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Gamete Biology |
State Key Laboratory of Reproductive Biology,3 Institute of Zoology, Graduate School of the Chinese Accademy of Sciences, Chinese Academy of Sciences, Beijing 100080, China
Department of Molecular Biology,4 University of Texas Southwestern Medical Center, Dallas, Texas 75390
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunits at different stages of oocyte and early embryo development. The 20S proteasome subunits were accumulated in the germinal vesicle, around the condensed chromosomes at prometaphase, with spindle at metaphase I and II, the region between the separating chromosomes, and especially the midbody at anaphase I and telophase I, the pronucleus, and the nucleus in early embryonic cells. In conclusion, our results suggest that the UPP is important at multiple steps of pig oocyte meiosis, fertilization, and early embryonic mitosis and that it may play its roles by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.
fertilization, gamete biology, kinases, meiosis, ovum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protein kinases, such as mitogen-activated protein kinase (MAPK), maturation-promoting factor (MPF), Ca2+/calmodulin-dependent protein kinase II, protein kinase C, and Polo-like kinase (Plk), play important roles in modifying protein activity [3, 5–8]. Furthermore, degradation of some important proteins by the ubiquitin-proteasome pathway (UPP) has also been shown to participate in activation/inactivation of several signal transduction pathways related to oocyte meiotic processes [9]. Composed of 76 amino acids, ubiquitin is an 8.45-kDa protein that is highly conserved in all eukaryotes. The proteasome is a large, 26S, multicatalytic protease complex that degrades polyubiquitinated proteins to small peptides. It is composed of three subcomplexes: a 20S core particle that carries the catalytic activity and two 19S regulatory particles, yielding a dumbbell-shaped complex. The 20S core particle is composed of subunits 1–7ß1–7ß1–71–7 [10]. Proteins critical to regulation of the cell-cycle progression, including cyclins, Plk, Cdks, c-mos proto-oncogene products, securin, Cut2, and Ase1, are degraded by the UPP at specific cell-cycle points [11, 12]. Degradation of a protein via the UPP involves two discrete and successive steps: tagging of the substrate by covalent attachment of multiple ubiquitin molecules, and degradation of the tagged protein by the 26S proteasome complex with release of free and reusable ubiquitin [10]. Anaphase-promoting complex/cyclosome (APC/C) functions as a cell cycle-regulated ubiquitin ligase that mediates protein ubiquitination [13].
Currently, three major cell-cycle transitions (entry into S phase, separation of sister chromatids, and exit from mitosis) are known to require the degradation of specific proteins [13]. Mitotic entry is regulated by MPF, a kinase complex composed of the regulatory subunit cyclin B and the catalytic subunit p34cdc2 kinase. Mitotic [14] or meiotic [15] exit requires MPF inactivation, which is achieved by cyclin B destruction via the UPP. As an E3 ubiquitin ligase, Ret Finger Protein-Like 4 targets cyclin B1 for proteasomal degradation, which is a key aspect for oocyte meiotic cell-cycle control and the crucial oocyte-to-embryo transition [16]. However, almost all data concerning the relationship between cyclin degradation and the metaphase-anaphase transition at exit from the M phase are from mitotic cells, and it is not known whether the same mechanisms are involved in the metaphase-anaphase transition during meiosis I and meiosis II.
It has been shown that the 26S proteasome changes during Xenopus oocyte maturation and early development, suggesting that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles of the early embryo [17, 18]. In toad and starfish oocytes, inhibition of proteasomal proteolytic activity prevented GV breakdown (GVBD) [19, 20]. Inhibition of GVBD was also observed with microinjection of antiproteasome subunit antibodies into the starfish oocytes [21–24]. Ubiquitin-mediated cyclin B degradation [25, 26] was characterized in clam oocytes entering the first meiotic division [27]. Furthermore, in Xenopus, cyclin B degradation was initiated by the 26S proteasome on egg activation induced by treatment with the calcium ionophore A23187 [15, 28]. Other investigators showed that the intracellular calcium mobilization regulated the activity of 26S proteasome during the metaphase-anaphase transition in the ascidian meiotic cell cycle [29].
