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The Role of RhoA in the Germinal Vesicle Breakdown of Mouse Oocytes
Biochemical and Biophysical Research Communications 273, 997–1002 (2000) doi:10.1006/bbrc.2000.3052, available online at http://www.idealibrary.com on
The Role of RhoA in the Germinal Vesicle Breakdown of Mouse Oocytes Yong Pil Cheon,* ,1,2 Sung Woo Kim,* ,2 Soo-Jung Kim,* Young-Il Yeom,† Chaejoon Cheong,‡ and Kwon-Soo Ha* ,3 *Biomolecule Research Team, Korea Basic Science Institute, Taejon 305-333, Korea; †Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology, Taejon 305-333, Korea; and ‡Magnetic Resonance Research Team, Korea Basic Science Institute, Taejon 305-333, Korea
Received June 7, 2000
We have investigated a new role of RhoA in the germinal vesicle breakdown (GVBD) of mouse oocytes. First, RhoA was identified by immunostaining and ADPribosylation in germinal vesicle (GV) stage-oocytes. RhoA was mainly localized in the ooplasmic area, but rarely detected in germinal vesicle. Incubation of oocyte extract with C3 transferase induced a strong ADPribosylation at about 25 kDa. Incubation of GV-stage oocytes in culture medium induced the spontaneous maturation to GVBD by about 78 and 87% of total oocytes at 1 and 3 h, respectively. However, microinjection of C3 transferase into GV-stage oocytes significantly inhibited GVBD at 1 (GVBD ⴝ 29%) and 3 h (GVBD ⴝ 49%). To study the role of reactive oxygen species (ROS) in the oocyte maturation, the level of intra-oocyte ROS was measured using a ROS-specific fluorescent dye H 2DCFDA during the oocyte maturation. Spontaneous maturation of GV-stage oocytes induced a significant increase of ROS at 3 h by about twofold over the control level and then the increased level was maintained until 6 h. However, microinjection of C3 transferase inhibited the production of intra-oocyte ROS. Incubation with ROS scavengers, N-acetyl-L-cysteine and catalase, blocked the ROS increase. The ROS scavengers also significantly inhibited GVBD, as did C3 transferase. Thus, it was proposed that RhoA was involved in the GVBD, possibly by the production of ROS in mouse oocytes. © 2000 Academic Press
Key Words: mouse oocyte; RhoA; reactive oxygen species; C3 transferase; GVBD.
This work was supported in part by the grant from the Hyupdong Program of the Ministry of Science and Technology (98-N3-01-01-A-04). 1 Current address. Center for Biomedical Research, Population Council, Rockefeller University, 1230 York Avenue, New York, NY 10021. 2 Cheon, Y. P. and Kim, S. W. contributed equally to this work. 3 Senior Researcher of Korea Basic Science Institute. To whom correspondence should be addressed. Fax: ⫹82-42-865-3419. E-mail: [email protected].
Fully-grown mouse oocytes are arrested at the prophase stage of the first meiotic division and reinitiate meiotic maturation by being released from the follicles (1, 2). The spontaneous oocyte maturation is characterized by germinal vesicle breakdown (GVBD) and chromosome condensation. After completion of meiosis I, the oocytes are again arrested at metaphase II until fertilization. It has been reported that the meiotic maturation of mouse oocytes is regulated by several factors, including mitogen-activated protein (MAP) kinase (1, 3), cAMP (2), intracellular Ca 2⫹ (4, 5), and cyclin B (6). The role of MAP kinase has been reported in the oocyte maturation of various animals including mouse (3), porcine (7), and Xenopus (8). The roles of cdc2 and cyclin B have been also reported in the reinitiation and progression of meiotic maturation in mouse oocytes (6, 9). However, detailed mechanisms of meiotic maturation are not clearly understood in mouse oocytes. It has been suggested that ROS is involved in the two-cell block of mouse embryos (10 –12). Culture of mouse embryos in the presence of superoxide dismutase or thioredoxin significantly increased the blastulation rate (10, 11). Contrary to the inhibitory role of ROS in the embryo development, there has been a recent report indicating a possible role of reactive oxygen species (ROS) in the maturation of rat oocytes (13). Incubation of oocyte-cumulus complexes of rats with various anti-oxidants inhibited GVBD for up to 8 h and the inhibitory effect was reversible. Now, it is accepted that reactive oxygen species (ROS) act as an important second messenger in intracellular signaling in mammalian cells (14). ROS is required for the activation of various enzymes such as NF-B, phospholipase A 2 and D and MAP kinase, in response to agonists (15–18). ROS is also known to mediate the formation of stress fibers (19, 20) and increase of intracellular Ca 2⫹ (21–23). However, there is no report on
