motility, morphology, polarity, cell division [9,10], and Rho-induced actin stress fiber and focal adhesion formation [7,11,12]. The roles of ROCKs are related to their phosphorylation of multiple proteins, including myosin light chain (MLC) phosphatase, lipase-modulator (LIM) kinases, adducin, and ezrin-radixin-moesin proteins. Rho-associated protein kinase phosphorylates MLC phosphatase and inhibits its activity [13]. Inhibiting ROCK results in increased MLC phosphorylation and actomyosin-based contractility, which contribute to smooth muscle contraction, stress fiber formation, and focal adhesions [14]. In addition, ROCK phosphorylates LIM kinase-1 (LIMK1) [15] and LIM kinase-2 (LIMK1) [16], which enhances their ability to phosphorylate Cofilin [17]. LIMK phosphorylation by ROCK and the subsequent increased phosphorylation of Coflin by LIMK contribute to Rho-mediated reorganization of the actin cytoskeleton [17]. Y-27632 is a well-known specific selective inhibitor of ROCKs in an ATP-competitive manner. Y-27632 contains a 4-aminopyridine ring, and affects ROCK to block phenylephrine-induced contraction of aortic strips in rabbits [18] and stress fiber formation in Swiss 3T3 cells [19]. Y-27632 has been widely used as a ROCK inhibitor to evaluate the roles of ROCK kinases in a variety of cell and animal models [19–22]. However, the roles of ROCK on the asymmetric division of mammalian oocytes are still unclear. The present study was aimed to investigate the function and regulatory mechanism of ROCK on the meiosis of porcine oocytes, extending the understanding regarding the regulators of oocyte asymmetric division, as to date only a few molecules have been identified as being involved in this process. Here, we examined the roles of ROCK using porcine oocytes as a model. Compared with mouse oocytes, firstly, the size of porcine oocytes is much larger, whereas the relative size of the first polar body is smaller, and the spindle is much smaller, which means that a longer distance for actinmediated spindle movement during porcine oocyte meiosis. Secondly, it takes 42 to 48 hours for porcine oocytes to complete the meiotic maturation, which are much longer than mouse oocytes. Accordingly, the window of spindle movement is correspondingly longer. Therefore, given the aspects of space and time, porcine oocyte is a good model for
studying the roles of actin on spindle migration during meiosis. Furthermore, data from porcine oocytes could provide the information about the conservative roles of ROCK among different species. Thus, in this study, using a special inhibitor of ROCK, we found that Y-27632 significantly affected porcine oocyte maturation. We also showed that ROCK inhibition caused aberrant actin expression and the failure of spindle positioning, which might have contributed to a failure of porcine oocyte polar body extrusion. Therefore, ROCK may regulate actin-mediated spindle positioning to participate in the porcine oocyte maturation. 2. Materials and methods 2.1. Antibodies and chemicals Basic maturation culture medium was TCM 199 (Sigma, St. Louis, MO, USA). Rabbit polyclonal anti-ROCK antibody was from Santa Cruz (Santa Cruz, CA, USA). PhalloidinTetramethylrhodamine, Phalloidin-fluorescein isothiocyanate (FITC), Lectin-FITC, and mouse monoclonal anti-a-tubulinFITC antibody were from Sigma. Alexa Fluor 488 and 568 antibodies were from Invitrogen (Carlsbad, CA, USA). Y-27632 was from Calbiochem (Darmstadt, Germany). All other chemicals and reagents were from Sigma-Aldrich unless otherwise stated (St. Louis, MO, USA). 