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In studies of sex discrimination in fish in the early 1900s, a specific antigen in the blood of gravid females was identified using immunological methods. At present, this specific antigen is known as vitellogenin, the major precursor of egg yolk protein that is synthesized in the female liver and is secreted into the blood to be incorporated into the egg. Recently, protein and gene analyses have revealed the presence of several vitellogenin variants. In addition, in the 1980s, choriogenin was identified as a novel precursor of egg envelope proteins that is secreted into the blood in response to stimulation by estrogen, similarly to vitellogenin. These two proteins not only play key roles in the process of oogenesis, but they are also used as effective biomarkers for assessing the impact of estrogen-like endocrine-disrupting chemicals (environmental hormones) in aquatic ecosystems. [ABSTRACT FROM AUTHOR]

Abstract: The dispersed fluorescence spectra have been measured for the 7AI–(H2O) n (n =1–3) clusters in a supersonic free jet expansion by exciting various vibronic bands in the S1 state. There has been controversy about the occurrence of the excited-state proton transfer in the n =2,3 clusters. The absence of visible fluorescence from the tautomer forms of 7AI–(H2O) n (n =1–3) indicates that the full tautomerization due to photoinduced excited-state proton transfer does not occur during the lifetime of the S1 state of the normal form each cluster. [Copyright &y& Elsevier]

Objective: To investigate whether circulating high density lipoprotein (HDL) concentrations influence inflammatory cytokine release during abdominal operations. Setting: University Hospital, Japan. Patients: 30 patients who underwent curative operations for gastric cancer. Main outcome measures: Serum HDL and cytokine concentrations were measured preoperatively, and portal and systemic blood samples were taken for the analysis of cytokine and endotoxin concentrations during operations. Results: Portal blood interleukin-6 (IL-6) values of patients with low serum HDL concentrations (n = 6) (< 0.9 mmol/L) were significantly (p < 0.01) higher than those in patients with normal or high HDL concentrations (n = 24) (≥ 0.9 mmol/L) (80.63 (74.62) pg/ml (mean (SD)) compared with 10.25 (9.61) pg/ml.). Moreover, an inverse correlation (r =-0.43, p < 0.05) was found between portal blood IL-6 concentrations and circulating HDL concentrations. Conclusions: We suggest that serum HDL concentrations influence IL-6 release in response to abdominal operations. Circulating HDL particles may reduce endotoxin-induced IL-6 release in humans. [ABSTRACT FROM AUTHOR]

