Diisopropyl fluorophosphate (DFP), a serine protease inhibitor, inhibited the activity of 26S proteasome in goldfish oocytes and arrested the oocytes at two distinct stages of maturation. One was prior to the migration of germinal vesicle (GV) toward the animal pole, and the other was between the attachment of GV to the oocyte plasma membrane and GV breakdown (GVBD). The first DFP-sensitive period corresponded to the period during which the activity of proteasomes increased, but maturation-promoting factor (MPF) was still inactive. During the second DFP-sensitive period, MPF was abruptly activated, although the proteasome activity detectable in the oocyte cytosol extracted by ultracentrifugation reached the lowest level during this period. These results suggest the requirement of 26S proteasome for at least two stages of oocyte maturation, the early stage before GV migration and the later stage, including MPF activation immediately before GVBD.
Oocyte maturation is triggered by maturation-inducing hormone (MIH), which acts on the oocyte surface and induces the activation of maturation-promoting factor (MPF) in the oocyte cytoplasm (Nagahama et al., 1995). Although MPF- induced oocyte maturation is insensitive, MIH-induced oocyte maturation is prevented by various protease inhibitors. Protease inhibitors originated in microorganisms such as leupeptin, antipain and chymostatin, block 1-methyladenine-induced oocyte maturation in starfish (Kishimoto et al., 1982). The same series of inhibitors and a synthetic chymotrypsin inhibitor (N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) also block progesterone-induced oocyte maturation in the frog (Guerrier et al., 1977; Ishikawa et al., 1989). These results suggest that protease activity is involved in the early step of oocyte maturation from MIH reception on the oocyte surface to MPF activation in the oocyte cytoplasm.
A non-lysosomal multicatalytic protease named proteasome has been identified in various eukaryotes. Proteasomes possess three distinct proteolytic activities (chymotrypsin-like, trypsin-like and peptidylglutamyl peptidase activities), and are sensitive to serine and thiol protease inhibitors (Hough et al., 1987; Orlowsky, 1990). In frog oocytes, both the proteasome activity and oocyte maturation are inhibited by serine protease inhibitors such as TPCK and diisopropyl fluorophosphate (DFP) (Azuma et al., 1991; Takahashi et al., 1994), suggesting that proteasomes are probably responsible for inducing oocyte maturation. The involvement of proteasomes in oocyte maturation has also been suggested by the inhibition of starfish oocyte maturation with trypsin inhibitors (Takagi Sawada et al., 1989, 1992).
We purified and characterized 20S and 26S proteasomes from goldfish oocyte cytosol (Tokumoto et al., 1995a, b). During the course of that study, we showed that goldfish oocyte cytosol mainly contains 26S proteasome. In the present study, we examined the inhibitory effects of DFP, a proteasome inhibitor, on oocyte maturation in goldfish. We found that DFP arrests oocytes at the stage before the migration of germinal vesicle (GV), when the activity of the 26S proteasome (active form) increases abruptly after MIH exposure. These findings suggest the commitment of 26S proteasome to the early events of goldfish oocyte maturation. We also demonstrated the inhibition of oocyte maturation by DFP applied just before germinal vesicle breakdown (GVBD), and discuss the involvement of 26S proteasome in the later events of oocyte maturation through the activation of MPF.
MATERIALS AND METHODS
Oocyte preparation and in vitro culture
Goldfish were purchased commercially and raised at 15°C. Ovaries were isolated from sacrificed females and placed in fresh goldfish Ringer's solution (125 mM NaCl, 2.4 mM KCl, 0.28 mM MgSO4, 0.89 mM MgCl2, 2.4 mM CaCl2, 2 mM HEPES, 5.6 mM glucose, 100 IU/ml penicillin, 0.2 mg/ml streptomycin, pH 7.5) and washed three times with the same solution. The maturation of full-grown immature oocytes was induced in vitro by incubating ovarian fragments (each containing 20–40 oocytes) in goldfish Ringer's solution containing 1 μg/ml of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β- DP), an MIH of goldfish (Nagahama et al., 1995) at room temperature with gentle agitation. To assess maturation processes, GV was morphologically examined under a binocular microscope after fixing the oocytes in clearing solution (Lessman and Kavumpurath, 1984). Nuclear state (%) at each time points was determined in twenty oocytes.