2.2. Oocyte isolation and IVC Animal care and use were in accordance with the Animal Research Institute Committee guidelines of Nanjing Agriculture University, China. Porcine ovaries were obtained from a local slaughterhouse, and transported to the laboratory within 3 hours after death in sterile saline (0.9% NaCl) containing 500 IU/mL of penicillin and 500 IU/mL of streptomycin at 30 C to 35 C. After washing twice in sterile PBS, cumulus oocyte complexes (COCs) were harvested from follicles (3–5 mm in diameter) by aspiration with a 20-ga needle. Oocytes were picked from the subnatant precipitate of follicular fluid, then washed twice with Dulbecco’ s PBS and modified medium 199 containing 0.1% (wt/vol) polyvinyl alcohol, 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 with intact cumulus cells and evenly granulated ooplasm were selected for further study. Cumulus oocyte complexes were cultured in 500 mL of a defined maturation medium consisting of 90% (vol/vol) modified M199, 10 ng/mL of EGF (mouse EGF; Sigma), 0.57 mM cysteine (Sigma), 10 IU/mL of hCG, 10 IU/mL of pregnant mare's serum gonadotropin, and 10% (vol/vol) pig follicular fluid. Oocytes were cultured for 26 or 44 hours in a four-well dish (NUNC, Roskilde, Denmark) at 38.5 C in a 5% CO2 atmosphere. 2.3. Y-27632 treatment For Y-27632 treatment, a stock solution of Y-27632 was diluted in DMSO to 5 mM and stored at 20 C. At the beginning of each culture, the stock solution was diluted with maturation medium 199 for final concentrations of 50
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Fig. 1. ROCK localization in porcine oocytes. (A) Subcellular ROCK localization during porcine oocyte meiotic maturation. Immunofluorescent staining was used to determine ROCK subcellular localization in porcine oocytes. In addition to spindle localization, ROCK also accumulated at the cortex and in the cytoplasm of oocytes from GVBD to the MII stage. Green, ROCK; blue, chromatin. Bar ¼ 20 mm. (B) Immunofluorescent staining for colocalization of a-tubulin with ROCK in porcine oocytes. This shows the colocalization of ROCK with spindles in a porcine oocyte at the MII stage. Red, ROCK; green, tubulin; blue, chromatin. Bar ¼ 20 mm. (C) Immunofluorescent staining for colocalization of actin with ROCK in oocytes. This shows a similar localization of ROCK with actin at the MI stage. Green, ROCK; red, actin; blue, chromatin. Bar ¼ 20 mm. (D) Immunofluorescent staining for ROCK in oocytes after Y-27632 treatment during porcine oocyte maturation. With Y-27632 treatment, ROCK was minimally expressed. Green, ROCK; blue, chromatin. Bar ¼ 20 mm. GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II; ROCK, Rho-associated protein kinase.
and 100 mM. These Y-27632 concentrations were determined from preliminary experiments (data not shown), and only those concentrations that had a significant effect on oocyte maturation were used. The COCs or denuded oocytes (DOs) were cultured with or without Y-27632 in vitro. After culture for 44 hours, the developmental competence of matured oocytes was evaluated by determining the rate of the first polar body extrusion during meiosis. Controls were cultured with pure DMSO at the same concentration under the same schedule. The COCs were denuded of their cumulus cells by gentle pipetting with 0.1% (wt/vol) hyaluronidase (Sigma). Oocytes with clearly extruded polar bodies were judged as matured oocytes. The occurrence of the first polar body extrusion in oocytes was examined after removing cumulus cells. 2.4. Confocal microscopy For immunofluorescent staining, oocytes were fixed with 4% paraformaldehyde (in PBS) for 30 minutes, and then treated with a membrane permeabilization solution (1% Triton X-100 in PBS) at room temperature for 8 to 12 hours. After 1 hour in blocking buffer (1% BSA in PBS), oocytes were then incubated overnight at 4 C or 5 hours at
room temperature with a rabbit polyclonal anti-ROCK antibody (1:50) or anti-a-tubulin-FITC (1:200) for 2 hours or with 10 mg/mL of phalloidin-Tetramethylrhodamine at room temperature for 1 hour. After three washes (2 minutes each) in wash buffer (0.1% Tween 20 and 0.01% Triton X-100 in PBS), oocytes were labeled with Alexa Fluor 488/568 goatantirabbit IgG (1:100) at room temperature for 1 hour (for ROCK). These samples were costained with Hoechst 33342 (Sigma, St. Louis, MO, USA) (10 mg/mL in PBS) for 10 minutes, mounted on glass slides, and then examined with a confocal laser-scanning microscope (Zeiss LSM 700 META, Germany).