Viviparous rockfishes (Sebastes spp.) are ecologically and economically important fisheries and aquaculture species. Because the males lack obvious morphological characteristics related to reproductive status, it is challenging to select semen donors for artificial insemination. We previously identified lipocalin-type prostaglandin D 2 synthase homolog protein (LPGDShp) in the urine of male black rockfish (Sebastes schlegelii). Subsequently, LPGDShp-like protein (hereafter, lipocalin-like protein) was immunologically detected in the serum of two Sebastes species, black rockfish and white-edged rockfish (S. taczanowskii). In the present study, an enzyme-linked immunosorbent assay (ELISA) was developed to explore the potential of serum lipocalin-like protein as a biomarker for selecting mature males. The ELISA measured lipocalin-like protein in the sera of black rockfish and white-edged rockfish over an assay range between 0.391 and 12.5 ng/mL. In male black rockfish, serum levels of lipocalin-like protein were higher during the copulation season, and they were higher than levels in females during both the copulation and non-copulation seasons. In male white-edged rockfish sampled nearly monthly for a year, serum lipocalin-like protein levels showed synchronous dynamics with gonadosomatic index (GSI). Histological examination of testis revealed that the serum levels were elevated in the late/functional maturation stages, as compared to early/mid-maturation and resting stages. In both rockfish species, while serum levels of lipocalin-like protein were positively correlated with GSI in males, no such trend was observed in females. These results indicate, for the first time, that serum lipocalin-like protein can be employed as a hematological biomarker to detect the reproductive status of male rockfishes. • An enzyme-linked immunosorbent assay was developed for a lipocalin-like protein. • Serum levels of the protein were quantified in two rockfish species. • Levels of the protein were elevated in males during the copulation season. • Lipocalin-like protein positively correlated with male gonadosomatic index. • Lipocalin-like protein is a candidate biomarker of reproductive male Sebastes. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor-binding proteins (IGFBPs) regulate the activity of insulin-like growth factor (IGF)-1. Among the three major circulating IGFBPs in salmonids, IGFBP-1b is an inhibitor of IGF activity induced under catabolic conditions. IGFBP-1b is considered to quickly sequester IGF-1 from the circulation. However, the level of circulating IGFBP-1b present in its unoccupied free form is unknown. Here, we aimed to develop a non-equilibrium ligand immunofunctional assay (LIFA) to evaluate IGF-binding capacity of circulating intact IGFBP-1b. Purified Chinook salmon IGFBP-1b, its antiserum, and europium-labeled salmon IGF-1 were used as the assay components. In the LIFA, IGFBP-1b was first captured by the antiserum, allowed to bind to the labeled IGF-1 for 22 h at 4 °C, and quantified its IGF-binding capacity. Serial dilutions of the standard and serum were prepared simultaneously within a certain concentration range (1.1–12.5 ng/ml). In underyearling masu salmon, IGF-binding capacity of intact IGFBP-1b was higher in fasted fish than in fed fish. Transferring Chinook salmon parr to seawater also increased IGF-binding capacity of IGFBP-1b, most likely due to osmotic stress. In addition, there was a strong relationship between total IGFBP-1b levels and its IGF-binding capacity. These results suggest that IGFBP-1b expressed under stress is mostly present in the free form. On the contrary, during smoltification of masu salmon, IGF-binding capacity of IGFBP-1b in the serum was relatively low and less related to the total IGFBP-1b level, suggesting its functional difference under certain physiological conditions. These results indicate that estimating both total IGFBP-1b level and its IGF-binding capacity is useful for evaluating the catabolic status and unraveling the regulation of IGF-1 activity by IGFBP-1b. [Display omitted] • Ligand immunofunctional assay was developed for salmon IGBP-1b. • IGF-binding capacity of IGFBP-1b increased by fasting and stress. • Most IGFBP-1b is present as unoccupied under catabolic conditions. • IGFBP-1b may distribute IGF-1 to different tissues during smoltification. [ABSTRACT FROM AUTHOR]

Abstract: We compared the response of plasma insulin-like growth factor-I (IGF-I) to growth hormone (GH) administration under two different salinities to test the hypothesis that environmental salinity alters the “activity” of the GH–IGF-I axis. In July, postsmolt coho salmon reared in fresh water (FW) were transferred to either FW or half seawater (1/2 SW) (15 ppt) tank. During the experiment, water temperature was maintained at 10 °C for both salinities; photoperiod was adjusted to that of Seattle (48°N), and fish were not fed. Two days after transfer, fish were injected once with porcine GH (pGH) at a dose of 2 or 8 μg/g body weight. Liver and blood samples were collected 1, 2 and 3 days after injection. Liver GH receptor (GHR) mRNA expression was analyzed by quantitative real-time RT-PCR, and plasma IGF-I, 41-kDa IGF-binding protein (main carrier of IGF-I) and pGH were quantified by radioimmunoassays. Transfer to 1/2 SW resulted in transient increases in basal levels of liver GHR mRNA and 41 kDa IGF-binding protein (IGFBP) but not IGF-I. The GH injection increased liver GHR mRNA, plasma IGF-I and 41-kDa IGFBP in fish in both FW and 1/2 SW. However, the time course and magnitude of the response differed between salinities. Fish in FW receiving 8 μg/g pGH had the highest IGF-I levels (63.7±6.8 ng/ml) one day after injection, whereas fish in 1/2 SW showed a peak (88.8±14.3 ng/ml) two days after injection of the same dose. It is speculated that the prolonged response to GH by fish in 1/2 SW may be due to slower disappearance of pGH from the circulation in fish in 1/2 SW. The transient increase in basal liver GHR mRNA may also contribute to a greater response for fish in 1/2 SW. These results suggest that salinity is capable of altering the “activity” of the GH–IGF-I axis in salmon. [Copyright &y& Elsevier]