Preparation of oocyte cytosol
Oocyte cytosol was extracted as follows. Thirty oocytes manually isolated from ovarian fragments were washed in goldfish Ringer's solution. Excess medium was removed, then 150 μl of fresh Ringer's solution was added. The oocytes were crushed by ultracentrifugation (150,000 × g, 30 min, 4°C) and clear supernatant was collected.
Assay of proteasome activity
The activity of the 26S proteasome in oocyte cytosol was measured using the fluorogenic substrate, Suc-LLVY-MCA (Peptide Institute) in the absent SDS, as described previously (Tokumoto and Ishikawa, 1993; Tokumoto et al., 1993).
Purification of 26S proteasome from goldfish oocyte cytosol
The 26S proteasome was purified from goldfish oocyte cytosol as described previously (Tokumoto et al., 1995b).
RESULTS AND DISCUSSION
Changes in GV morphology and proteasome activity during goldfish oocyte maturation
During normal maturation, GV breaks down beneath the animal pole of the oocyte after migrating from the center to the animal pole. Changes in GV during oocyte maturation are classified as follows: GV located at the center of the oocyte (GVI), GV migrating to the animal pole (GVM), GV attached to the oocyte plasma membrane (GVA), and GVBD (Tokumoto et al., 1993).
The activity of the 26S proteasome in the cytosol extracted from goldfish oocytes remarkably changed during oocyte maturation. The proteasome activity increased transiently within 1 hr of exposure to 17α,20β-DP, then gradually decreased to the lowest level at 5 hr thereafter. The oocytes started GV migration after the transient increase in proteasome activity, and completed GVBD, when the proteasome activity reached the lowest level. The 26S proteasome activity elevated again at 8 hr after completion of GVBD (Fig. 1).
Effect of DFP on proteasome activity
The 26S proteasome activity increased transiently after exposure to 17α,20β-DP (Fig. 1). To examine whether this increase is required for inducing oocyte maturation, we tested the effect of the proteasome inhibitor, DFP, on oocyte maturation. Although we showed that DFP inhibits proteasome activity in frog oocytes (Takahashi et al., 1994), the effects of DFP on proteasomes in goldfish oocytes remained unknown. We first examined this point. When goldfish oocytes were externally exposed to DFP, the proteasome activity in the cytosol extracted from the treated oocytes decreased in accordance with the dose (Fig. 2A). In the cytosol fraction from non-DFP treated oocytes incubated with various concentrations of DFP, the proteasome activity was inhibited with similar dose-dependence (Fig. 2B). Furthermore, the 26S proteasome purified from goldfish ovary (Tokumoto et al., 1995b) was inhibited by similar concentrations of DFP to those that inhibited the 26S proteasome activity in the oocytes and in the cytosol (Fig. 2C). The concentration required for 50% inhibition in each experiment ranged between 1 to 2 mM. These results confirmed that DFP permeates the oocyte cytoplasm, where it inhibits 26S proteasome activity.
Effects of DFP on oocyte maturation
Oocytes were incubated with various concentrations of DFP for 1 hr, then with 17α,20β-DP in the absence of DFP. Oocyte maturation was dose-dependently inhibited, being arrested at either GVI or GVA, but never at GVM (Fig. 3). To determine when oocytes are sensitive to DFP, 1 mM DFP was pulse-applied every 1 hr during the incubation, and the state of GV was examined 8 hr after exposure. The inhibitory effect of DFP was detected at two periods, before GVM (1–3 hr after the hormone treatment) and between GVM and GVA (4–5 hr after the hormone treatment) (Fig. 4). The first DFP- sensitive period corresponded to the period when the 26S proteasome activity increased after hormone exposure, whereas it was low during the second DFP-sensitive period (Fig. 1).