The proteasome has recently been detected in human and rat oocytes [4, 30, 31], but its subcellular localization during oocyte meiotic maturation, fertilization, and early embryo development has not been well documented. In rat, proteasomal catalytic activity is essential for the decrease in MPF activity and completion of the first meiotic division [31]. Limited data showed that the UPP was also functional in pig oocyte cell-cycle control and mitochondrial inheritance [32, 33]. To our knowledge, however, no systematic studies concerning the roles of the UPP in mammalian species have been reported. Especially, the relationship between the UPP and two vital kinases, MAPK/p90rsk and MPF, in meiotic cell cycle of mammals is unclear.
In the present study, we investigated 1) the subcellular localization of 20S proteasome subunit at different stages of pig oocyte meiotic maturation, fertilization, and early embryo mitosis; 2) the roles of the UPP and its relationship with MAPK/p90rsk cascade and MPF in hypoxanthine (HX)-maintained meiotic arrest; and 3) the roles of the UPP and its regulation on MAPK/p90rsk cascade and MPF during electrical activation and in vitro fertilization of mature pig oocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two potent, reversible, and cell-permeable proteasome inhibitors, ALLN (N-Acetyl-Leu-Leu-Nle-CHO) and MG-132 (Z-Leu-Leu-Leu-CHO), were obtained from Calbiochem (La Jolla, CA). ALLN and MG-132 were prepared as 100 and 10 mM stocks, respectively, in dimethyl sulfoxide (DMSO) and stored at –20°C in a dark box. Polyclonal rabbit anti-mouse p90rsk antibody, monoclonal mouse anti-pERK1/2 antibody, polyclonal rabbit anti-human ERK2 antibody, and polyclonal rabbit anti-human cyclin B1 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals or media components were embryo-culture or cell-culture grade and were obtained from Sigma (St. Louis, MO) unless otherwise noted.
For HX-maintained meiotic arrest of pig oocytes, TCM-199 (Gibco, Grand Island, NY) supplemented with 4 mM HX, 0.23 mM sodium pyruvate, 2 mM glutamine, 3 mg/ml of lyophilized crystallized BSA (Calbiochem), 75 µg/ml of potassium penicillin G, and 50 µg/ml of streptomycin sulfate was used. This medium is termed HX medium. The medium used for normal maturation culture of oocytes was TCM-199 supplemented with 75 µg/ml of potassium penicillin G, 50 µg/ml of streptomycin sulfate, 0.57 mM cysteine, 0.5 µg/ml of FSH, 0.5 µg/ml of LH, and 10 ng/ml of epidermal growth factor.
In Vitro Maturation of Pig Oocytes
Ovaries were collected from gilts at a local slaughterhouse and transported to the laboratory within 1 h. Oocytes were aspirated from antral follicles (diameter, 2–6 mm) with an 18-gauge needle fixed to a 20-ml disposable syringe. After three washes with maturation medium, oocytes with compact cumulus and evenly granulated ooplasm were selected for maturation culture. Denuded oocytes (DOs) were obtained by treatment the CEOs with 300 IU/ml of hyaluronidase (Sigma) and repeated pipetting. The DOs and CEOs were then washed twice and cultured in drug-free maturation medium or HX medium. A group of 25 oocytes was cultured in a 100-µl drop of medium at 39°C in an atmosphere of 5% CO2 and saturated humidity.
After a 44-h maturation culture, oocytes were freed of cumulus cells by treatment with 300 IU/ml of hyaluronidase and repeated pipetting. The mature DOs were then washed twice in TCM-199 medium and used for either electrical activation or in vitro fertilization.
Electrical Activation
Oocytes matured in HX-free medium were induced to undergo parthenogenetic activation by physical (electrical pulse) stimulation. The method of electrical activation was essentially the same as that reported by Fan et al. [5, 6]. Briefly, after washing three times in the electroporation medium (0.28 M mannitol, 0.05 mM CaCl2, 0.1 mM MgSO4, and 0.01% [w/v] BSA), cumulus-free oocytes were put in a fusion chamber. An 80-µsec, direct-current pulse at 120 V/mm was applied to oocytes. The oocytes were then washed three times and cultured in NCSU-23 medium containing 0.4% BSA. Before electrical stimulation, the matured oocytes were pretreated with 50 µM ALLN or 50 µM MG-132 for 30 min, and the inhibitors were always included in the medium from culture.