In salmon, at least three insulin-like growth factor binding proteins (IGFBPs) with molecular masses of 41, 28, and 22 kDa exist in serum. The 41-kDa IGFBP is up-regulated by growth hormone treatment and down-regulated by fasting, suggesting that it is a homolog of IGFBP-3. We purified the 41-kDa IGFBP from chinook salmon serum by IGF-I affinity chromatography followed by reversed-phase high pressure liquid chromatography. Purified IGFBP appeared as doublet bands on electrophoresis and was N-glycosylated. Analysis of partial N-terminal amino acid sequence revealed that salmon 41-kDa IGFBP has the cysteine rich domain conserved among IGFBP family. In a binding assay using 125I-salmon IGF-I, purified 41-kDa IGFBP specifically bound salmon IGF-I, human IGF-I and human IGF-II, but neither Long R3IGF-I nor salmon insulin, showing that binding characteristics of the salmon IGFBP are similar to those of mammalian IGFBPs. Although the partial amino acid sequence of 41-kDa IGFBP showed highest homologies with zebrafish and seabream IGFBP-2, the highly conserved nature of the N-terminus makes it impossible to identify the type of IGFBP from partial sequence data. However, based on physiological responses, molecular weight and type of glycosylation, the 41-kDa IGFBP is most similar to mammalian IGFBP-3. [Copyright &y& Elsevier]

Examines the role of vitamin E constituent in enhancing antioxidant protection in marine organisms. Impact of vitamin E on aerobic oxidation; Determination of the chromonal structure of marine-derived tocopherol (MDT); Evaluation of the antioxidant activities of MDT.

• Enzyme-linked immunosorbent assays were developed for three vitellogenin subtypes in marbled sole. • Estradiol treatment induced three vitellogenin subtypes in immature soles. • Three vitellogenins were quantified in sera of maturing soles inhabiting in "reference site" coastal areas. • The dominant vitellogenin subtype was C-type in male, while was Ab-type in female. Non-competitive, enzyme-linked immunosorbent assays (ELISAs) for three distinct sole vitellogenins (VtgAa, VtgAb and VtgC) were designed using their purified lipovitellin (Lv) products and corresponding digoxigenin-labeled, anti-Lv polyclonal antibodies, primarily for employment in monitoring estrogenic pollution of the environment. The working range of the ELISAs was from 0.97 to 1,000 ng/mL for all Vtg subtypes. Each ELISA appeared to be specific to the targeted Vtg subtype. Intra- and inter-assay coefficients of variation in the developed ELISAs were lower than 10%. Three Vtg subtypes were induced in serum of immature fish by estradiol-17β (E2) injection (0.5 mg/kg body weight). All Vtg subtypes were induced one day after the injection, reaching peak levels (Lv equivalents) within three days, as follows: 39.1 ± 28.9 μg/mL (VtgAa), 57.9 ± 30.7 μg/mL (VtgAb) and 12.6 ± 4.8 μg/mL (VtgC). In wild-caught males, VtgAa, VtgAb and VtgC were detected in ranges from 0.26 to 1.21, 0.19 to 8.69, and 0.17 to 53.50 μg/mL, respectively, over various sampling periods. In vitellogenic females sampled in January, the average level of VtgAb (8,744.43 ± 733.93 μg/mL) was significantly higher than for VtgAa (150.33 ± 22.35 μg/mL) or VtgC (57.08 ± 6.00 μg/mL); thus VtgAb appeared to be the most dominant Vtg subtype. The present study entails the first report on development of subtype-specific Vtg ELISAs in marbled sole, which empowers us to detect and monitor estrogenic contamination in aquatic environments inhabited by this species. [ABSTRACT FROM AUTHOR]

