Mika Tokumoto

Ocular and Cerebellar Defects in Zebrafish Induced by Overexpression of the LIM Domains of the Islet-3 LIM/Homeodomain Protein

Islet-3 is an LIM/homeodomain protein that is expressed specifically in the eyes and the presumptive tectum in the central nervous system of zebrafish (Danio rerio) embryos. Overexpression of the protein (LIM(Isl-3)) consisting only of the Islet-3 LIM domains in embryos specifically prevented formation of the optic vesicles; caused abnormal termination of the expression of wnt1, engrailed2, and pax2 in the mesencephalic and metencephalic region between 14 hr and 20 hr postfertilization; and severely impaired morphogenetic movement in this region between 20 hr and 26 hr, which should normally lead to formation of the cerebellar primordium. Such defects were all rescued by simultaneous overexpression of Islet-3, suggesting that LIM(Isl-3) acted as a specific dominant-negative variant of Islet-3. These data, combined with the results of mosaic analyses, suggest that Islet-3 is activated by putative LIM-binding cofactors and functions to promote evagination of the optic vesicles and to maintain reciprocal interaction between the mesencephalon and the mesencephalic-metencephalic boundary essential for normal development of this region.

Induction and inhibition of oocyte maturation by EDCs in zebrafish

BackgroundOocyte 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.MethodsIn 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.ResultsAmong 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.ConclusionThese results suggest that EDCs act as agonists or antagonists in the induction of oocyte maturation in fish.

In Vivo Induction of Oocyte Maturation and Ovulation in Zebrafish

The maturation of fish oocytes is a well-characterized system induced by progestins via non-genomic actions. In a previous study, we demonstrated that diethylstilbestrol (DES), a non-steroidal estrogen, induces fish oocyte maturation via the membrane progestin receptor (mPR). Here, we attempted to evaluate the effect of DES as an environmental endocrine disrupting chemical (EDC) upon fish oocyte maturation using live zebrafish. DES triggered oocyte maturation within several hours in vivo when administrated directly into the surrounding water. The natural teleost maturation-inducing hormone, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20beta-DHP) also induced oocyte maturation in vivo. Steroids such as testosterone, progesterone or 17alpha-hydroxyprogesterone were also effective in vivo. Further studies indicated that externally applied 17,20beta-DHP even induced ovulation. In contrast to 17,20beta -DHP, DES induced maturation but not ovulation. Theoretically this assay system provides a means to distinguish pathways involved in the induction of ovulation, which are known to be induced by genomic actions from the pathway normally involved in the induction of oocyte maturation, a typical non-genomic action-dependent pathway. In summary, we have demonstrated the effect of EDCs on fish oocyte maturation in vivo. To address the effects, we have explored a conceptually new approach to distinguish between the genomic and non-genomic actions induced by steroids. The assay can be applied to screens of progestin-like effects upon oocyte maturation and ovulation for small molecules of pharmacological agents or EDCs.

Identification of the Xenopus 20S proteasome α4 subunit which is modified in the meiotic cell cycle

The proteasomes are large, multi-subunit particles that act as the proteolytic machinery for most of the regulated intracellular protein degradation in eukaryotic cells. To investigate the regulatory mechanism for the 26S proteasome in cell-cycle events, we purified this proteasome from immature and mature oocytes, and compared its subunits. Immunoblot analysis of 26S proteasomes showed a difference in the subunit of the 20S proteasome. A monoclonal antibody, GC3beta, cross-reacted with two bands in the 26S proteasome from immature oocytes (in G2-phase); however, the upper band was absent in the 26S proteasome from mature oocytes (in M-phase). These results suggest that changes in the subunits of 26S proteasomes are involved in the regulation of the meiotic cell cycle. Here we describe the molecular cloning of one of the alpha subunits of the 20S proteasome from a Xenopus ovarian cDNA library using an anti-GC3beta monoclonal antibody. From the screening, two types of cDNA are obtained, one 856bp, the other 984bp long. The deduced amino-acid sequences comprise 247 and 248 residues, respectively. These deduced amino-acid sequences are highly homologous to those of alpha4 subunits of other vertebrates. Phosphatase treatment of 26S proteasome revealed the upper band to be a phosphorylated form of the lower band. These results suggest that a part of the alpha4 subunit of the Xenopus 20S proteasome, alpha4_xl, is phosphorylated in G2-phase and dephosphorylated in M-phase.

