Properties of gene knockdown system by vector-based siRNA in zebrafish.

RNA interference (RNAi) has emerged as a powerful tool to silence specific genes. Vector-based RNAi systems have been developed to downregulate targeted genes in a spatially and temporally regulated fashion both in vitro and in vivo. The zebrafish (Danio rerio) is a model animal that has been examined based on a wide variety of biological techniques, including embryonic manipulations, forward and reverse genetics, and molecular biology. However, a heritable and tissue-specific knockdown of gene expression has not yet been developed in zebrafish. We examined two types of vector, which produce small interfering RNA (siRNA), the direct effector in RNAi system; microRNA (miRNA) process mimicking vectors with a promoter for RNA polymerase II and short hairpin RNA (shRNA) expressing vector through a promoter for RNA polymerase III. Though gene-silencing phenotypes were not observed in the miRNA process mimicking vectors, the transgenic embryos of the second vector (Tg(zU6-shGFP)), shRNA expressing vector for enhanced green fluorescence protein, revealed knockdown of the targeted gene. Interestingly, only the embryos from Tg(zU6-shGFP) female but not from the male fish showed the downregulation. Comparison of the quantity of siRNA produced by each vector indicates that the vectors tested here induced siRNA, but at low levels barely sufficient to silence the targeted gene.

Characterization of multiple membrane progestin receptor (mPR) subtypes from the goldfish ovary and their roles in the induction of oocyte maturation.

Oocyte maturation (OM) in goldfish is induced by the maturation inducing hormone (MIH) via its membrane receptor. Previously, we described the cloning of the membrane progesterone receptor alpha (mPRα or paqr7b) cDNA from a goldfish ovarian cDNA library and obtained experimental evidence that the mPRα protein is an intermediary in MIH induction of OM in goldfish. Three mPR subtypes have been identified in fish by cDNA cloning or by in silico analysis of genome sequence databases. In order to investigate the potential roles of the mPR subtypes in oocyte maturation, we cloned additional mPRs from a goldfish ovarian cDNA library. RACE amplification, and screening of the cDNA library identified one ß (paqr8) and two γ subtypes (paqr5) (hereafter referred to as γ-1 and γ-2), respectively. Tissue distribution of mPR subtypes showed differential expression pattern. However, in addition to mPRα, the ß, γ-1 and γ-2 subtypes were also expressed in follicle-enclosed oocytes. Cell lines expressing the ß, γ-1 and γ-2 genes were established and their steroid binding properties compared. The ß subtype exhibited higher binding affinity than the γ subtypes for 17,20ß-DHP, the MIH in goldfish. Microinjection of goldfish oocytes with a morpholino antisense oligonucleotide to mPRß blocked the induction of oocyte maturational competence, whereas injection of antisense oliogonucleotides to mPRγ-1 and γ-2 were ineffective. These results suggest that the goldfish mPRß protein acts as an intermediary during MIH induction of OM in goldfish, in a manner similar to that described previously for mPRα.

Two-Step Mechanism of Cyclin B Degradation Initiated by Proteolytic Cleavage with the 26 S Proteasome in Fish.

To complete meiosis II, cyclin B is degraded in a short period by the inactivation of M-phase promoting factor (MPF). Previously, we showed that the destruction of cyclin B was initiated by the ubiquitin-independent proteolytic activity of the 26 S proteasome through an initial cut in the N-terminus of cyclin (at K57 in the case of goldfish cyclin B). We hypothesized that this cut allows cyclin to be ubiquitinated for further destruction by the ubiquitin-dependent proteolytic pathway, which leads to MPF inactivation. In this study, we aimed to identify the ubiquitination site for further degradation. The destruction of cyclin B point mutants in which lysine residues in a lysine-rich stretch following the cut site of cyclin B had been mutated was analyzed. All the lysine point mutants except K57R (a point mutant in which K57 was substituted with arginine) were susceptible to proteolytic cleavage by the 26 S proteasome. However, the degradation of the K77R and K7677R mutants in Xenopus egg extracts was significantly slower than the degradation of other mutants, and a 42 kDa truncated form of cyclin B was detected during the onset of the degradation of these mutants. The truncated form of recombinant cyclin B, an N-terminal truncated cyclin BΔ57 produced as cut by the 26 S proteasome, was not further cleaved by the 26 S proteasome but rather degraded in Xenopus egg extracts. The injection of the K57R, K77R and K7677R cyclin B proteins stopped cleavage in Xenopus embryos. From the results of a series of experiments, we concluded that cyclin B degradation involves a two-step mechanism initiated by initial ubiquitin-independent cleavage by the 26 S proteasome at lysine 57 followed by its ubiquitin-dependent destruction by the 26 S proteasome following ubiquitination at lysine 77.