• Recombinant rainbow trout IGFBP-2b was produced. • TR-FIA for salmon/trout IGFBP-2b was established. • Serum IGFBP-2b levels were positively correlated with growth rates. • TR-FIA for IGFBP-2b is useful to evaluate growth status of salmonids. Insulin-like growth factor (IGF)-1 promotes the growth of vertebrates, and its binding proteins (IGFBPs) regulate the activity of circulating IGF-1. Three IGFBPs, IGFBP-2b, -1a, and -1b, were consistently detected in the circulatory system of salmonids. IGFBP-2b is thought to be the main carrier of IGFs and promoter of IGF-1-mediated growth in salmonids. Currently, there are no immunoassays for detecting IGFBP-2b. In this study, we developed a time-resolved fluoroimmunoassay (TR-FIA) for IGFBP-2b detection in salmonid fishes. To establish TR-FIA, we produced two recombinant trout (rt) IGFBP-2bs expressed, one with thioredoxin (Trx) and a histidine (His) tag, and the other with His-tag only. We labeled both recombinant proteins with europium (Eu). Only Eu-Trx.His.rtIGFBP-2b cross-reacted with anti-IGFBP-2b, and the addition of increasing amounts of Trx.His.rtIGFBP-2b replaced the binding, indicating its utility as a tracer and assay standard. The addition of unlabeled salmon IGF-1 did not affect the binding of the standard or sample. Serial dilution curves of sera from rainbow trout, Chinook salmon, and chum salmon were parallel to those of the standard. The assay range (ED 80 –ED 20) of the TR-FIA was 60.4 to 251.3 ng/ml, and its minimum detection limit of this assay was 21 ng/ml. The intra- and inter-assay coefficients of variation were 5.68% and 5.65%, respectively. Circulating IGFBP-2b levels in fed rainbow trout were higher than those in fasted fish and were correlated with individual growth rates. This TR-FIA is useful for further exploring the physiological responses of circulating IGFBP-2b and evaluating the growth status of salmonids. [ABSTRACT FROM AUTHOR]

Highlights • Chemiluminescent immunoassays were developed for three vitellogenins in grey mullet. • Vitellogenins were quantified in maturing fish inhabiting in potential control "clean" areas. • The dominant vitellogenin subtype was C-type in male, while was Ab-type in female. • 17α-ethinylestradiol exposure induced three vitellogenin subtypes in immature mullets. Abstract Chemiluminescent immunoassays (CLIAs) were developed for each of three subtypes of vitellogenin (VtgAa, VtgAb and VtgC) in grey mullet, primarily for use in monitoring estrogenic pollution of the environment. The working range of VtgAa-CLIA and VtgAb-CLIA was from 0.975 to 1,000 ng/ml, while that of VtgC-CLIA was from 0.487 to 1,000 ng/ml. Each CLIA appeared to be specific to the targeted Vtg subtype. Intra- and inter-assay coefficients of variation in the developed CLIAs were lower than 10%. In male serum, VtgAa, VtgAb and VtgC were detected in ranges from 0.01 to 0.38, 0.02 to 1.01, and 0.01 to 3.12 μg/ml, respectively, during various sampling periods. In vitellogenic females (October), serum VtgAb levels (1,192.05 ± 237.81 μg/ml) were significantly higher than levels of the other two Vtg subtypes (120.82 ± 30.42 and 119.23 ± 16.95 μg/ml for VtgAa and VtgC, respectively). When immature mullet were fed diets containing 17α-ethinylestradiol (EE2) at three different doses (0.4, 40 and 4,000 ng/g body weight), all Vtg subtypes were induced by 40 ng/g and 4,000 ng/g EE2. The VtgC (610.30 ± 150.18 μg/ml) was most highly expressed among the three Vtgs in fish fed 40 ng/g EE2, while VtgAb (33.25 ± 13.58 mg/ml) was highest in expression in fish fed 4,000 ng/g EE2. The present study provided practical subtype-specific Vtg assays for the first time in grey mullet, providing the necessary means to evaluate estrogenic activities in aquatic environments. [ABSTRACT FROM AUTHOR]