Involvement of 26S proteasome in the early phase of oocyte maturation
As found in the oocytes exposed to DFP (Fig. 3), those incubated with 1 mM DFP during the first DFP-sensitive period were arrested at either GVI or GVA (Fig. 4), indicating that the first protease-dependent critical point exists between GVI and GVM and that if oocytes pass this point, they can reach GVA without interruption. Between GVI and GVM, oocytes have relatively high 26S proteasome activity and are sensitive to DFP. Thus, 26S proteasome activity should be necessary for oocytes to pass this early step of maturation, including the GV migration.
Possible involvement of 26S proteasome in the later phase of oocyte maturation
When oocytes were treated with 1 mM DFP at the second DFP-sensitive period, GV continued to migrate toward the animal pole and attached to the oocyte plasma membrane, but did not break down (Fig. 4). This result indicates that the period between GVA and GVBD is also protease-dependent. MPF is abruptly activated during this period (Hirai et al., 1992; Yamashita et al., 1992, 1995; Katsu et al., 1993). Moreover, an injection of active MPF into immature goldfish oocytes induced GVBD without the preceding migration of GV towards the animal pole, resulting in GVBD in the center of the oocytes (Yamashita et al., unpublished observation). This finding indicated that when MPF is precociously activated, GV will be broken down irrespective of its position within the oocyte. Therefore, it is most likely that the point between GVA and GVBD is mainly controlled by MPF.
DFP-induced arrest of oocyte maturation should be caused by the failure to activate MPF. We examined whether or not the 26S proteasome is involved in MPF activation. Since proteasome activity in the extracted cytoplasm is low during this period (Fig. 1), the 26S proteasome may not be positively involved in MPF activation. DFP inhibits various serine proteases, leaving the possibility that proteases other than proteasomes play a role in inducing oocyte maturation during this period via the activation of MPF. However, it should be noted that the proteasome activity referred to in this study was that found in oocyte cytosol extracted by ultracentrifugation. It is uncertain whether or not the level of 26S proteasome activity in intact oocytes is really low during the second DFP- sensitive period. Proteasomes are colocalized in nucleus and cytoplasm (Tanaka et al., 1992) and accumulate into GV before GVBD in amphibian oocytes (Arrige et al., 1988; Gautier et al., 1988). Cell cycle-dependent accumulation of proteasome into the nucleus is also detected in ascidian embryos (Kawahara and Yokosawa, 1992). Therefore, it is likely that the decrease in the 26S proteasome activity in the extracted cytosol before GVBD is due to transportation of the 26S proteasome to GV, which will be easily precipitated by centrifugation. Thus its contents, including proteasomes were not sufficiently extracted. In other words, substantial 26S proteasome activity may be present in GV before GVBD.
In mammalian cultured cells (Pines and Hunter, 1991) and starfish oocytes (Ookata et al., 1992), MPF accumulates into the nucleus just before its breakdown. Proteasomes also accumulate in the nucleus prior to nuclear envelope breakdown in liver cells (Tanaka et al., 1992) and in amphibian oocytes (Gautier et al., 1988). Although not confirmed in the same organism, the simultaneous accumulation of proteasomes and MPF to GV before GVBD implies their interaction within it, which may be prerequisite for subsequent GVBD. Detailed examinations on the localization and the activity of proteasome during oocyte maturation are required to clarify the involvement of proteasomes in MPF activation.
The level of 26S proteasome activity increases again after GVBD. The timing of the second peak apparently corresponds to that of the first polar body extrusion, between the first and second meiotic metaphase. We reported that in goldfish oocytes, H1 histone kinase (MPF) activity transiently decreased when the first polar body was eliminated (Yamashita et al., 1992). Thus, it is possible that activation of the 26S proteasome activity in this period is responsible for the decrease in MPF activity, probably by inducing the degradation of the regulatory subunit of MPF, cyclin B.
This work was supported by grants from the Saneyoshi Scholarship Foundation (0724 to T.T.), by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan (07283104 to Y.N.), and by a Grant-in-Aid (Bio Media Program) from the Ministry of Agriculture, Forestry and Fisheries (BMP 96-II-2-6). This study was performed under the NIBB Cooperative Research Program (95–112 and 96–120 to T.T.).