A major substrate for MPF: cDNA cloning and expression of polypeptide chain elongation factor 1γ from goldfish (Carassius auratus)

One of the eukaryotic polypeptide chain elongation factors, EF-1 g n i complex, is involved in polypeptide chain elongation via the GDP/GTP exchange activity of EF-1 f. In the complex, EF-1 n has been reported to be a major substrate for maturation promoting factor (MPF). Here, we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, EF-1 n from the goldfish ovary. The cloned cDNA was 1490 u bp in length and encoded 442 amino acids. The deduced amino acid sequence was highly homologous to EF-1 n from other species. Although, the phosphorylation site identified in Xenopus EF-1 n was not conserved in the goldfish homologue, phosphorylation analysis showed that the goldfish EF-1 n was phosphorylated by MPF. We concluded that EF-1 n is a substrate for MPF during oocyte maturation in goldfish.

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

BackgroundDuring 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.Resultsp48 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.Conclusionp48 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 a 20S Proteasome α2 Subunit from Goldfish (Carassius auratus) and Its Expression Analysis

Abstract Proteasomes are large, multisubunit particles that act as the proteolytic machinery for most regulated intracellular protein breakdown in eukaryotic cells. The core proteinase of this complex, the 20S proteasome, is comprised of four stacked rings with seven subunits each. The outer two rings are made up of seven, distinct α-type subunits, while the two inner rings are composed of seven, different β-type subunits. Here we present the cloning, sequencing and expression analysis of Carassius auratus, α2_ca, which encodes one of the proteasome α subunits from goldfish ovary. The cloned cDNA is 838 bp long and encodes 234 amino acids. The deduced amino acid sequence is highly homologous to α2 subunits from other vertebrates. The expression of mRNA for α2_ca occurs at very high levels in ovary and muscle and moderately high levels in testis, brain and gill. It was also shown that protein content was different from mRNA expression levels.

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

BackgroundThe 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.ResultsWe 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).ConclusionsSix 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.

Molecular cloning of cDNA encoding a cyclin-selective ubiquitin carrier protein (E2-C) from Carassius auratus (goldfish) and expression analysis of the cloned gene

Destruction of cyclin B is required for exit from mitosis and meiosis. A cyclin‐specific ubiquitinating system, including cyclin‐selective ubiquitin carrier protein (E2‐C), is thought to be responsible for cyclin B destruction. Here we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, E2‐C which encodes the cyclin‐selective ubiquitin carrier protein from goldfish ovary. The cloned cDNA is 677 bp long and encodes 172 amino acids. The deduced amino acid sequence is highly homologous to E2‐C from other species. Recombinant goldfish E2‐C possesses ubiquitinating activity against cyclin B. The expression of mRNA for E2‐C was similar to that of mRNA for cyclin B, occurring at very high level in the ovary. The similarity of the expression pattern of E2‐C and cyclin B suggests that E2‐C mediates a cyclin‐specific ubiquitination.

Sonophotocatalysis of endocrine disrupting chemicals

Sonolysis and photolysis often exhibit synergistic effects in the degradation of organic molecules. An assay of fish oocyte maturation provides an appropriate experimental system to investigate the hormonal activities of chemical agents. Oocyte maturation in fish is triggered by maturation-inducing hormone (MIH), which acts on receptors on the oocyte surface. A synthetic estrogen, diethylstilbestrol (DES), possesses inducing activity of fish oocyte maturation, and a widely used biocide, pentachlorophenol (PCP), exhibits a potent inhibitory effect on fish oocyte maturation. In this study, the effects of the combined treatment by sonolysis with photolysis (sonophotocatalysis) to diminish the hormonal activity of DES and the maturation preventing activity of PCP was examined. By sonophotocatalysis, hormonal activity of DES was completely lost within 30 min and the inhibiting activity of PCP was lost within 120 min. These results demonstrated that sonophotocatalysis is effective for diminishing the endocrine-disrupting activity of chemical agents.