Transcription of vitellogenin ( vtg ) genes are initiated when estradiol-17β (E 2 )-estrogen receptor (ER) complexes bind estrogen response elements (ERE) located in the gene promoter region. Transcriptional regulation of dual vtg subtypes (major salmonid A-type vtg : vtgAs ; minor C-type vtg : vtgC ) by E 2 was investigated under co-expression of a potential major transcriptional factor, erα1 , in cutthroat trout. Two forms of trout vtgAs promoters (1 and 2) and one vtgC promoter were sequenced. These promoters structurally differ based on the number of EREs present. The vtgAs promoter 1 exhibited the highest maximal transcriptional activity by in vitro gene reporter assays. The concentration of E 2 that induces 50% of gene reporter activity (half-maximal effective concentrations, EC 50 ) was similar among all vtg promoters and also to the EC 50 of E 2 administered to induce vtg transcription in vivo . This study revealed a difference in transcriptional properties of multiple vtg promoters for the first time in a salmonid species, providing the basis to understand mechanisms underlying regulation of vitellogenesis via dual vtg gene expression. [ABSTRACT FROM AUTHOR]

To evaluate potential involvement of clathrin in endocytosis of vitellogenin (Vtg) by teleost oocytes, cDNAs encoding clathrin heavy chain ( cltc ) were cloned from ovaries of cutthroat trout. Quantitative PCR revealed three types of cltc ( cltc-a1 , cltc-a2 , cltc-b ) to be expressed in 10 different tissues including the ovary. The cltc-a1 alone exhibited a significant decrease in ovarian expression during vitellogenesis; this was correlated with a corresponding decrease in transcripts encoding the major Vtg receptor (Vtgr). No development-related changes in ovarian cltc-a2 or cltc-b transcript levels were observed. In situ hybridization revealed a strong ctlc signal in pre-vitellogenic oocytes, but not in vitellogenic oocytes. Western blotting using a rabbit antiserum (a-Cltc) raised against a recombinant Cltc preparation detected a polypeptide band with an apparent mass of ~ 170 kDa in vitellogenic ovary extracts. Immunohistochemistry using a-Cltc revealed Cltc to be uniformly distributed throughout the ooplasm of perinucleolus stage oocytes, translocated to the periphery of lipid droplet stage oocytes, and localized to the oolemma during vitellogenesis. These patterns of cltc /Cltc distribution and abundance during oogenesis, which are identical to those previously reported for vtgr /Vtgr in this species, constitute the first empirical evidence that cltc-a1 /Cltc-a1 is involved in Vtg endocytosis via the Vtgr in teleost fish. [ABSTRACT FROM AUTHOR]

• High molecular-weight urinary proteins (HMWups) were characterized in Sebastes. • The HMWups were lipocalin-type prostaglandin D 2 synthase (l -PGDS) homologs. • l -PGDS homolog transcripts were dominantly expressed in the testis and kidney. • The l -PGDS homologs produced in kidney are potential sources of HMWups. • Developed molecular tools enable study of their potential pheromonal activities. Black rockfish (Sebastes schlegelii) and its relatives are viviparous marine fish. Males produce urinary proteins during the copulation season; however, the identity of these proteins was unknown. In this study, we focused on high-molecular-weight urinary proteins (HMWups) in male black rockfish. The HMWups were identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS) of urine. In silico analyses of RNA-seq data predicted the tissue distribution of candidate HMWup transcripts and their gene structures. Candidate cDNAs were cloned and a recombinant protein of a major candidate was prepared. Western blotting of urine using an antiserum against the recombinant protein was performed to reconfirm the LC-MS/MS results. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry were employed to validate the prediction by RNA-seq and identify the cells producing HMWups, respectively. LC-MS/MS, in conjunction with Western blotting and cDNA cloning, identified the HMWups as lipocalin-type prostaglandin D 2 synthase (l -PGDS) homologs. RNA-seq analyses and qRT-PCR revealed that the l -PGDS homolog transcripts were dominantly expressed in the testis and male kidney; Sertoli cells and epithelial cells in the renal tubules were immunoreactive. These results indicated that major protein components in the urine of male black rockfish are l -PGDS homologs, potentially produced by the renal tubules in the kidney. Male rockfish (genus Sebastes) are thought to release unknown pheromone substances during mating behavior. The knowledge and tools obtained in this study empower research into the role(s) of HMWups in pheromone systems underlying rockfish reproduction. No protein-type teleost pheromone has heretofore been discovered. [ABSTRACT FROM AUTHOR]