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the direct evidence presenting the role of ROS in the oocyte maturation. It is well known that RhoA activates various cellular responses including stress fiber formation, focal adhesion complex, and cell proliferation in fibroblasts (for reviews, see 24 and 25). Recently, it has been reported that RhoA is involved in the production of intracellular ROS in fibroblasts (20, 22, 23, 26). There have been reports suggesting the possible role of Rho in the development of mouse embryos (27, 28). Microinjection of C3 transferase into metaphase II-arrested mouse oocytes inhibited emission of the second polar body and cleavage to the 2-cell stage induced by insemination (27). In addition, inhibition of Rho with C3 transferase prevented polarization of eight-cell blastomeres in the mouse (28). However, there is no report on the role of RhoA in the meiotic maturation of mouse oocytes. In this report, we present evidence suggesting new roles of RhoA and ROS in the GVBD of mouse oocytes. RhoA was identified in mouse oocytes by two different methods, immunostaining and ADP-ribosylation. The role of RhoA in the oocyte maturation was investigated by microinjection of C3 transferase. C3 transferase is known to inhibit the activity of RhoA by ADPribosylation at Asp 41 (24). Microinjection of C3 transferase into GV-stage oocytes significantly inhibited GBVD. The level of intra-oocyte ROS was increased during the GVBD and the increase was blocked by C3 transferase. In addition, ROS scavengers, NAC and catalase, inhibited the production of ROS and GVBD. MATERIALS AND METHODS Collection and culture of oocytes. ICR (SPF condition, Korea Research Institute of Chemical Technology, Korea) mice (6 – 8 weeks old) were injected with five units of gonadotropin from pregnant Mares’ serum to enhance multiple follicular development. After killing mice, their ovaries were removed and transferred to BWW medium (94.6 mM NaCl, 4.78 mM KCl, 1.19 mM KH 2PO 4, 1.19 mM MgSO 4 䡠 7H 2O, 1.71 mM calcium lactate, 21.58 mM sodium lactate, 0.33 mM sodium pyruvate, 25.07 mM NaHCO 3, 100 units/ml penicillin, 100 g/ml streptomycin, pH 7.4, and 0.4% BSA) supplemented with 200 g/ml dibutyryl cyclic AMP to prevent from undergoing GVBD. Oocytes were collected by puncturing ovarian follicles with a 26-gauge needle under a dissecting microscope and then surrounding cumulus cells were removed by pipetting gently with a fine-bore pipette in BWW medium. Germinal vesicle (GV)-stage oocytes (cumulus cell-free) were carefully washed to remove dibutyryl cyclic AMP and then cultured in 40 l drops of BWW medium under mineral oil for 18 h at 37°C in a humidified CO 2-controlled (5%) incubator. Sometimes, oocytes were incubated with 30 mM NAC or 500 unit/ml catalase from Aspergillus niger in BWW medium. Then, the number of oocytes at GV- and GVBD-stage was scored with a differential interference contrast microscope (Carl Zeiss, Germany). Immunofluorescence. Oocytes were fixed with 3.7% (v/v) formaldehyde in Dulbecco’s phosphate-buffered saline (DPBS) (26) for 60 min and permeabilized with 0.2% (v/v) Triton X-100 in DPBS for 60 min. The oocytes were incubated for 60 min with a blocking solution (20 mM Tris, pH 7.5 containing 3% BSA, 1% horse serum, 138 mM NaCl, and 0.1% Triton X-100). Then, the oocytes were incubated with monoclonal anti-RhoA (Santa Cruz Biotech., 1:100, v/v) for 60 min
and further incubated with FITC-conjugated anti-mouse IgG (Sigma, 1:200, v/v) for 60 min. The stained samples were observed with a laser scanning confocal microscope (LSM510, Carl Zeiss). The samples were excited with a 488 nm Ar laser and the images were filtered with a longpass 515 nm filter. ADP-ribosylation of RhoA. ADP-ribosylation of RhoA was carried out according to the procedures of Leem et al. (29). Briefly, GV-stage oocytes (cumulus cell-free) were suspended in 50 l of lysis buffer (1 mM EDTA, 1.5 mM MgCl 2, 1 mM PMSF, 10 g/ml leupeptin, 10 g/ml pepstatin A, 10 g/ml aprotinin, and 10 mM Tris, pH 7.5) and disrupted by sonication for 15 s (3 ⫻ 5 s). After centrifugation at 16,000 rpm for 10 min, 20 l (12.5 g) of supernatant was mixed with 10 l of 3⫻ reaction buffer (60 mM Tris, pH 7.5 containing 3 mM EDTA, 3 mM MgCl 2, 3 mM dithiothreitol, 30 mM thymidine, 0.6 M NAD, 1 g C3 transferase, and 0.2 Ci [␣- 32P]NAD). C3 transferase was prepared by expressing the gene in E. coli according to the procedures of Leem et al. (29). Following incubation at 37°C for 30 min, the reaction was stopped by adding 10 l of 4⫻ SDS sample buffer. Resulting samples were then subjected to a 12% SDS–PAGE (30) and subsequent autoradiography. Microinjection of C3 transferase. GV-stage oocytes (cumulus cellfree) were washed with BWW medium to remove dibutyryl cyclic AMP and transferred to 40 l drops of BWW medium under mineral oil. Then, about 10 pl of 1.9 mg/ml C3 transferase in DPBS was injected into oocytes by using a differential interference contrast microscope (Carl Zeiss, Germany) equipped with a micromanipulator (5171, Eppendorf, Germany) and a manual microinjector (Sutter, USA). A quantitative direct pressure system was used for microinjection, as previously described with some modification (31), in which a small volume of mineral oil in a micropipette permitted controlled injection of picoliter quantities. Sometimes, an automatic microinjector (5246, Eppendorf, Germany) was used. Injections were completed within 15 min after removing dibutyryl cyclic AMP. Injected oocytes were then cultured in BWW medium. Measurement of intra-oocyte ROS. The level of ROS in oocytes was determined by the procedures of Koo et al. (20). Briefly, oocytes were cultured in BWW medium for the indicated times and then incubated with 20 M H 2DCFDA (Molecular Probes, USA) for the last 5 min. Following washing with BWW medium, the oocytes were immediately observed with laser scanning confocal microscopes (LSM410 or LSM510, Carl Zeiss, Germany). The samples were excited with a 488 nm argon laser and resulting images were filtered with a longpass 515 nm filter. The results were expressed as the relative fluorescence intensity (fold stimulation) from the ratio of fluorescence intensity of treated oocytes to that of control oocytes.