Nodal/Bozozok-independent induction of the dorsal organizer by zebrafish cell lines.

Formation of the dorsal organizer (Spemann organizer) is an important process in early vertebrate development. In zebrafish, two molecular cascades--Bozozok/Dharma (Boz) and Nodal signaling--act in parallel to induce the dorsal organizer. However, the complete molecular mechanism regulating this event remains unclear. Here we report that zebrafish cell lines derived from various developmental stages can induce a secondary axis when they are implanted into the mid-blastula but not the early gastrula. The implanted cells themselves did not differentiate, but instead induced ectopic expression of dorsal organizer markers in cells around the implanted cells and induced notochord formation in the secondary axis. These results indicate that cultured cell lines have the ability to induce a secondary axis through the initiation of dorsal organizer activity. However, ectopic expression of boz and sqt were not observed in cultured cells. In addition, implanted cell lines could induce the dorsal organizer even in maternal-zygotic one-eyed pinhead mutants, which are not responsive to Nodal signaling. Finally, the Nodal signaling pathway was not activated following implantation of cultured cells. Collectively, these data suggest that zebrafish cell lines induce the dorsal organizer independent of the boz and Nodal signaling pathways.

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.

Interactions of diethylstilbestrol (DES) and DES analogs with membrane progestin receptor-alpha and the correlation with their nongenomic progestin activities.

Progestin induction of oocyte maturation (OM) in fish is a useful model for investigating endocrine disruption of nongenomic steroid actions. Although diethylstilbestrol (DES) analogs have been shown to mimic the actions of progestins to induce meiotic maturation of goldfish and zebrafish oocytes, their molecular mechanisms of action remain unclear. The ability of these endocrine-disrupting chemicals (EDCs) to interact with the progestin receptor mediating OM was investigated in receptor binding assays using plasma membranes from goldfish ovaries and breast cancer cells transfected with goldfish membrane progestin receptor (mPR)-alpha. Membranes prepared from both ovaries and mPRalpha-transfected cells showed high-affinity, saturable, displaceable, single binding sites specific for the goldfish maturation-inducing steroid, 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-DHP). DES and DES analogs (dipropionate-DES and hexestrol), which induce OM in goldfish, bound to the receptor and caused concentration-dependent displacement of [3H]-17,20beta-DHP, whereas dimethyl ether-DES had no affinity for the receptor. Scatchard plot analysis of specific 17,20beta-DHP binding in the presence of different amounts of DES showed that DES binding is of the noncompetitive type. The activities of DES and DES analogs to induce meiotic maturation of goldfish oocytes were examined in an in vitro bioassay. Whereas a concentration-dependent induction of OM was observed in response to DES, dipropionate-DES, and hexestrol, dimethyl ether-DES did not show any OM-inducing activity. The close correspondence between binding of DES and its analogs to the mPRalpha protein and their OM-inducing activities suggests a mechanism of endocrine disruption mediated by binding to mPRalpha resulting in its activation, thereby mimicking the nongenomic action of the progestin 17,20beta-DHP.

Molecular cloning and expression of cDNA coding for four spliced isoforms of casein kinase Ialpha in goldfish oocytes.

Casein kinase I (CKI) is a member of the serine/threonine protein kinases and located in a separate group within the superfamily of eukaryotic protein kinases. CKI isoforms regulate several checkpoints of the cell cycle and meiosis. In higher eukaryotes, CKIalpha has four isoforms produced through the alternative splicing of two short inserts. Here, we report the cloning, sequencing and expression of four alternatively spliced isoforms of CKIalpha from goldfish ovary. The cloned cDNAs were 2099-3002-bp long and classified as CKIalpha, CKIalphaS, CKIalphaL and CKIalphaLS. It was revealed that two major (3.0 and 2.0 kb) messages were strongly expressed in the ovary. Four isoforms are expressed in previtellogenic to vitellogenic oocytes. In the huge nucleus of the oocyte, referred to as the germinal vesicle, CKIalphaS is dominant and CKIalphaL is expressed at a detectable level. Immunoblot analysis revealed that CKIalpha and CKIalphaS are major products in both immature and mature oocytes. These two isoforms were expressed in a tissue-dependent manner.