One or more distinct forms of the nuclear estrogen receptor (ER) have been isolated from many vertebrates to date. To better understand the molecular evolution of ERs, we cloned and characterized er cDNAs from the inshore hagfish, Eptatretus burgeri , a modern representative of the most primitive vertebrates, the agnathans. Two er cDNAs, er1 and er2 , were isolated from the liver of a reproductive female hagfish. A phylogenetic analysis placed hagfish ER1 into a position prior to the divergence of vertebrate ERs. Conversely, hagfish ER2 was placed at the base of the vertebrate ERβ clade. The tissue distribution patterns of both ER subtype mRNAs appeared to be different, suggesting that each subtype has different physiological roles associated with estrogen actions. An estrogen responsive-luciferase reporter assay using mammalian HEK293 cells was used to functionally characterize these hagfish ERs. Both ER proteins displayed estrogen-dependent activation of transcription. These results clearly demonstrate that the hagfish has two functional ER subtypes. [ABSTRACT FROM AUTHOR]

Fish egg yolk is largely derived from vitellogenins, which are synthesized in the liver, taken up from the maternal circulation by growing oocytes via receptor-mediated endocytosis and enzymatically processed into yolk proteins that are stored in the ooplasm. Lipid droplets are another major component of fish egg yolk, and these are mainly composed of neutral lipids that may originate from maternal plasma lipoproteins. This review aims to briefly summarize our current understanding of the molecular mechanisms underlying yolk formation in fishes. A hypothetical model of oocyte growth is proposed based on recent advances in our knowledge of fish yolk formation. [ABSTRACT FROM AUTHOR]

In salmon plasma/serum, three major insulin-like growth factor binding proteins (IGFBPs) are consistently detected at 22-, 28- and 41-kDa. The 22-kDa form has been identified as IGFBP-1b and shown to increase under catabolic conditions. We developed a competitive time-resolved fluoroimmunoassay (TR-FIA) for salmon IGFBP-1b. Purified salmon IGFBP-1b was used for biotin-labeling, assay standard and antiserum production. The TR-FIA did not cross-react with the 41-kDa form (IGFBP-2b) but showed 3% cross-reactivity with the 28-kDa form (IGFBP-1a). It measured IGFBP-1b levels as low as 0.4 ng/ml, and ED 80 and ED 20 were 0.9 and 24.6 ng/ml, respectively. There appears to be little interference by IGF-I. Using the TR-FIA, serum IGFBP-1b levels were measured in individually-tagged underyearling masu salmon fed or fasted for 5 weeks, or fasted for 3 weeks followed by refeeding for 2 weeks. Fasting for 3 weeks significantly increased circulating IGFBP-1b levels, while it returned to the basal levels after prolonged fasting for additional 2 weeks. Serum IGFBP-1b level negatively correlated with body weight, condition factor, specific growth rate and serum IGF-I level. During parr–smolt transformation of masu salmon, average circulating IGFBP-1b levels were the highest in May. There was a positive correlation between serum IGFBP-1b and IGF-I, which is in contrast to that in the fasting/feeding experiment. IGFBP-1b also showed a positive relationship with gill Na + , K + -ATPase activity. These results suggest that the relationship between circulating IGFBP-1b and IGF-I during smoltification differs from that during fasting and IGFBP-1b may play a role in the development of hypoosmoregulatory ability. [ABSTRACT FROM AUTHOR]