RESULTS Identification of RhoA in Mouse Oocytes In order to investigate the possible role of RhoA in the GVBD of mouse oocytes, first, we have identified RhoA in mouse oocytes by immunostaining and ADPribosylation. For immunostaining, GV-stage oocytes were stained with monoclonal anti-RhoA and observed by a laser scanning confocal microscope. As shown in Fig. 1a, RhoA was mainly localized in the ooplasm, but rarely detected in the germinal vesicle. Interestingly, RhoA was detected as small particles, even though the significance is not understood. GVBD-stage oocytes were also stained with monoclonal anti-RhoA and observed with a confocal microscope to study any changes in the distribution of RhoA during the oocyte maturation. However, RhoA was observed mainly in the cyto-
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C3 transferase significantly inhibited GVBD at 1 (GVBD ⫽ 28%) and 3 h (GVBD ⫽ 51%) (Fig. 2). No cytotoxic effect of C3 transferase on the oocytes was observed during the incubation time. Cytotoxicity was visually assessed by shrinkage of ooplasm and oocyte fragmentation. Interestingly, continuous incubation of oocytes injected with C3 transferase showed an almost normal rate of GVBD maturation at 18 h (GVBD ⫽ 87.5%), indicating that the inhibitory effect of C3 transferase on GVBD was transient. These results suggested that RhoA was required for the early maturation of mouse oocytes to the GVBD stage. Increase of ROS during Oocyte Maturation
FIG. 1. Identification of RhoA by immunostaining (a) and ADPribosylation (b) in mouse oocytes. (a) GV-stage oocytes were incubated with control (A and B) or monoclonal anti-RhoA antibody (C and D) and then further incubated with FITC-conjugated anti-mouse IgG as explained under Materials and Methods. The stained oocytes were observed with a confocal microscope. A and C are fluorescent images of RhoA, and B and D are overlays of fluorescent and transmitted images. The bar is 20 m. (b) Proteins were extracted from GV-stage oocytes and ADP-ribosylation reaction was carried out using [ 32P]NAD and C3 transferase as explained under Materials and Methods. The labeled proteins were then subjected to SDS– PAGE and subsequent autoradiography.
Since it has been recently reported that RhoA was essential for the production of intracellular H 2O 2 in fibroblasts (20, 22, 23, 26), we have investigated the possible role of ROS in the oocyte maturation to the GVBD-stage. To test the possibility, we have studied the changes of intra-oocyte ROS using a cell-permeable ROS-sensitive fluorophore H 2DCFDA. First, GV-stage oocytes were incubated in BWW medium for 3 h, stained with H 2DCFDA, and then observed with a confocal microscope. As shown in Fig. 3a, incubation of the
plasmic area without any significant changes in the distribution (data not shown). The identity of RhoA in mouse oocytes was further confirmed by ADP-ribosylation with C3 transferase. It is well known that C3 transferase inhibits the activity of RhoA by ADP-ribosylation at Asp 41 (24). Incubation of oocyte extract with C3 transferase induced a strong ADP-ribosylation band at about 25 kDa protein (Fig. 1b). A similar ADP-ribosylation band was also obtained with the protein extracted from Swiss 3T3 fibroblasts (data not shown). These results suggested that mouse oocytes contain RhoA, which is inhibited by exogenous C3 transferase, during the maturation. Inhibition of GVBD by C3 Transferase in Oocyte Maturation To study the possible role of RhoA in the oocyte maturation, C3 transferase was microinjected into GVstage oocytes, incubated for the indicated times, and then the number of oocytes at the stage of GV and GVBD was scored under an inverted microscope. GVBD was determined by the disappearance of GV. As shown in Fig. 2, in the control group (uninjected), about 85% oocytes reached GVBD-stage within 3 h after in vitro culture in BWW medium. DPBS-injected oocytes, as an injection control group, showed a similar rate of GVBD to the un-injected group. However, injection of
FIG. 2. Inhibitory effect of C3 transferase on the GVBD of mouse oocytes. GV-stage oocytes were microinjected with DPBS or C3 transferase (C3) and incubated for 18 h in BWW medium as explained under Materials and Methods. The number of oocytes at GV-, GVBD-stages was counted at 1, 3 and 18 h. The number of oocytes used for uninjected control (Con), DPBS injection (DPBS), and C3 transferase injection (C3) were 100, 82, and 62, respectively. The values represent percentage of oocytes at each stage. Data are expressed as the means ⫾ S.D from four independent experiments (**, P ⬍ 0.001).
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Regulation of ROS Production by RhoA Next, we have investigated the possible role of RhoA in the regulation of ROS production. To test the possibility, GV-stage oocytes were microinjected with C3 transferase, incubated for 3 h, and DCF fluorescence was observed with a confocal microscope. Relative ROS level was obtained by processing DCF images acquired from 31–37 oocytes. As shown in Fig. 4, the oocytes injected with DPBS showed a significant increase of ROS by about twofold over the control level. However, microinjection of C3 transferase completely inhibited the production of intra-oocyte ROS, which was consistent with the previous reports in fibroblasts (20, 22, 23, 26). These results suggested that RhoA was essential for the production of intra-oocyte ROS during the GVBD of mouse oocytes. Role of ROS in Oocyte Maturation
FIG. 3. Inhibitory effect of NAC on ROS production (a), and time-course changes of ROS during the resumption of oocyte maturation. (a) GV-stage oocytes (A) were incubated with control (B) or 30 mM NAC (C) for 3 h, and stained with 20 M H 2 DCHDA for the last 5 min as explained under Materials and Methods. The oocytes were then observed with a confocal microscope. (b) GV-stage oocytes were incubated with (open circle) or without (closed circle) 200 g/ml dibutyryl cyclic AMP (cAMP) for the indicated times and stained with 20 M H 2DCHDA for the last 5 min. The ROS level (fold stimulation) was determined by processing DCF images from 21–24 oocytes as described under Materials and Methods. Data are expressed as the means ⫾ S.D from four independent experiments (*, P ⬍ 0.01; **, P ⬍ 0.001). The numbers in parentheses represent the number of oocytes analyzed.
