Induction and inhibition of oocyte maturation by EDCs in zebrafish

BACKGROUND: Oocyte maturation in lower vertebrates is triggered by maturation-inducing hormone (MIH), which acts on unidentified receptors on the oocyte surface and induces the activation of maturation-promoting factor (MPF) in the oocyte cytoplasm. We previously described the induction of oocyte maturation in fish by an endocrine-disrupting chemical (EDC), diethylstilbestrol (DES), a nonsteroidal estrogen. METHODS: In this study, stimulatory and inhibitory effects of EDCs and natural steroids on oocyte maturation were examined in zebrafish. For effective agents, some details about the mechanism in induction or inhibition of maturation were examined. Possible groups of DES interacting with the MIH receptor are discussed based on relative potency of steroids to induce maturation. RESULTS: Among agents tested, tamoxifen (TAM) and its metabolite 4-hydroxytamoxifen (4-OHT) showed stimulatory activity similar to DES. The time courses of the change in germinal vesicle breakdown and an intracellular molecular event (the synthesis of cyclin B) induced by TAM were indistinguishable from those induced by MIH. In contrast, pentachlorophenol (PCP) had a potent inhibitory effect on MIH-induced oocyte maturation. PCP inhibited not only MIH-induced maturation but also DES- and TAM-induced maturation. Methoxychlor also inhibited maturation when oocytes were pre-treated with this agent. CONCLUSION: These results suggest that EDCs act as agonists or antagonists in the induction of oocyte maturation in fish

Identification of α-type subunits of the Xenopus 20S proteasome and analysis of their changes during the meiotic cell cycle

BACKGROUND: The 26S proteasome is the proteolytic machinery of the ubiquitin-dependent proteolytic system responsible for most of the regulated intracellular protein degradation in eukaryotic cells. Previously, we demonstrated meiotic cell cycle dependent phosphorylation of α4 subunit of the 26S proteasome. In this study, we analyzed the changes in the spotting pattern separated by 2-D gel electrophoresis of α subunits during Xenopus oocyte maturation. RESULTS: We identified cDNA for three α-type subunits (α1, α5 and α6) of Xenopus, then prepared antibodies specific for five subunits (α1, α3, α5, α6, and α7). With these antibodies and previously described monoclonal antibodies for subunits α2 and α4, modifications to all α-type subunits of the 26S proteasome during Xenopus meiotic maturation were examined by 2D-PAGE. More than one spot for all subunits except α7 was identified. Immunoblot analysis of 26S proteasomes purified from immature and mature oocytes showed a difference in the blots of α2 and α4, with an additional spot detected in the 26S proteasome from immature oocytes (in G2-phase). CONCLUSIONS: Six of α-type subunits of the Xenopus 26S proteasome are modified in Xenopus immature oocytes and two subunits (α2 and α4) are modified meiotic cell cycle-dependently

Regulated interaction between polypeptide chain elongation factor-1 complex with the 26S proteasome during Xenopus oocyte maturation

BACKGROUND: During Xenopus oocyte maturation, the amount of a 48 kDa protein detected in the 26S proteasome fraction (p48) decreased markedly during oocyte maturation to the low levels seen in unfertilized eggs. The results indicate that the interaction of at least one protein with the 26S proteasome changes during oocyte maturation and early development. An alteration in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycle, in the early embryo. In this study, we identified p48. RESULTS: p48 was purified by conventional column chromatography. The resulting purified fraction contained two other proteins with molecular masses of 30 (p30) and 37 (p37) kDa. cDNAs encode elongation factor-1γ and δ were obtained by an immuno-screening method using polyclonal antibodies against purified p48 complex, which recognized p48 and p37. N-terminal amino acid sequence analysis of p30 revealed that it was identical to EF-1β. To identify the p48 complex bound to the 26S proteasome as EF-1βγδ, antibodies were raised against the components of purified p48 complex. Recombinant EF-1 β,γ and δ were expressed in Escherichia coli, and an antibody was raised against purified recombinant EF-1γ. Cross-reactivity of the antibodies toward the p48 complex and recombinant proteins showed it to be specific for each component. These results indicate that the p48 complex bound to the 26S proteasome is the EF-1 complex. MPF phosphorylated EF-1γ was shown to bind to the 26S proteasome. When EF-1γ is phosphorylated by MPF, the association is stabilized. CONCLUSION: p48 bound to the 26S proteasome is identified as the EF-1γ. EF-1 complex is associated with the 26S proteasome in Xenopus oocytes and the interaction is stabilized by MPF-mediated phosphorylation

Molecular cloning of cDNA encoding APC11, a catalytic component of anaphase-promoting-complex (APC/C), from goldfish (Carassius auratus) and establishment of in vitro ubiquitinating system

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In Vivo Induction of Oocyte Maturation and Ovulation in Zebrafish

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Involvement of 26S Proteasome in Oocyte Maturation of Goldfish Carassius auratus

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Involvement of 26S Proteasome in Oocyte Maturation of Goldfish Carassius auratus

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