In Vitro Fertilization
In vitro fertilization of pig oocytes was performed using semen obtained from a local farm and in a manner essentially the same as that reported by Fan et al. [5]. Oocytes matured in HX-free medium were used for in vitro fertilization, and DOs were obtained as described above. Before insemination, the matured oocytes were pretreated with 50 µM ALLN or 50 µM MG-132 for 30 min. Pig sperm washed twice in Dulbecco PBS containing 0.1% BSA were capacitated in modified tris-buffered medium (mTBM) containing 2 mM caffeine and 0.2% BSA and then incubated with pig oocytes in inhibitor-free mTBM for 5–6 h. Inseminated oocytes were washed three times in NCSU-23 containing 0.4% BSA and the inhibitors to remove loosely attached sperm and then cultured in the same medium.
Nuclear Status Examination
Orcein staining was conducted as described by Sun et al. [34] with minor modification. Denuded oocytes were mounted on slides, fixed in acetic acid:ethanol (1:3 [v/v]) for at least 48 h, stained with 1% orcein, and examined with a phase-contrast microscope.
Western Blot Analysis
For detection of p90rsk and active ERK1/2, proteins from 50 oocytes were collected in SDS sample buffer and heated to 100°C for 4 min. After cooling on ice and centrifuging at 12 000 x g for 3 min, samples were frozen at –20°C until use. The total proteins were separated by SDS-PAGE with a 4% stacking gel and a 10% separating gel for 30 min at 90 V and for 2.5 h at 120 V, respectively, and then electrophoretically transferred onto nitrocellulose membrane for 2.5 h at 200 mA and 4°C. Then, the membrane was blocked overnight at 4°C in tris-buffered saline (pH 7.4, 20 mM Tris, 137 mM NaCl) with 0.1% Tween-20 (TBST) containing 5% low-fat milk. To detect both p90rsk and active ERK1/2, blots were cut in two parts containing the proteins above and below the 68-kDa molecular weight marker and then incubated separately for 2 h in TBST with 1:500 polyclonal rabbit anti-mouse p90rsk antibodies for the upper part of the membrane and with 1:500 mouse anti-human pERK1/2 antibody for the lower part of the membrane. After three washes of 10 min each in TBST, the upper and lower parts of the membrane were incubated for 1 h at 37°C with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Ig) G and HRP-conjugated goat anti-mouse IgG diluted 1: 1000 in TBST, respectively. The membranes were washed three times in TBST and then processed using the enhanced chemiluminescence (ECL) detection system (Amersham, Little Chalfont, UK).
For reprobing, the membrane was washed in stripping buffer (100 mM ß-mercaptoethanol, 20% SDS, and 62.5 mM Tris, [pH 6.7] at 50°C for 30 min to strip off bound antibody after ECL detection. The membrane was reprobed with polyclonal rabbit anti-ERK2 antibody diluted 1:500 using the same procedure as described above. All experiments were repeated at least three times.
For detection of cyclin B1, proteins from 100 oocytes were extracted, separated, and transferred onto the nitrocellulose membrane as mentioned above. The membrane was blocked with 5% low-fat milk in TBST for 1 h at 37°C and then incubated overnight with polyclonal rabbit anti-human cyclin B1 antibody diluted 1:200 at 4°C. The steps of second antibody binding, washing, and ECL processing were the same as those described for the ERK2 detection.
Confocal Microscopy
After removing the zona pellucida in acidic Tyrode medium (pH 2.5), oocytes were fixed with 4% paraformaldehyde in PBS (pH 7.4) for at least 30 min at room temperature. Cells were permeabilized with 1% Triton X-100 overnight at 37°C, followed by blocking in PBS containing 1% BSA for 1 h and incubation overnight at 4°C with polyclonal mouse anti-human 20S proteasome subunits antibody (Zymed, South San Francisco, CA) diluted 1:50 in blocking solution. After three washes in PBS containing 0.1% Tween 20 and 0.01% Triton X-100 (washing solution) for 5 min each, the oocytes were labeled with fluorescein isothiocyanate-conjugated goat anti-mouse IgG diluted 1:100. Nuclear status of oocytes was evaluated by staining with 10 µg/ml of propidium iodide for 10 min. Following extensive washing, samples were mounted between a coverslip and a glass slide supported by four columns of a mixture of Vaseline and paraffin (9: 1). The slides were sealed with nail polish. Cells were observed under a Leica confocal laser scanning microscope (TCS-NT; Leica, Wetzlar, Germany) on the same day. Nonspecific staining was determined by substituting primary antibodies with normal rabbit IgG. Each experiment was repeated three times, and at least 20 oocytes were examined each time.