mouse oocytes for 3 h caused a significant increase in the DCF fluorescence. However, pre-incubation with NAC, a ROS scavenger, blocked the fluorescence increase. Incubation with A. niger catalase also blocked the ROS increase (data not shown), indicating that the fluorescence increase was mainly caused by the production of H 2O 2. Next, time-course changes of ROS level during oocyte maturation were also determined by culturing GV-stage oocytes for various time periods and measuring the DCF fluorescence from 21–24 oocytes. As shown in Fig. 3b, incubation of GV-stage oocytes in BWW medium induced a small increase at 1 h and then the maximal increase at 3 h. Then, the increased level was maintained until 6 h. However, there was no significant change in the ROS level, when GV-stage oocytes were incubated with BWW medium containing dibutyryl cyclic AMP to block the oocyte maturation. Thus, it was suggested that ROS was produced during the early maturation of mouse oocytes.
Our previous results showed the role of RhoA in the GVBD of mouse oocytes and the ROS production during the oocyte maturation. Thus, we have tested whether ROS played an important role in the GVBD of mouse oocytes by the incubation with two ROS scavengers, NAC and A. niger catalase. The scavengers inhibited the ROS production at 3 h (Fig. 3a). To test the possibility, GV-stage oocytes were incubated with NAC or catalase for 3 h, and the rate of GVBD was determined. As shown in Fig. 5, NAC significantly inhibited GVBD at 1 and 3 h, as did C3 transferase. Similar inhibitory effects were also observed by cata-
FIG. 4. Inhibitory effect of C3 transferase on the production of ROS in mouse oocytes. GV-stage oocytes were injected with DPBS or C3 transferase (C3) for 3 h, labeled with 20 M H 2DCHDA for the last 5 min and then observed by a confocal microscope. The ROS level (fold stimulation) was determined as explained in the legend of Fig. 3b. Data are expressed as the means ⫾ S.D from four independent experiments (**, P ⬍ 0.001). The numbers in parentheses represent the number of oocytes analyzed.
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FIG. 5. Inhibitory effect of NAC and catalase on the GVBD of mouse oocytes. GV-stage oocytes were incubated with control (Con), 30 mM NAC (NAC) or 500 unit/ml catalase (Cat) in BWW medium for 3 h and then the number of oocytes at GV-, GVBD-stages was scored as explained in the legend of Fig. 2. The numbers of oocytes for control, NAC and catalase were 75, 73, and 72, respectively. Data are means ⫾ S.D from four independent experiments (*, P ⬍ 0.01; **, P ⬍ 0.001).
lase. Considering the role of RhoA in the production of intra-oocytes ROS, it was suggested that RhoA was involved in the spontaneous GVBD of mouse oocytes, possibly by the ROS production. DISCUSSION In this report, we have presented a new role of RhoA in the resumption of spontaneous maturation in mouse oocytes. RhoA was identified by immunostaining with monoclonal anti-RhoA and ADP-ribosylation with C3 transferase. Microinjection of C3 transferase significantly inhibited GVBD. The level of intra-oocyte ROS increased during the resumption of oocyte maturation and the ROS increase was completely inhibited by the injection of C3 transferase. In addition, ROS scavengers, NAC and catalase, which blocked the ROS increase, largely inhibited GVBD. Thus, our results provided evidences suggesting that RhoA was required for GVBD by the production of intra-oocyte ROS in mouse oocytes. It was interesting that RhoA was involved in the GVBD of mouse oocytes since the role of RhoA has never been reported in the maturation of mouse oocytes. RhoA was mainly localized in the cytoplasmic
area and the inhibition of RhoA activity by C3 transferase largely inhibited GVBD. It is well known that RhoA activates stress fiber formation, cell proliferation and several enzymes including phosphoinositide 3-kinase, phosphatidylinositol-4-phosphate 5-kinase, and phospholipase D in fibroblasts (24, 25). The role of Rho has been reported in the development of mouse embryos (27, 28). Microinjection of C3 transferase inhibited the development of mouse embryos into the two-cell stage (27) and the polarization of eight-cell blastomeres (28). Thus, it can be suggested that RhoA has important roles in the oocyte maturation and embryonic development in the mouse. It is likely that ROS is important in the spontaneous resumption of oocyte maturation. Recently, it has been reported that GVBD was reversibly inhibited by the incubation with antioxidants including nordihydroguaiaretic acid and 2-tert-butyl-4-hydroxyanisole in rat oocytes (13). These results indicated that ROS may be required for the oocyte maturation. Consistent with the report, our results showed that the inhibition of ROS production by C3 transferase largely blocked GVBD. In addition, ROS scavengers, NAC and catalase, also inhibited the GVBD. In contrast, inhibitory effect of ROS has been reported in the development of mouse embryo. It has been reported that ROS was involved in the two-cell block of mouse embryos (11, 12). Incubation of mouse pronuclear embryos with superoxide dismutase significantly increased blastulation rate in mouse embryos (10). Thus, it is possible to suggest that ROS has important roles in the maturation of mouse oocytes and development of embryos, even though its roles could be different. RhoA played an essential role in the ROS production in mouse oocytes. Recently, we have reported that the production of intracellular H 2O 2 was dependent on the activity of RhoA in fibroblasts (20, 22, 23, 26). The elevation of intracellular H 2O 2 in response to lysophosphatidic acid or phosphatidic acid was blocked by scrape-loading of C3 transferase in Rat-2 fibroblasts (22, 26). C3 transferase also inhibited the production intracellular ROS in response to epidermal growth factor or transforming growth factor- in fibroblasts (20, 23). In consistence with the previous reports, microinjection of C3 transferase, which induced a strong ADPribosylation in mouse oocytes, completely inhibited the production of intracellular ROS during the oocyte maturation. Thus, it is likely that RhoA is an essential regulator in the production of ROS in mouse oocytes as well as fibroblasts. REFERENCES