Identification of a protein kinase which phosphorylates a subunit of the 26S proteasome and changes in its activity during meiotic cell cycle in goldfish oocytes.

The proteasome is involved in the progression of the meiotic cell cycle in fish oocytes. We reported that the alpha4 subunit of the 26S proteasome, which is a component of the outer rings of the 20S proteasome, is phosphorylated in immature oocytes and dephosphorylated in mature oocytes. To investigate the role of the phosphorylation, we purified the protein kinase from immature oocytes using a recombinant alpha4 subunit as substrate. A protein band which well corresponded to the kinase activity was identified as casein kinase Ialpha (CKIalpha). Two-dimensional (2D) PAGE analysis showed that part of the alpha4 subunit was phosphorylated by CKIalpha in vitro. This spot was detected in purified immature 26S proteasome but not in mature 26S proteasome, demonstrate that the alpha4 subunit is phosphorylated by CKIalpha meiotic cell cycle dependently.

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.

Destruction of cyclin B is required for exit from mitosis and meiosis. A cyclin-degrading system, including anaphase-promoting-complex/cyclosome (APC/C), has been shown to be responsible for cyclin B destruction. Here we present the cloning, sequencing, and expression analysis of goldfish (Carassius auratus) APC11, which encodes the catalytic component of APC/C from goldfish ovary. The cloned cDNA is 348 bp long and encodes 88 amino acids. The deduced amino acid sequence is highly homologous to APC11 from other species. The expression of mRNA for APC11 was ubiquitous among tissues, as opposed to that of mRNA for E2-C, which occurred at a very high level in the ovary. Recombinant goldfish APC11 possesses ubiquitinating activity against cyclin B. We established an in vitro ubiquitinating system of proteins composed of purified recombinant E1, E2-C, and APC11 from goldfish. The reconstructed system for these ubiquitinating enzymes makes it feasible to elucidate the molecular mechanism of cyclin B degradation.

Generation of Transparent Zebrafish with Fluorescent Ovaries: A Living Visible Model for Reproductive Biology.

The transparent zebrafish enables researchers to study the morphology and distribution of cells and tissues in vivo. To capture the dynamic processes of germ cell proliferation and juvenile ovarian development in zebrafish in vivo, we established transgenic (TG) lines to allow us to monitor the changes in the ovaries of living fish. The original transgenic line with ovarian fluorescence was occasionally established. Although the cDNA integrated in the strain was constructed for the expression of enhanced green fluorescent protein (EGFP) driven by the medaka ß-actin promoter, expression of EGFP is restricted to the oocytes and gills in adult fish. Mutant strains with transparent bodies, roy and ruby, were isolated in zebrafish. In this study, we crossed the TG strain with fluorescent ovary with transparent strains and established the TG (ß-actin:EGFP);ruby strain. The strain is highly transparent, and the oocytes are easily observed in living fish. We identified a fluorescent tissue that might contain the undifferentiated germ cells close to the cloaca in the strain. This strain can be used for analysis of ovarian development in vivo.

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.

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-1gamma and delta 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-1beta. To identify the p48 complex bound to the 26S proteasome as EF-1betagammadelta, antibodies were raised against the components of purified p48 complex. Recombinant EF-1 beta,gamma and delta were expressed in Escherichia coli, and an antibody was raised against purified recombinant EF-1gamma. 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-1gamma was shown to bind to the 26S proteasome. When EF-1gamma is phosphorylated by MPF, the association is stabilized. CONCLUSION: p48 bound to the 26S proteasome is identified as the EF-1gamma. EF-1 complex is associated with the 26S proteasome in Xenopus oocytes and the interaction is stabilized by MPF-mediated phosphorylation.