Experimental Design
Roles of the UPP in the meiotic maturation of pig oocytes
The subcellular localization of 20S proteasome subunit during pig oocyte meiotic maturation was studied by confocal microscopy. To study the possible roles of the UPP during pig oocyte meiotic maturation of HX-maintained, meiosis-arrested pig oocytes, CEOs or DOs were cultured for 36 h in HX medium with or without different concentrations of the proteasome-inhibitor ALLN or MG-132. At the end of the culture, oocytes were harvested for nuclear status examination and Western blot analysis. As a negative control, oocytes were cultured in HX medium containing 0.1% or 0.2% DMSO. To study the effect of UPP inhibitors on oocyte meiotic maturation after different times of treatment, CEOs or DOs were cultured in HX medium containing 50 µM ALLN or 50 µM MG-132, and the oocytes were collected at 0, 12, 16, 20, 24, and 36 h for nuclear status examination and Western blot analysis. To analyze the possible regulation of UPP on MAPK/p90rsk cascade during oocyte meiotic maturation, pig CEOs or DOs were cultured in HX medium containing 50 µM ALLN or 50 µM MG-132 plus 50 µM U0126 and 50 µM ALLN or 50 µM MG-132 plus 50 µM U0126 for 36 h. At the end of the culture, oocytes were harvested for nuclear status examination and Western blot analysis. To study the possible regulation of the UPP on MPF activation during meiotic resumption of oocytes, pig CEOs or DOs were cultured in HX medium containing 50 µM ALLN and 30 µM roscovitine or 50 µM MG-132 and 30 µM roscovitine for 36 h. At the end of the culture, oocytes were harvested for nuclear status examination and Western blot analysis of cyclin B1, the regulatory subunit of MPF.
Roles of the UPP in the process of pig oocyte activation and early embryos
The subcellular localization of 20S proteasome subunit during oocyte activation and early embryo cleavage was studied by confocal microscopy. The roles of the UPP in the activation of MII-arrested pig oocytes were analyzed. In vitro-matured pig oocytes were first removed of cumulus cells and then washed twice in maturation medium. Before electrical activation and in vitro fertilization, oocytes were pretreated in maturation medium containing 50 µM ALLN or 50 µM MG-132 for 30 min, and 50 µM ALLN or 50 µM MG-132 was always included in the medium for culture after electrical activation or insemination. Extrusion of the second polar body (PB2) and pronuclear formation were examined at 14 and 20 h, respectively, after activation. To investigate the possible regulation of the UPP on MAPK/p90rsk cascade and MPF activity, oocytes were collected at 0, 6, 10, 14, and 20 h after activation with or without the drug treatments mentioned above. The expression of ERK2 and p90rsk as well as the phosphorylation level of ERK1/2 were detected by Western blot analysis. To detect the expression of cyclin B1, oocytes were collected at 0 and 2 h after electrical activation or at 0 and 6 h after insemination for Western blot analysis.
Statistical Analysis
All percentages from three repeated experiments are expressed as mean ± SEM, and the number of oocytes observed is provided in parentheses. All frequencies were subjected to arcsine transformation. The transformed data were statistically compared by ANOVA using SPSS software (SPSS Inc., Chicago, IL) followed by the Student-Newman-Keuls test. Differences at P < 0.05 were considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Subunit During Oocyte Meiotic Maturation
The distribution of 20S proteasome subunit during pig oocyte meiotic maturation is shown in Figure 1. The specimens were stained with propidium iodide to visualize the DNA and confirm the stage of meiotic maturation. The localization of 20S proteasome subunit varied at different developmental stages. In GV-stage oocytes, the 20S proteasome subunits mainly accumulated in the GV (Fig. 1A). Shortly after GVBD (
24 h after maturation culture in our system), the 20S proteasome subunit was concentrated to the periphery of condensed chromosomes (Fig. 1B). In metaphase I, with the organization of chromosomes to the equatorial plate, prominent staining of 20S proteasome subunit was observed around the aligned chromosomes, putatively the position of the metaphase I spindle (Fig. 1C). At anaphase I, the staining of 20S proteasome subunit concentrated around the separating homologous chromosomes, and a more intense staining was detected at the region between the separating homologous chromosomes (Fig. 1D). At telophase I, the 20S proteasome subunit accumulated in the region between the separated homologous chromosomes and associated with the midbody between the emitted PB1 and the oocytes (Fig. 1E). At MII, the localization of 20S proteasome subunit was similar to that at metaphase I and mainly concentrated around the aligned chromosomes, putatively the position of the MII spindle (Fig. 1F).