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The Role of RhoA in the Germinal Vesicle Breakdown of Mouse Oocytes Yong Pil Cheon,* ,1,2 Sung Woo Kim,* ,2 Soo-Jung Kim,* Young-Il Yeom,† Chaejoon Cheong,‡ and Kwon-Soo Ha* ,3 *Biomolecule Research Team, Korea Basic Science Institute, Taejon 305-333, Korea; †Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology, Taejon 305-333, Korea; and ‡Magnetic Resonance Research Team, Korea Basic Science Institute, Taejon 305-333, Korea
Received June 7, 2000
We have investigated a new role of RhoA in the germinal vesicle breakdown (GVBD) of mouse oocytes. First, RhoA was identified by immunostaining and ADPribosylation in germinal vesicle (GV) stage-oocytes. RhoA was mainly localized in the ooplasmic area, but rarely detected in germinal vesicle. Incubation of oocyte extract with C3 transferase induced a strong ADPribosylation at about 25 kDa. Incubation of GV-stage oocytes in culture medium induced the spontaneous maturation to GVBD by about 78 and 87% of total oocytes at 1 and 3 h, respectively. However, microinjection of C3 transferase into GV-stage oocytes significantly inhibited GVBD at 1 (GVBD ⴝ 29%) and 3 h (GVBD ⴝ 49%). To study the role of reactive oxygen species (ROS) in the oocyte maturation, the level of intra-oocyte ROS was measured using a ROS-specific fluorescent dye H 2DCFDA during the oocyte maturation. Spontaneous maturation of GV-stage oocytes induced a significant increase of ROS at 3 h by about twofold over the control level and then the increased level was maintained until 6 h. However, microinjection of C3 transferase inhibited the production of intra-oocyte ROS. Incubation with ROS scavengers, N-acetyl-L-cysteine and catalase, blocked the ROS increase. The ROS scavengers also significantly inhibited GVBD, as did C3 transferase. Thus, it was proposed that RhoA was involved in the GVBD, possibly by the production of ROS in mouse oocytes. © 2000 Academic Press
Key Words: mouse oocyte; RhoA; reactive oxygen species; C3 transferase; GVBD.
This work was supported in part by the grant from the Hyupdong Program of the Ministry of Science and Technology (98-N3-01-01-A-04). 1 Current address. Center for Biomedical Research, Population Council, Rockefeller University, 1230 York Avenue, New York, NY 10021. 2 Cheon, Y. P. and Kim, S. W. contributed equally to this work. 3 Senior Researcher of Korea Basic Science Institute. To whom correspondence should be addressed. Fax: ⫹82-42-865-3419. E-mail: [email protected].
Fully-grown mouse oocytes are arrested at the prophase stage of the first meiotic division and reinitiate meiotic maturation by being released from the follicles (1, 2). The spontaneous oocyte maturation is characterized by germinal vesicle breakdown (GVBD) and chromosome condensation. After completion of meiosis I, the oocytes are again arrested at metaphase II until fertilization. It has been reported that the meiotic maturation of mouse oocytes is regulated by several factors, including mitogen-activated protein (MAP) kinase (1, 3), cAMP (2), intracellular Ca 2⫹ (4, 5), and cyclin B (6). The role of MAP kinase has been reported in the oocyte maturation of various animals including mouse (3), porcine (7), and Xenopus (8). The roles of cdc2 and cyclin B have been also reported in the reinitiation and progression of meiotic maturation in mouse oocytes (6, 9). However, detailed mechanisms of meiotic maturation are not clearly understood in mouse oocytes. It has been suggested that ROS is involved in the two-cell block of mouse embryos (10 –12). Culture of mouse embryos in the presence of superoxide dismutase or thioredoxin significantly increased the blastulation rate (10, 11). Contrary to the inhibitory role of ROS in the embryo development, there has been a recent report indicating a possible role of reactive oxygen species (ROS) in the maturation of rat oocytes (13). Incubation of oocyte-cumulus complexes of rats with various anti-oxidants inhibited GVBD for up to 8 h and the inhibitory effect was reversible. Now, it is accepted that reactive oxygen species (ROS) act as an important second messenger in intracellular signaling in mammalian cells (14). ROS is required for the activation of various enzymes such as NF-B, phospholipase A 2 and D and MAP kinase, in response to agonists (15–18). ROS is also known to mediate the formation of stress fibers (19, 20) and increase of intracellular Ca 2⫹ (21–23). However, there is no report on