2.5. Fluorescence intensity analysis To analyze actin fluorescence intensity, samples of control oocytes and treated oocytes were mounted on the same glass slide. ImageJ software (v1.47) 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 made for the cell membrane and cytoplasm. The average values of all measurements were used to compare the final average intensities between control and treated oocytes.
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Fig. 2. Y-27632 effects on porcine oocyte meiotic competence. (A) Judgment standard for porcine oocyte maturation that involves the diffusion of COCs and polar body extrusion. For controls, most COCs were surrounded by more than five layers of intact cumulus cells, whereas weak diffusion was observed after treatment with Y-27632 at 50 and 100 mM. (B) Y-27632 treatment resulted in reduced meiotic competence for porcine COCs in a dose-dependent manner. Compared with controls, the first polar body extrusion rate was significantly lower with Y-27632 treatment at 50 and 100 mM (P < 0.01). Meiotic competence with 50 mM was significantly better than with 100 mM (P < 0.05). (C) Y-27632 treatment results in a decreased first polar body extrusion rate for porcine DOs in a dose-dependent manner. The first polar body extrusion rate of controls was significantly higher as compared with Y-27632 treatment (P < 0.05). COC, cumulus oocyte complex; DO, denuded oocyte.
2.6. Statistical analysis
3.2. Effects of Y-27632 on porcine oocyte maturation
Each experiment was repeated three times. A total of 60 oocytes were cultured with or without Y-27632. At least 30 oocytes were examined for each experimental condition, and the results were expressed as means standard errors of the mean. Statistical comparisons were made by ANOVA followed by Duncan multiple comparisons tests. A value of Pless than 0.05 was considered significant.
The COCs were treated with Y-27632 for 44 hours to inhibit endogenous ROCK expression during maturation. As shown in Figure 2A, compared with controls that most COCs were surrounded by more than five layers of intact cumulus cells, the expansion of COCs was weaker after treatment with 50 mM Y-27632. Similar effects were observed after incubation with 100 mM Y-27632. Furthermore, the first polar body extrusion of COCs decreased in a dose-dependent manner (Fig. 2B). For controls, most oocytes had extruded polar bodies, and were arresting at the MII stage after culture for 44 hours in vitro; the rate of the first polar body extrusion was 73.59 3.00% (n ¼ 132). However, the rate decreased to 50.54 1.27% (n ¼ 112; P < 0.01) when treated with 50 mM Y-27632, and the vast majority of oocytes failed to extrude polar bodies after 100 mM Y-27632 treatment, with a significantly reduced rate of 32.26 1.90% (n ¼ 138; P < 0.01). These results show that porcine oocyte maturation is affected by Y-27632 treatment. A concentration of 100 mM Y-27632 was adopted for subsequent experiments. Denuded oocytes were studied with Y-27632 to exclude the effects of cumulus cells on the oocyte maturation (Fig. 2C). For controls, the rate of the first polar body extrusion was 56.52 4.14% (n ¼ 75 DOs), which was significantly higher than that of 100 mM Y-27632 treatment groups (24.22 1.14%; n ¼ 81 DOs; P < 0.05). This indicates that Y-27632 has a direct effect on porcine oocyte nuclear maturation. Thus, COCs were used to investigate the
3. Results 3.1. Intracellular localization of ROCK during porcine oocyte meiosis We examined subcellular localization of ROCK at different developmental stages of porcine meiotic maturation. Rho-associated protein kinase was observed at spindles from GVBD to MII stage (Fig. 1A). In addition, ROCK also accumulated in the cortex and the cytoplasm of porcine oocytes during GVBD to the MII stage. To confirm this localization pattern, we performed double staining for ROCK and a-tubulin or actin. As shown in Figure 1B, ROCK colocalized with spindle at MII stage. Also, ROCK colocalized with actin at the cortex (Fig. 1C). Furthermore, we tested ROCK localization in Y-27632-treated oocytes. Compared with control oocytes, the expression of ROCK was significantly reduced at the spindles after treatment (Fig. 1D).