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Effect of UPP Inhibitors on Meiotic Resumption, MAPK/ p90rsk Phosphorylation, and Cyclin B1 Synthesis in Oocytes Cultured in HX medium
To characterize the functional roles of the UPP in meiotic resumption, the selective proteasome-inhibitors ALLN and MG-132 were used. As shown in Figures 2 and 3, the GVBD rates of the DOs and CEOs were increased in a dose- (Fig. 2) and time-dependent (Fig. 3) manner by ALLN or MG-132 in HX-maintained meiotic arrest model. After CEOs (Fig. 2A) had been cultured in HX medium for 36 h, the GVBD rate in 50 µM ALLN-treated CEOs (89% ± 2%) or 50 µM MG-132-treated CEOs (86% ± 5%) was significantly higher than in the inhibitor-free group (6% ± 1%). The GVBD rate in 50 µM ALLN-treated DOs (86% ± 3%) or 50 µM MG-132-treated DOs (92% ± 2%) was also significantly higher than in the inhibitor-free group (9% ± 4%) (Fig. 2B).
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We further examined the roles of the UPP and its relationship with MAPK/p90rsk cascade and MPF in HX-maintained meiotic arrest of pig oocytes by using the MAPK kinase (MEK) inhibitor U0126 and the MPF inhibitor roscovitine. As shown in Figure 4, 50 µM U0126 or 30 µM roscovitine can effectively inhibit the stimulation of MG-132 and ALLN on GVBD in both CEOs (Fig. 4A) and DOs (Fig. 4B).
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Effects of UPP Inhibitors on MAPK/p90rsk Phosphorylation and Cyclin B1 Accumulation in Oocytes Cultured in HX Medium
Taking consideration of the stimulation of GVBD by ALLN and MG-132 in pig oocytes, we proposed that ALLN and MG-132 might affect these processes by regulating MAPK/p90rsk and cyclin B1, the regulatory subunit of MPF. Phosphorylation of p90rsk was assessed by examining its electrophoretic mobility shift on SDS-PAGE, and phosphorylation of ERK1/2 was evaluated by both mobility shift and a specific antibody against phospho-ERK1/ 2. As shown in Figures 5 and 6, the phosphorylation of MAPK/p90rsk and the expression level of cyclin B1 in CEOs (Fig. 5A) and DOs (Fig. 5B) were significantly higher in HX medium containing ALLN or MG-132 than in GV-stage oocytes (Fig. 5, lane 1) and in control oocytes (cultured in drug-free HX medium) after 36 h (Fig. 5, lane 2). The MEK inhibitor U0126 (Fig. 5, lanes 5 and 6) and the MPF inhibitor roscovitine (Fig. 5, lanes 7 and 8) completely inhibited the phosphorylation of MAPK/p90rsk and the accumulation of cyclin B1 stimulated by ALLN and MG-132. Furthermore, the phosphorylation of MAPK and the level of cyclin B1 were increased in a dose- (Fig. 6) and time-dependent (Fig. 7) manner in ALLN and MG-132-treated CEOs (Fig. 6, A and A' and Fig. 7, A and A') and DOs (Fig. 6, B and B' and Fig. 7, B and B').