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the direct evidence presenting the role of ROS in the oocyte maturation. It is well known that RhoA activates various cellular responses including stress fiber formation, focal adhesion complex, and cell proliferation in fibroblasts (for reviews, see 24 and 25). Recently, it has been reported that RhoA is involved in the production of intracellular ROS in fibroblasts (20, 22, 23, 26). There have been reports suggesting the possible role of Rho in the development of mouse embryos (27, 28). Microinjection of C3 transferase into metaphase II-arrested mouse oocytes inhibited emission of the second polar body and cleavage to the 2-cell stage induced by insemination (27). In addition, inhibition of Rho with C3 transferase prevented polarization of eight-cell blastomeres in the mouse (28). However, there is no report on the role of RhoA in the meiotic maturation of mouse oocytes. In this report, we present evidence suggesting new roles of RhoA and ROS in the GVBD of mouse oocytes. RhoA was identified in mouse oocytes by two different methods, immunostaining and ADP-ribosylation. The role of RhoA in the oocyte maturation was investigated by microinjection of C3 transferase. C3 transferase is known to inhibit the activity of RhoA by ADPribosylation at Asp 41 (24). Microinjection of C3 transferase into GV-stage oocytes significantly inhibited GBVD. The level of intra-oocyte ROS was increased during the GVBD and the increase was blocked by C3 transferase. In addition, ROS scavengers, NAC and catalase, inhibited the production of ROS and GVBD. MATERIALS AND METHODS Collection and culture of oocytes. ICR (SPF condition, Korea Research Institute of Chemical Technology, Korea) mice (6 – 8 weeks old) were injected with five units of gonadotropin from pregnant Mares’ serum to enhance multiple follicular development. After killing mice, their ovaries were removed and transferred to BWW medium (94.6 mM NaCl, 4.78 mM KCl, 1.19 mM KH 2PO 4, 1.19 mM MgSO 4 䡠 7H 2O, 1.71 mM calcium lactate, 21.58 mM sodium lactate, 0.33 mM sodium pyruvate, 25.07 mM NaHCO 3, 100 units/ml penicillin, 100 g/ml streptomycin, pH 7.4, and 0.4% BSA) supplemented with 200 g/ml dibutyryl cyclic AMP to prevent from undergoing GVBD. Oocytes were collected by puncturing ovarian follicles with a 26-gauge needle under a dissecting microscope and then surrounding cumulus cells were removed by pipetting gently with a fine-bore pipette in BWW medium. Germinal vesicle (GV)-stage oocytes (cumulus cell-free) were carefully washed to remove dibutyryl cyclic AMP and then cultured in 40 l drops of BWW medium under mineral oil for 18 h at 37°C in a humidified CO 2-controlled (5%) incubator. Sometimes, oocytes were incubated with 30 mM NAC or 500 unit/ml catalase from Aspergillus niger in BWW medium. Then, the number of oocytes at GV- and GVBD-stage was scored with a differential interference contrast microscope (Carl Zeiss, Germany). Immunofluorescence. Oocytes were fixed with 3.7% (v/v) formaldehyde in Dulbecco’s phosphate-buffered saline (DPBS) (26) for 60 min and permeabilized with 0.2% (v/v) Triton X-100 in DPBS for 60 min. The oocytes were incubated for 60 min with a blocking solution (20 mM Tris, pH 7.5 containing 3% BSA, 1% horse serum, 138 mM NaCl, and 0.1% Triton X-100). Then, the oocytes were incubated with monoclonal anti-RhoA (Santa Cruz Biotech., 1:100, v/v) for 60 min
and further incubated with FITC-conjugated anti-mouse IgG (Sigma, 1:200, v/v) for 60 min. The stained samples were observed with a laser scanning confocal microscope (LSM510, Carl Zeiss). The samples were excited with a 488 nm Ar laser and the images were filtered with a longpass 515 nm filter. ADP-ribosylation of RhoA. ADP-ribosylation of RhoA was carried out according to the procedures of Leem et al. (29). Briefly, GV-stage oocytes (cumulus cell-free) were suspended in 50 l of lysis buffer (1 mM EDTA, 1.5 mM MgCl 2, 1 mM PMSF, 10 g/ml leupeptin, 10 g/ml pepstatin A, 10 g/ml aprotinin, and 10 mM Tris, pH 7.5) and disrupted by sonication for 15 s (3 ⫻ 5 s). After centrifugation at 16,000 rpm for 10 min, 20 l (12.5 g) of supernatant was mixed with 10 l of 3⫻ reaction buffer (60 mM Tris, pH 7.5 containing 3 mM EDTA, 3 mM MgCl 2, 3 mM dithiothreitol, 30 mM thymidine, 0.6 M NAD, 1 g C3 transferase, and 0.2 Ci [␣- 32P]NAD). C3 transferase was prepared by expressing the gene in E. coli according to the procedures of Leem et al. (29). Following incubation at 37°C for 30 min, the reaction was stopped by adding 10 l of 4⫻ SDS sample buffer. Resulting samples were then subjected to a 12% SDS–PAGE (30) and subsequent autoradiography. Microinjection of C3 transferase. GV-stage oocytes (cumulus cellfree) were washed with BWW medium to remove dibutyryl cyclic AMP and transferred to 40 l drops of BWW medium under mineral oil. Then, about 10 pl of 1.9 mg/ml C3 transferase in DPBS was injected into oocytes by using a differential interference contrast microscope (Carl Zeiss, Germany) equipped with a micromanipulator (5171, Eppendorf, Germany) and a manual microinjector (Sutter, USA). A quantitative direct pressure system was used for microinjection, as previously described with some modification (31), in which a small volume of mineral oil in a micropipette permitted controlled injection of picoliter quantities. Sometimes, an automatic microinjector (5246, Eppendorf, Germany) was used. Injections were completed within 15 min after removing dibutyryl cyclic AMP. Injected oocytes were then cultured in BWW medium. Measurement of intra-oocyte ROS. The level of ROS in oocytes was determined by the procedures of Koo et al. (20). Briefly, oocytes were cultured in BWW medium for the indicated times and then incubated with 20 M H 2DCFDA (Molecular Probes, USA) for the last 5 min. Following washing with BWW medium, the oocytes were immediately observed with laser scanning confocal microscopes (LSM410 or LSM510, Carl Zeiss, Germany). The samples were excited with a 488 nm argon laser and resulting images were filtered with a longpass 515 nm filter. The results were expressed as the relative fluorescence intensity (fold stimulation) from the ratio of fluorescence intensity of treated oocytes to that of control oocytes.