We compared profiles of serum insulin-like growth factor (IGF)-I levels during smoltification of masu salmon reared under different environments, hatcheries and growth histories. Masu salmon from the Kenichi River in Hokkaido showed a sharp increase in serum IGF-I from April to May, followed by a peak of gill Na,K-ATPase (NKA) activity. Fish at Kumaishi Hatchery had an IGF-I profile similar to that of the river fish, while the increase in gill NKA was lower. At Shimamaki Hatchery, interval feeding during winter appeared to suppress the spring IGF-I peak. At Kumaishi Hatchery, a difference in size during smoltification affected IGF-I levels at release, but the numbers of adults that returned to the release site were not significantly different. In the following year, three release groups differing in winter size and/or spring growth (Large-High, Large-Low and Small-High) were created. Large-High and Small-High fish showed a higher IGF-I peak than Large-Low fish in April, while smolt-to-adult return of Large-High fish was highest. These results suggest that in smolting masu salmon in freshwater, circulating IGF-I level alone is not a predictor of long-term survival in seawater. However, since growth history in freshwater affected the smolt-to-adult return, optimizing rearing conditions is a critical component of hatchery releases for masu salmon. [ABSTRACT FROM AUTHOR]

Despite its key role in transportation of triacylglycerides in blood, the distribution, localisation and molecular weight variants of apolipoprotein B (Apob) in teleost fish have essentially escaped study. To address this, a specific short-finned eel ( Anguilla australis) Apob antiserum was produced by an immunised rabbit, purified and partially characterised. Localisation of Apob at both the mRNA (in situ hybridisation) and protein (immunohistochemistry) levels mirrored that of mammals; thus immunostaining was confined to the interstitial spaces of the liver and the vascular core of the intestinal villi. Immunostaining of proteins by Western blotting, followed by high-resolution LC-MS, indicated that peptide sequence coverage of Apob in low-density lipoproteins spanned the full-length protein. We conclude that only full-length Apob is produced by eels and that both liver and intestine are key sites for its synthesis. [ABSTRACT FROM AUTHOR]

Molecular characterization of a major allergen from salmon roeSalmon roe has a high allergic potency and often causes anaphylaxis in Japan. The major allergic protein of salmon roe is β′-component, which is a 35kDa vitellogenin fragment consisting of two subunits. To elucidate structural information and immunological characteristics, β′-component and the subunit components were purified from chum salmon (Onchorhincus keta) roe and vitellogenin-encoding mRNA was used to prepare β′-component subunit-encoding cDNA. This was PCR-amplified, cloned and sequenced and the deduced amino acid sequence compared with partial sequences of β′-component obtained by peptide mapping. The recombinant β′-component subunit was produced by bacterial expression in Escherichia coli and its IgE-binding ability was measured by ELISA using the sera of a patient allergic to salmon roe. This was then compared with that of the native β′-component with and without carboxymethylation. Following successful cloning of the cDNA encoding the β′-component subunit, 170 amino acid residues were deduced and matched with the amino acid sequences of 121 and 88 residues in the 16kDa and 18kDa subunits, respectively. The sequences of both β′-component subunits were almost identical, and the predicted secondary structure of the β′-component showed a high content of β-pleated sheets and no α-helices. There was no difference in IgE-binding ability between the native and recombinant β′-component subunits at the same protein concentration, regardless of carboxymethylation. In conclusion, β′-component is a homodimer protein composed of two isoform subunits having the same level of IgE-binding ability and, therefore, allergenic identity. [ABSTRACT FROM PUBLISHER]

Abstract: Teleost fish eggs contain a substantial yolk mass consisting of lipids and proteins that provides essential nutrients for embryonic and larval development. The polar lipid and protein components of the yolk are delivered to oocytes by circulating vitellogenins, however the source(s) of the neutral lipid remains unknown. We cloned a cDNA encoding an orthologue of low-density-lipoprotein receptor (LDLR) from the ovary of cutthroat trout, Oncorhynchus clarki (ct-Ldlr). Predominant expression of ct-ldlr mRNA was observed in the ovary and moderate expression was detected in intestine, gill and brain. The relative abundance of ct-ldlr transcripts was highest in early pre-vitellogenic ovaries and significantly decreased during vitellogenesis, followed by a slight increase during final maturation and in post-ovulatory follicles. In situ hybridization revealed an intense and evenly distributed localization of ct-ldlr transcripts in the ooplasm of pre-vitellogenic oocytes and these signals disappeared in vitellogenic follicles. Collectively, these results suggest that the Ldlr is involved in deposition of yolk lipids in cutthroat trout oocytes. The ct-ldlr transcripts also were detected in theca and granulosa cells, suggesting that this receptor may be involved in cholesterol uptake for ovarian steroidogenesis. This is the first report on partial characterization of an ldlr orthologue in any fish species. [Copyright &y& Elsevier]