Identification of alpha-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 alpha4 subunit of the 26S proteasome. In this study, we analyzed the changes in the spotting pattern separated by 2-D gel electrophoresis of alpha subunits during Xenopus oocyte maturation. RESULTS: We identified cDNA for three alpha-type subunits (alpha1, alpha5 and alpha6) of Xenopus, then prepared antibodies specific for five subunits (alpha1, alpha3, alpha5, alpha6, and alpha7). With these antibodies and previously described monoclonal antibodies for subunits alpha2 and alpha4, modifications to all alpha-type subunits of the 26S proteasome during Xenopus meiotic maturation were examined by 2D-PAGE. More than one spot for all subunits except alpha7 was identified. Immunoblot analysis of 26S proteasomes purified from immature and mature oocytes showed a difference in the blots of alpha2 and alpha4, with an additional spot detected in the 26S proteasome from immature oocytes (in G2-phase). CONCLUSIONS: Six of alpha-type subunits of the Xenopus 26S proteasome are modified in Xenopus immature oocytes and two subunits (alpha2 and alpha4) are modified meiotic cell cycle-dependently.

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

One of the eukaryotic polypeptide chain elongation factors, EF-1 beta gamma delta complex, is involved in polypeptide chain elongation via the GDP/GTP exchange activity of EF-1 alpha. In the complex, EF-1 gamma 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 gamma from the goldfish ovary. The cloned cDNA was 1490 bp in length and encoded 442 amino acids. The deduced amino acid sequence was highly homologous to EF-1 gamma from other species. Although, the phosphorylation site identified in Xenopus EF-1 gamma was not conserved in the goldfish homologue, phosphorylation analysis showed that the goldfish EF-1 gamma was phosphorylated by MPF. We concluded that EF-1 gamma is a substrate for MPF during oocyte maturation in goldfish.

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.

Cloning and identification of a membrane progestin receptor in goldfish ovaries and evidence it is an intermediary in oocyte meiotic maturation.

Previously, a cDNA clone encoding a protein that satisfies the criteria for its designation as a membrane progestin receptor, mPRalpha, was discovered in spotted seatrout ovaries. Moreover, preliminary evidence was obtained for a role for mPRalpha in maturation-inducing hormone (MIH) induction of oocyte maturation in this species. Here, we describe the cloning of the mPRalpha cDNA from a goldfish ovarian cDNA library. Northern blot analysis indicates the presence of a major 2.6kb transcript in ovaries that encodes a 354 amino acid protein which shows high sequence identity with seatrout (81%), zebrafish (93%), and human (55%) mPRalphas. Western blot analysis using a polyclonal goldfish mPRalpha antibody shows a major immunoreactive band of the predicted molecular weight (40kDa) in goldfish ovarian membranes. Computer modeling predicts that the deduced protein has seven transmembrane domains, typical of G protein-coupled receptors. Treatment of full grown, late vitellogenic stage follicle-enclosed oocytes in vitro with gonadotropin increased mPRalpha protein levels. A correlation between mPRalpha protein levels and the ability of oocytes to undergo GVBD in response to the MIH (maturational competence) was observed after treatment with gonadotropin. Microinjection of goldfish oocytes with a morpholino antisense oligonucleotide to mPRalpha blocked both the induction of oocyte maturational competence and mPRalpha protein upregulation by gonadotropin. These results with the goldfish mPRalpha protein are similar to those obtained previously with spotted seatrout, further supporting the hypothesis that the mPRalpha acts as an intermediary in MIH induction of oocyte maturation in teleosts.

Diethylstilbestrol induces fish oocyte maturation.

An endocrine-disrupting chemical, diethylstilbestrol (DES), a nonsteroidal estrogen, triggers oocyte maturation in fish. The morphology (the time course of the change in germinal vesicle breakdown) and an intracellular molecular event (the de novo synthesis of cyclin B) induced by DES were indistinguishable from those induced by a natural maturation-inducing hormone, 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-DHP). A synergistic action of DES on 17,20beta-DHP-induced oocyte maturation was observed. Both 17,20beta-DHP- and DES-induced oocyte maturation was inhibited by an antibody against the maturation-inducing hormone receptor. The structural requirement for the action of DES is discussed based on results obtained with DES analogs.