RESULTS Identification of RhoA in Mouse Oocytes In order to investigate the possible role of RhoA in the GVBD of mouse oocytes, first, we have identified RhoA in mouse oocytes by immunostaining and ADPribosylation. For immunostaining, GV-stage oocytes were stained with monoclonal anti-RhoA and observed by a laser scanning confocal microscope. As shown in Fig. 1a, RhoA was mainly localized in the ooplasm, but rarely detected in the germinal vesicle. Interestingly, RhoA was detected as small particles, even though the significance is not understood. GVBD-stage oocytes were also stained with monoclonal anti-RhoA and observed with a confocal microscope to study any changes in the distribution of RhoA during the oocyte maturation. However, RhoA was observed mainly in the cyto-
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C3 transferase significantly inhibited GVBD at 1 (GVBD ⫽ 28%) and 3 h (GVBD ⫽ 51%) (Fig. 2). No cytotoxic effect of C3 transferase on the oocytes was observed during the incubation time. Cytotoxicity was visually assessed by shrinkage of ooplasm and oocyte fragmentation. Interestingly, continuous incubation of oocytes injected with C3 transferase showed an almost normal rate of GVBD maturation at 18 h (GVBD ⫽ 87.5%), indicating that the inhibitory effect of C3 transferase on GVBD was transient. These results suggested that RhoA was required for the early maturation of mouse oocytes to the GVBD stage. Increase of ROS during Oocyte Maturation
FIG. 1. Identification of RhoA by immunostaining (a) and ADPribosylation (b) in mouse oocytes. (a) GV-stage oocytes were incubated with control (A and B) or monoclonal anti-RhoA antibody (C and D) and then further incubated with FITC-conjugated anti-mouse IgG as explained under Materials and Methods. The stained oocytes were observed with a confocal microscope. A and C are fluorescent images of RhoA, and B and D are overlays of fluorescent and transmitted images. The bar is 20 m. (b) Proteins were extracted from GV-stage oocytes and ADP-ribosylation reaction was carried out using [ 32P]NAD and C3 transferase as explained under Materials and Methods. The labeled proteins were then subjected to SDS– PAGE and subsequent autoradiography.
Since it has been recently reported that RhoA was essential for the production of intracellular H 2O 2 in fibroblasts (20, 22, 23, 26), we have investigated the possible role of ROS in the oocyte maturation to the GVBD-stage. To test the possibility, we have studied the changes of intra-oocyte ROS using a cell-permeable ROS-sensitive fluorophore H 2DCFDA. First, GV-stage oocytes were incubated in BWW medium for 3 h, stained with H 2DCFDA, and then observed with a confocal microscope. As shown in Fig. 3a, incubation of the
plasmic area without any significant changes in the distribution (data not shown). The identity of RhoA in mouse oocytes was further confirmed by ADP-ribosylation with C3 transferase. It is well known that C3 transferase inhibits the activity of RhoA by ADP-ribosylation at Asp 41 (24). Incubation of oocyte extract with C3 transferase induced a strong ADP-ribosylation band at about 25 kDa protein (Fig. 1b). A similar ADP-ribosylation band was also obtained with the protein extracted from Swiss 3T3 fibroblasts (data not shown). These results suggested that mouse oocytes contain RhoA, which is inhibited by exogenous C3 transferase, during the maturation. Inhibition of GVBD by C3 Transferase in Oocyte Maturation To study the possible role of RhoA in the oocyte maturation, C3 transferase was microinjected into GVstage oocytes, incubated for the indicated times, and then the number of oocytes at the stage of GV and GVBD was scored under an inverted microscope. GVBD was determined by the disappearance of GV. As shown in Fig. 2, in the control group (uninjected), about 85% oocytes reached GVBD-stage within 3 h after in vitro culture in BWW medium. DPBS-injected oocytes, as an injection control group, showed a similar rate of GVBD to the un-injected group. However, injection of
FIG. 2. Inhibitory effect of C3 transferase on the GVBD of mouse oocytes. GV-stage oocytes were microinjected with DPBS or C3 transferase (C3) and incubated for 18 h in BWW medium as explained under Materials and Methods. The number of oocytes at GV-, GVBD-stages was counted at 1, 3 and 18 h. The number of oocytes used for uninjected control (Con), DPBS injection (DPBS), and C3 transferase injection (C3) were 100, 82, and 62, respectively. The values represent percentage of oocytes at each stage. Data are expressed as the means ⫾ S.D from four independent experiments (**, P ⬍ 0.001).
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Regulation of ROS Production by RhoA Next, we have investigated the possible role of RhoA in the regulation of ROS production. To test the possibility, GV-stage oocytes were microinjected with C3 transferase, incubated for 3 h, and DCF fluorescence was observed with a confocal microscope. Relative ROS level was obtained by processing DCF images acquired from 31–37 oocytes. As shown in Fig. 4, the oocytes injected with DPBS showed a significant increase of ROS by about twofold over the control level. However, microinjection of C3 transferase completely inhibited the production of intra-oocyte ROS, which was consistent with the previous reports in fibroblasts (20, 22, 23, 26). These results suggested that RhoA was essential for the production of intra-oocyte ROS during the GVBD of mouse oocytes. Role of ROS in Oocyte Maturation
FIG. 3. Inhibitory effect of NAC on ROS production (a), and time-course changes of ROS during the resumption of oocyte maturation. (a) GV-stage oocytes (A) were incubated with control (B) or 30 mM NAC (C) for 3 h, and stained with 20 M H 2 DCHDA for the last 5 min as explained under Materials and Methods. The oocytes were then observed with a confocal microscope. (b) GV-stage oocytes were incubated with (open circle) or without (closed circle) 200 g/ml dibutyryl cyclic AMP (cAMP) for the indicated times and stained with 20 M H 2DCHDA for the last 5 min. The ROS level (fold stimulation) was determined by processing DCF images from 21–24 oocytes as described under Materials and Methods. Data are expressed as the means ⫾ S.D from four independent experiments (*, P ⬍ 0.01; **, P ⬍ 0.001). The numbers in parentheses represent the number of oocytes analyzed.
mouse oocytes for 3 h caused a significant increase in the DCF fluorescence. However, pre-incubation with NAC, a ROS scavenger, blocked the fluorescence increase. Incubation with A. niger catalase also blocked the ROS increase (data not shown), indicating that the fluorescence increase was mainly caused by the production of H 2O 2. Next, time-course changes of ROS level during oocyte maturation were also determined by culturing GV-stage oocytes for various time periods and measuring the DCF fluorescence from 21–24 oocytes. As shown in Fig. 3b, incubation of GV-stage oocytes in BWW medium induced a small increase at 1 h and then the maximal increase at 3 h. Then, the increased level was maintained until 6 h. However, there was no significant change in the ROS level, when GV-stage oocytes were incubated with BWW medium containing dibutyryl cyclic AMP to block the oocyte maturation. Thus, it was suggested that ROS was produced during the early maturation of mouse oocytes.