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Subcellular Localization of 20S Proteasome Subunit During Pig Oocyte Parthenogenetic Activation, Fertilization, and Early Embryo Cleavage
The distribution of 20S proteasome subunit during pig oocyte parthenogenetic activation, fertilization, and early embryo development is shown in Figure 8. The 20S proteasome subunit was detected with an intense staining in one pronucleus (resulted from electrical activation) (Fig. 8A), two pronuclei (Fig. 8, B and C), or three pronuclei (Fig. 8D) (resulted from polyspermy during in vitro fertilization). As with the pronuclear envelope breakdown (i.e., syngamy), 20S proteasome subunit in the female and male pronuclei also appeared to be fused (Fig. 8E). At the anaphase of 1-cell embryos, the 20S proteasome subunit concentrated around the separating chromosomes (Fig. 8F). In 2-cell (Fig. 8G) and 3-cell (Fig. 8H) embryos, the staining of 20S proteasome subunit accumulated in the nuclei.
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Effect of UPP Inhibitors on PB2 Emission and Pronuclear Formation During Electrical Activation and In Vitro Fertilization
Before electrical activation or in vitro fertilization, the in vitro-matured, MII-arrested pig oocytes were pretreated in maturation medium containing 50 µM ALLN or 50 µM MG-132 for 30 min. As shown in Figure 9, A and B, ALLN and MG-132 inhibited PB2 emission and pronuclear formation during electrical activation and in vitro fertilization of pig oocytes. The percentage of PB2 emission was 83% ± 1% in electrically activated pig oocytes (Fig. 9A), whereas the percentage of PB2 emission was only 23% ± 2% and 24% ± 1% in 50 µM MG-132 and 50 µM ALLN-treated oocytes, respectively (Fig. 9A). The percentage of pronuclear formation was 83% ± 1% in electrically activated pig oocytes (Fig. 9A), whereas the percentage of pronuclear formation was only 26% ± 3% and 23% ± 2%, respectively, in 50 µM MG-132 and 50 µM ALLN-treated oocytes (Fig. 9A). The results from in vitro fertilization were similar to those in electrical activation of pig oocytes (Fig. 9B).
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Regulation of UPP Inhibitors on MAPK/P90rsk Phosphorylation and Cyclin B1 Accumulation During Electrical Activation and In Vitro Fertilization of Pig Oocytes
Before electrical activation or in vitro fertilization, the in vitro-matured, MII-arrested pig oocytes were first pretreated for 30 min in maturation medium containing 50 µM ALLN or 50 µM MG-132. As shown in Figure 9C, lane 1, cyclin B1 could be detected in the MII-arrested oocytes, whereas this protein could not be detected at 2 h after electrical activation (Fig. 9C, left, lane 2) or at 6 h (Fig. 9C, right, lane 2) after insemination. However, when the UPP was inhibited by 50 µM MG-132 (Fig. 9C, lane 3) or 50 µM ALLN (Fig. 9C, lane 4), the expression of cyclin B could still be detected at 2 h (Fig. 9C, lanes 3 and 4) after electrical activation or at 6 h (Fig. 9C, lanes 3 and 4) after insemination. Furthermore, as shown in Fig. 9D, the MAPK and p90rsk were kept phosphorylated at 0–10 h (Fig. 9D, lanes 1–3) after electrical activation or insemination (Fig. 9D, lanes 1–3) but were completely dephosphorylated at 14–20 h after electrical activation (Fig. 9D, lanes 4 and 5) or insemination (Fig. 9D, lanes 4 and 5). The contents of both kinases remained stable during the whole process of parthenogenetic activation or fertilization. In oocytes treated with 50 µM MG-132 ALLN (Fig. 9D, lanes 6 and 7) or 50 µM ALLN (Figs. 9D, lanes 8 and 9), the phosphorylation of MAPK and p90rsk could still be detected at 14 or 20 h after electrical activation (Fig. 9D, lanes 6–9) or insemination (Fig. 9D, lanes 6–9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we investigated the roles of the UPP in meiosis resumption and its regulation on MAPK/p90rsk cascade and MPF in pig oocytes meiotic arrest in HX medium. Germinal vesicle breakdown was inhibited by HX [36], whereas ALLN or MG-132 could overcome this inhibitory effect and induce GVBD in a dose- and time-dependent manner in pig CEOs and DOs. In lower eukaryotes, such as in toad and starfish oocytes, the inhibition of proteasomal proteolytic activity prevented GVBD, and potent inhibition of GVBD was also observed by microinjection of antiproteasome subunit antibodies [21]. The antibody-injected starfish oocytes failed to activate pre-MPF, because the dephosphorylation of phospho-Tyr15 in Cdc2 kinase was not observed even in the presence of 1-methyadenine, a maturation-inducing hormone in starfish [21]. We recently found that inhibition of the UPP also blocked GVBD of pig oocytes cultured in NCSU-37 medium that contains follicular fluid [33]. Others reported that the proteasome-inhibitors lactacystin and MG-132 did not interfere with GVBD of rat oocytes [31], but those authors did not show if the inhibitors could stimulate GVBD. These different results may be caused by those authors using the spontaneous maturation model whereas we employed the HX-maintained meiotic arrest model, which more closely resembles what happens in vivo. The accumulation of 20S proteasome subunits in the GV of pig oocytes, as revealed in the present study, provides further evidence for the possibility that the UPP is involved in GVBD. Thus, the inhibition of protein degradation mediated by the UPP may not inhibit GVBD. On the contrary, the UPP may be active in maintaining the meiotic arrest at the GV stage, and inhibition of the UPP stimulates GVBD in pig oocytes under physiological conditions.