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Fig. 3. Y-27632 effects on actin assembly in porcine oocytes. (A) Actin expression in oocytes was examined by immunofluorescent staining. Actin expression was significantly lower after Y-27632 treatment at the membrane and in the cytoplasm. Red, actin. (B) Average actin fluorescence intensity in porcine oocytes. Actin fluorescence intensity was significantly lower in Y-27632-treated oocytes both at the membrane (P < 0.01) and in the cytoplasm (P < 0.05). (C) Actin cap formation after Y-27632 treatment. Compared with controls, actin caps disappeared after Y-27632 treatment.
preliminary mechanisms of ROCK during porcine oocyte meiotic maturation.
P < 0.01). These results demonstrate that ROCK inhibition by Y-27632 results in a reduced actin expression.
3.3. Effects of Y-27632 on actin assembly in porcine oocytes
3.4. Effects of Y-27632 on spindle positioning in porcine oocytes
Next, we investigated the effects of Y-27632 on actin filament expression in porcine oocytes. As compared with control oocytes, actin expression decreased both at the membrane and in the cytoplasm of oocytes after Y-27632 treatment (Fig. 3A). In addition, using ImageJ software, we confirmed the change of actin expression by quantitative analysis of actin fluorescence intensity levels in oocytes. As shown in Figure 3B, for control oocytes, the actin fluorescence intensity (80.10 5.79% vs. 48.73 4.59%; P < 0.01) was significantly higher than that of treated oocytes at the membrane. Similar results were found in the cytoplasm that actin fluorescence signals were significantly reduced after Y-27632 treatment (25.59 2.19% vs. 16.85 1.76%;
As noted above, Y-27632-treated porcine oocytes failed to undergo nuclear maturation. Thus, we focused on the roles of Y-27632 on spindle positioning at MI stage. We treated oocytes with Y-27632 for 26 hours when most oocytes had reached the late MI stage. Using confocal microscopy, immunofluorescent images of spindles labeled with an anti-a-tubulin antibody were acquired to assess the spindle locations. As shown in Figure 4A, almost all of the spindles arrested at the central area of cytoplasm after treatment with 100 mM Y-27632 as compared with control oocytes that exhibited peripherally located spindles. Enlarged images also confirmed this (Fig. 4B).
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Fig. 4. Y-27632 effects on spindle positioning. (A) Porcine oocyte spindle positioning. Almost all spindles migrated to the cortex of oocytes; however, most oocytes exhibited central spindle positioning. (B) Enlarged images of spindle positioning. For controls, spindles migrated to the periphery of oocytes, whereas with Y-27632 treatment, spindles were arrested at central areas. (C) Distributions of porcine spindle positions after 26 hours of maturation culture. Compared with controls, there were significantly more MI oocytes with centrally located spindles among Y-27632-treated oocytes (P < 0.05). (D) Cortical granule-free domain (CGFD) formation during oocyte maturation after Y-27632 treatment. Y-27632 treatment disrupted CGFDs and cortical granules were uniformly distributed throughout the entire cortex. AT, anaphase and telophase; CG, cortical granule; DIC, differential interference contrast; MI, metaphase I; MII, metaphase II.
The rate of control oocytes with centrally located spindles in the MI stage was significantly lower than that of Y-27632-treated oocytes (33.15 6.78%, n ¼ 213 vs. 61.93 5.66%, n ¼ 213; P < 0.05), whereas the control oocytes that exhibited peripherally located spindles in the MI stage (35.69 4.85% vs. 24.92 5.99%) and in the anaphase and telophase/MII stage (31.16 7.57% vs. 13.15 2.90%) were significantly higher than treatment groups (Fig. 4C). Thus, ROCK inhibition by Y-27632 interferes with spindle migration, which results in centrally located spindle positioning. To further confirm the spindle positioning affected by ROCK, we investigated two characteristics of successful spindle positioning, actin cap formation, and CGFD. Microfilaments were enriched to form an actin cap in control oocytes, whereas a large proportion of oocytes had no actin caps formed after Y-27632 treatment (Fig. 4D). We also examined the formation of CGFD. In control oocytes, CGs were absent in the region close to chromosomes at the MI stage, whereas it was uniformly distributed throughout the entire cortex after Y-27632 treatment (Fig. 4D). These results suggest that ROCK inhibition disrupts the formation of actin caps and CGFD, which indicates spindle-positioning failure. 4. Discussion In the present study, using the special ROCK inhibitor Y-27632, we investigated the roles and preliminary mechanisms of ROCK on porcine oocyte meiotic maturation. The
