decrease in MPF activity during the first polar body extrusion. Meiotic maturation of fish oocytes is triggered by maturation-inducing hormone (MIH), which acts.
Biomedical Research 14 (4) 305-308, 1993
Changes in the activity and protein levels of p_roteasomes during oocyte maturation in goldfish (Carassius auratus)
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TOSHINOBU TOKUMOTO " , MASAKANE. YAMASHITA 2 , MICHIYASU YOSHIKUNI2 and YOSHITAKA NAGAHAMA2 ID6l)Ell“ll'I16I1[ of Molecular Biomechanics, The Graduate University for Advanced Studies, Okazaki 444, and 2Laboratory of Reproductive Biology, Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444, Japan ‘
ABSTRACT
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Changes in the activity and protein levels of proteasomes were determined in_ goldfish oocytes during meiotic maturation induced in vitro by 170:,20,8-dihydroxy-4-pregnen-3-one (l7a,20,6-DP), a naturally occurring maturation-inducing hormone in this species. Both the activity and protein levels of proteasome exhibited two peaks during the l7a,20,8-DP-induced oocyte maturation with a similar pattern of fluctuation. The first peak (a 2-fold increase) occurred before the migration of germinal vesicle (GV) (within l h after l70:,20,6-DP treatment), followed by a gradual decrease towards GV breakdown (GVBD), reaching the lowest level at 6 h. The second peak occurred immediately after GVBD (7 h after the treatment), followed by a sharp decrease at 8 h. The time of the second peak appears to correspond to the time of the first polar body extrusion, a time when maturation-promoting factor (MPF) activity was shown to decrease transiently in goldfish oocytes during the 17a,20,6’-DPinduced meiotic maturation. Thus, it is suggested that proteasomes are involved in the decrease in MPF activity during the first polar body extrusion.
Meiotic maturation of fish oocytes 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 (ll). Previous studies using various protease inhibitors suggest that proteases are involved in the MIH-induced meiotic maturation of frog (3, 5) and starfish (6, 14) oocytes. Recently, a non-lysosomal multicatalytic protease named proteasome has been identified in various eukaryotic sources (l3, 15). Proteasome possesses three distinct proteolytic activities (chymotrypsinlike, trypsin-like and peptidylglutamyl peptidase activities), and are sensitive to serine and thiol protease inhibitors (4, l3). In frog oocytes, serine protease inhibitors have been reported to prevent both the proteasome activity and oocyte maturation (l). It is thus suggested that proteasome is one of the most likely candidates for proteases responsible for the MIH-induced oocyte maturation. In this study, we examined changes in the activity and protein levels of proteasomes during the l7a,20,6’—
dihydroxy-4-pregnen-3-one (l70i,20)6’-DP, a natural MIH in fish)-induced oocyte maturation in vitro in goldfish (12). Oocyte maturation was induced in virro by incubating fragments of ovaries (each contains 20-40 oocytes) in goldfish Ringer’s solution (125 mM NaCl, 2.4 mM KCI, 0.28 mM MgCl2, 2.4 mM CaCl2, 2mM HEPES, 5.6 mM glucose, 100,000 IU/l penicillin, 0.2 g/l streptomycin, pH 7.5) containing lag/ml of l7a,20p’-DP, as described previously (17). Maturation processes were assessed by immersing the oocytes in a clearing solution (9), enabling easy microscopic examination for the presence or absence of a germinal vesicle. In goldfish, like in other vertebrates, the fully grown oocyte possesses a large nucleus (germinal vesicle) in meiotic prophase. The germinal vesicle of this stage is located centrally. The first visible event associated with l7a,20,6-DP-induced final maturation is the migration ofthe germinal vesicle to the animal pole where the micropile is situated; at this stage their germinal vesicle becomes visible
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