Many studies have implied that MAPK/p90rsk cascade and MPF are involved in initiating resumption of the meiotic arrest in mammals [3, 7, 37–39], but other studies have shown independent activation of MAPK and MPF during initiation of meiotic maturation in pig oocytes [40]. These contrary results may be caused by the different experimental models employed. It can be proposed that the stimulation of GVBD by ALLN and MG-132 could be related to activation of the MAPK/p90rsk cascade and MPF. Our results showed that phosphorylation of MAPK and p90rsk and expression of cyclin B1 were increased in a dose- and time-dependent manner by ALLN and MG-132 treatment of pig DOs and CEOs in HX medium. Furthermore, the MEK inhibitor U0126 or MPF inhibitor roscovitine completely overcame the stimulation of GVBD induced by ALLN or MG-132, and the phosphorylation of MAPK and p90rsk as well as the increase of cyclin B1 were also completely inhibited by U0126 and roscovitine. These results imply that the activation of MAPK/p90rsk cascade and MPF may be essential for the stimulation of GVBD by ALLN and MG-132, which are also consistent with other reports that the MEK-inhibitor PD 98059 prevents resumption of nuclear maturation in the majority of pig CEOs [39]. The stimulation of ERK and p90rsk phosphorylation by UPP inhibition in mammalian oocytes is, to our knowledge, reported for the first time in the present study, and it might be mediated by MPF (Cdc2/cyclin B complex). It has been well accepted that MPF and MAPK facilitate the activation of each other during oocyte maturation in vertebrates (for a recent review, see Fan and Sun [7]). Inhibition of the UPP pathway strengthened the accumulation of cyclin B, which led to the acceleration of Cdc2, MAPK, and p90rsk activation sequentially. However, other studies have also implied that although MAPK was activated around the same time as GVBD and MPF activation in porcine oocytes [41], MAPK activation was not required for GVBD induction in porcine oocytes and that the major roles of MAPK during porcine oocyte maturation are to promote GVBD by increasing MPF activity [37]. Overall, our results suggest that inhibition of the UPP by ALLN and MG-132 could promote the phosphorylation of MAPK/p90rsk via some unknown mechanisms and prevent the degradation of cyclin B1 and, subsequently, that it results in the MPF activation that is essential for inducing GVBD in pig oocytes.
In Figure 2, we noticed that 2 µM MG-132 caused approximately 60% stimulation of GVBD in CEOs; however, this concentration showed very little effect on ERK phosphorylation (Fig. 6). On the other hand, 2 µM ALLN, which enhanced GVBD by 15% only (Fig. 2), showed higher pERK than 2 µM MG-132. The Western blot results were not quantitative, so we cannot expect the density of bands to be comparable to the GVBD rate. In addition, whether a relationship exists between MAPK phosphorylation in oocytes and occurrence of GVBD is not clear (we are investigating this problem by oocyte GV centrifugation and RNA interference). Overall, in both ALLN and MG-132 treatments, the ERK phosphorylation was apparently stimulated after drug treatment in comparison with the drug-free control group. Furthermore, we only detected the phosphorylation of MAPK in oocytes, not in the cumulus cell. We think the contradiction may be related to the phosphorylation of MAPK in cumulus cells, because some reports have implied that the phosphorylation of MAPK in cumulus cell regulates oocyte GVBD [3]. This hypothesis (not included in the present study) is being further examined in our laboratory.