results showed that Y-27632 significantly affected porcine oocyte maturation. In addition, ROCK inhibition by Y-27632 resulted in a failure of spindle positioning and actin expression. Therefore, ROCK might play an essential role in porcine oocyte maturation. We first examined whether ROCK was expressed in porcine oocytes, and we found that ROCK had a specific localization during porcine oocyte maturation. Our results showed that ROCK colocalized with spindles in the cytoplasm and also colocalized with actin at the membranes, which were consistent with the previous studies that ROCK was primarily found in the cytoplasm and membranes in Cos-7 and National Institutes of Health 3T3 cells and confluent Madin-Darby canine kidney cells [8,23,24]. Because ROCK plays a crucial role in cytoskeletal dynamics [25] and actin is involved in the spindle migration during meiosis [26], the localization pattern of ROCK in porcine oocyte might suggest a possibility that ROCK is related to actin dynamics in porcine oocyte, and plays a significant role during porcine oocyte meiotic maturation. To verify our hypothesis, we disrupted ROCK activity by Y-27632 treatment. We found that the polar body extrusion of porcine oocytes failed after ROCK inhibition, indicating a failure of porcine oocyte maturation. A number of studies have shown that the small GTPase Rho participates in cytokinesis [27–29]. Rho-associated protein kinase is one of the effectors for Rho, and is also considered to be involved in the progression of cytokinesis in several models [30]. Another study also indicates that ROCK2 is essential for
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cytokinesis in Xenopus embryos and mammalian cells by regulating glial filament segregation [31]. Our results are consistent with the previous studies. Therefore, ROCK plays a significant role during porcine oocyte meiotic maturation. To find the possible reason of ROCK inhibition on porcine oocyte maturation defect, we first examined the actin expression, because the cytokinesis in animal cells involves the concerted efforts of the actin cytoskeleton [32,33]. The results showed that the inhibition of ROCK activity caused the degradation of actin expression and the barrier of clear actin cap formation. These results were similar to previous studies that ROCK is involved in cytoskeletal rearrangements and the formation of stress fibers [34,35]. Our results may suggest that ROCK regulates the actin assembly. Similar research has shown that the actin nucleation activity of formin-2 is responsible for actin filament assembly and actin-driven chromosomal motility, which lead to asymmetric division in meiotic oocytes [36]. Therefore, our results might reflect that ROCK regulates porcine oocyte meiosis by its effect on actin assembly. Recently, in mammalian oocyte, the progressive formation of an actin filament meshwork has been confirmed to regulate the spindle movement for polar body extrusion during asymmetric division [4]. Previous studies have established that several factors regulate actin assembly and polymerization, which are involved in spindle positioning. Formin-2 is responsible for actin filament assembly, and is critical for driving spindle migration in mouse oocytes [37]. The actin nucleator Arp2/3 complex promotes actin polymerization, and contributes to spindle migration and oocyte asymmetric division [38,39]. Moreover, Rho GTPase subfamily molecules are critical for regulating the actin cytoskeleton. For example, the small GTPase Cdc42 has been recently found to participate in spindle migration and asymmetric division [40,41]. In addition, another small GTPase, Rac1, is confirmed to play a similar role in spindle positioning [42,43]. Therefore, we next examined the spindle position after ROCK inhibition. Our results show that Y-27632-treated porcine oocytes exhibit spindle positioning defects. Rho-associated protein kinase inhibition results in centrally arrested spindles, and the disruption of actin cap and CGFD formation, which are in agreement with the previous studies. Therefore, ROCK regulates porcine oocyte polar body extrusion by affecting meiotic spindle positioning. 4.1. Conclusions In summary, we found that ROCK is an actin assembly regulator of meiotic spindle positioning in porcine oocytes. Our results also establish a possible underlying mechanism of ROCK for the polar body extrusion during porcine oocyte meiotic maturation. Acknowledgments This work was supported by the National Basic Research Program of China (2014CB138503), the Natural Science Foundation of Jiangsu Province (BK20130671), China; and the Biogreen 21 Program (PJ009594 & PJ00909801), RDA, Republic of Korea.
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