Our previous results showed the role of RhoA in the GVBD of mouse oocytes and the ROS production during the oocyte maturation. Thus, we have tested whether ROS played an important role in the GVBD of mouse oocytes by the incubation with two ROS scavengers, NAC and A. niger catalase. The scavengers inhibited the ROS production at 3 h (Fig. 3a). To test the possibility, GV-stage oocytes were incubated with NAC or catalase for 3 h, and the rate of GVBD was determined. As shown in Fig. 5, NAC significantly inhibited GVBD at 1 and 3 h, as did C3 transferase. Similar inhibitory effects were also observed by cata-
FIG. 4. Inhibitory effect of C3 transferase on the production of ROS in mouse oocytes. GV-stage oocytes were injected with DPBS or C3 transferase (C3) for 3 h, labeled with 20 M H 2DCHDA for the last 5 min and then observed by a confocal microscope. The ROS level (fold stimulation) was determined as explained in the legend of Fig. 3b. Data are expressed as the means ⫾ S.D from four independent experiments (**, P ⬍ 0.001). The numbers in parentheses represent the number of oocytes analyzed.
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FIG. 5. Inhibitory effect of NAC and catalase on the GVBD of mouse oocytes. GV-stage oocytes were incubated with control (Con), 30 mM NAC (NAC) or 500 unit/ml catalase (Cat) in BWW medium for 3 h and then the number of oocytes at GV-, GVBD-stages was scored as explained in the legend of Fig. 2. The numbers of oocytes for control, NAC and catalase were 75, 73, and 72, respectively. Data are means ⫾ S.D from four independent experiments (*, P ⬍ 0.01; **, P ⬍ 0.001).
lase. Considering the role of RhoA in the production of intra-oocytes ROS, it was suggested that RhoA was involved in the spontaneous GVBD of mouse oocytes, possibly by the ROS production. DISCUSSION In this report, we have presented a new role of RhoA in the resumption of spontaneous maturation in mouse oocytes. RhoA was identified by immunostaining with monoclonal anti-RhoA and ADP-ribosylation with C3 transferase. Microinjection of C3 transferase significantly inhibited GVBD. The level of intra-oocyte ROS increased during the resumption of oocyte maturation and the ROS increase was completely inhibited by the injection of C3 transferase. In addition, ROS scavengers, NAC and catalase, which blocked the ROS increase, largely inhibited GVBD. Thus, our results provided evidences suggesting that RhoA was required for GVBD by the production of intra-oocyte ROS in mouse oocytes. It was interesting that RhoA was involved in the GVBD of mouse oocytes since the role of RhoA has never been reported in the maturation of mouse oocytes. RhoA was mainly localized in the cytoplasmic
area and the inhibition of RhoA activity by C3 transferase largely inhibited GVBD. It is well known that RhoA activates stress fiber formation, cell proliferation and several enzymes including phosphoinositide 3-kinase, phosphatidylinositol-4-phosphate 5-kinase, and phospholipase D in fibroblasts (24, 25). The role of Rho has been reported in the development of mouse embryos (27, 28). Microinjection of C3 transferase inhibited the development of mouse embryos into the two-cell stage (27) and the polarization of eight-cell blastomeres (28). Thus, it can be suggested that RhoA has important roles in the oocyte maturation and embryonic development in the mouse. It is likely that ROS is important in the spontaneous resumption of oocyte maturation. Recently, it has been reported that GVBD was reversibly inhibited by the incubation with antioxidants including nordihydroguaiaretic acid and 2-tert-butyl-4-hydroxyanisole in rat oocytes (13). These results indicated that ROS may be required for the oocyte maturation. Consistent with the report, our results showed that the inhibition of ROS production by C3 transferase largely blocked GVBD. In addition, ROS scavengers, NAC and catalase, also inhibited the GVBD. In contrast, inhibitory effect of ROS has been reported in the development of mouse embryo. It has been reported that ROS was involved in the two-cell block of mouse embryos (11, 12). Incubation of mouse pronuclear embryos with superoxide dismutase significantly increased blastulation rate in mouse embryos (10). Thus, it is possible to suggest that ROS has important roles in the maturation of mouse oocytes and development of embryos, even though its roles could be different. RhoA played an essential role in the ROS production in mouse oocytes. Recently, we have reported that the production of intracellular H 2O 2 was dependent on the activity of RhoA in fibroblasts (20, 22, 23, 26). The elevation of intracellular H 2O 2 in response to lysophosphatidic acid or phosphatidic acid was blocked by scrape-loading of C3 transferase in Rat-2 fibroblasts (22, 26). C3 transferase also inhibited the production intracellular ROS in response to epidermal growth factor or transforming growth factor- in fibroblasts (20, 23). In consistence with the previous reports, microinjection of C3 transferase, which induced a strong ADPribosylation in mouse oocytes, completely inhibited the production of intracellular ROS during the oocyte maturation. Thus, it is likely that RhoA is an essential regulator in the production of ROS in mouse oocytes as well as fibroblasts. REFERENCES
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