In Figure 6, it seems that cyclin B1 is much less stimulated by ALLN in comparison to MG-132 in DOs; however, the two inhibitors showed the same stimulation of GVBD. The principal aim of these experiments was to compare the meiotic maturation of pig oocytes with or without UPP activity, so the effects of two inhibitors of the UPP, ALLN and MG-132, are only compared with the drug-free control group instead of with each other.
We also found that the UPP may be involved in metaphase-anaphase transition, because the oocytes were arrested at metaphase I after treatment with ALLN or MG-132 for 36 h (56%, n = 158). This result was similar to that obtained in rat oocytes, in which MG-132 caused the meiotic arrest at metaphase I [31]. Moreover, the accumulation of 20S proteasome subunits between the separating chromosomes at anaphase I and in the midbody between the PB1 and the oocyte at telophase I also suggested involvement of the UPP in metaphase-anaphase transition and the polar body emission.
The MII arrest of mammalian oocytes is maintained by the activity of cytostatic factor, which may include MPF and the MAPK/p90rsk cascade [42]. After mammalian oocyte activation, cyclin B1 is quickly degraded; thus, MPF is inactivated by a calcium-induced and APC/C-dependent degradation pathway, which is the prerequisite for the metaphase-anaphase transition [43]. However, the phosphorylation and expression levels of MAPK/p90rsk remain stable after oocyte activation until the initiation of pronuclear formation [6, 7]. Our previous study found that UPP inhibition blocked sperm penetration into oocytes. When oocytes were transferred to UPP inhibitor-containing medium soon after sperm penetration, to our surprise, PB2 emission and pronuclear formation occurred, probably because the protein ubiquitination and degradation happens so fast after sperm penetration and, once this occurs, fertilization events are not affected by the inhibitor [33]. In this experiment, we pretreated the oocytes with UPP inhibitors before insemination. The MII-arrested oocyte activation was inhibited by ALLN or MG-132, as indicated by inhibition of PB2 emission and pronuclear formation, suggesting the possibility that the UPP may become active very quickly as a result of pig oocyte activation, which in turn serves to mediate the degradation of cyclin B1 and, thus, the inactivation of MPF and, indirectly, MAPK/p90rsk dephosphorylation. Our Western blot results showed that the phosphorylation of MAPK and p90rsk was maintained until 20 h after activation and that the expression of cyclin B1 could also be detected at 2 h after electrical activation or at 6 h after insemination in oocytes cultured in NCSU-23 medium containing proteasome inhibitor, strongly suggesting that the inhibition of the degradation of some proteins by ALLN or MG-132, especially cyclin B1, may be important for maintaining the activity of MPF and the phosphorylation of MAPK and p90rsk and, eventually, for maintaining the MII arrest of pig oocytes.
The accumulation of 20S proteasome subunit in pronucleus suggests that this localization is vital for the pronuclear functions. Others have reported that the proteasome-inhibitor MG-132 could alter the orderly progression of DNA synthesis during the S phase in HeLa cells and lead to replication of DNA, suggesting the existence of proteasome-dependent mechanisms regulating the orderly progression of the S phase [44]. Moreover, the accumulation of 20S proteasome subunits in the region between the separating chromosomes at anaphase of 1-cell embryo and the nucleus of 2- and 3-cell embryos also suggests that the UPP may be important in regulation of the early embryonic mitosis.
Taken together, our results suggest that the UPP regulates MPF and MAPK/p90rsk cascade and is involved in multiple steps of pig oocyte meiotic maturation, activation, and early embryonic mitosis, including the initiation of meiotic resumption, metaphase-anaphase transition, polar body emission, release of MII arrest, and early embryonic development.
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FOOTNOTES |
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2 Correspondence: Qing-Yuan Sun, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China. FAX: 8610 6256 5689; sunqy1{at}yahoo.com
Received: 2 February 2004.
First decision: 19 February 2004.
Accepted: 19 April 2004.
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