Abstract: A cDNA encoding a vitellogenin receptor with 8 ligand binding repeats (vtgr) was cloned from ovaries of the cutthroat trout, Oncorhynchus clarki. In situ hybridization and quantitative PCR analyses revealed that the main site of vtgr mRNA expression was the oocytes. Expression was strongly detected in perinucleous stage oocytes, gradually decreased as oocytes grew, and became hardly detectable in vitellogenic oocytes. A rabbit antibody (a-Vtgr) was raised against a recombinant Vtgr protein in order to immunologically detect and localize Vtgr within the ovarian follicles. Western blotting using a-Vtgr detected a bold band with an apparent mass of ~95–105kDa in an ovarian preparation that also bound Sakhalin taimen, Hucho perryi, vitellogenin in ligand blots. Immunohistochemistry using a-Vtgr revealed that the Vtgr was uniformly distributed throughout the ooplasm of perinucleolus stage oocytes, subsequently translocated to the periphery of lipid droplet stage oocytes, and became localized to the oolemma during vitellogenesis. We provide the first characterization of Vtgr at both the transcriptional and the translational levels in the cutthroat trout, and our results suggest that this receptor is involved in uptake of Vtg by oocytes of this species. [Copyright &y& Elsevier]

Abstract: Two subtypes of insulin-like growth factor binding protein (IGFBP)-1 are present in salmon blood and they are both up-regulated under catabolic conditions such as stress. The present study examined effects of fasting and re-feeding on IGFBP-1a (28-kDa form) and IGFBP-1b (22-kDa form) both at mRNA and protein levels along with IGF-I and RNA/DNA ratio in yearling masu salmon. Fish were individually tagged and assigned to one of three treatments: Fed, Fasted or Re-fed. Circulating IGF-I levels significantly decreased after fasting for 5weeks and were positively correlated with individual growth rates. Liver igf-1 mRNA levels were not affected by the treatment. Muscle RNA/DNA ratio did not respond to fasting nor showed correlations with growth rates. Circulating IGFBP-1a and IGFBP-1b increased during fasting and decreased after re-feeding. Both serum levels were inversely correlated with growth rates, while IGFBP-1b had consistent negative relationships with growth rates. Fasting/re-feeding also affected their mRNA levels in the liver. These results suggest that circulating IGF-I and IGFBP-1b could serve as positive and negative indices of growth, respectively, in masu salmon. Different sensitivities of IGBP-1a and IGFBP-1b may be useful to assess a broad range of catabolic conditions when they are combined. [Copyright &y& Elsevier]

Abstract: We established profiles of insulin-like growth factor (IGF)-I mRNA in the liver, gill and white muscle and circulating IGF-I during smoltification of hatchery-reared masu salmon, and compared with that of gill Na+,K+-ATPase (NKA) activity. Gill NKA activity peaked in May and dropped in June. Liver igf1 mRNA was high in March and decreased to low levels thereafter. Gill igf1 increased from March, maintained its high levels during April and May and decreased in June. Muscle igf1 mRNA levels were relatively high during January and April when water temperature was low. Serum IGF-I continuously increased from March through June. Serum IGF-I during March and May showed a positive correlation with NKA activity, although both were also related to fish size. These parameters were standardized with fork length and re-analyzed. As a result, serum IGF-I and gill igf1 were correlated with NKA activity. On the other hand, samples from desmoltification period (June) that had high serum IGF-I levels and low NKA activity disrupted the relationship. Expression of two IGF-I receptor (igf1r) subtypes in the gill decreased in June, which could account for the disruption by preventing circulating IGF-I from acting on the gill and retaining it in the blood. The present study suggests that the increase in gill NKA activity in the course of smoltification of masu salmon was supported by both endocrine and local IGF-I, and the decrease during desmoltification in freshwater was due at least in part to the down-regulation of gill IGF-I receptors. [Copyright &y& Elsevier]

Abstract: A fish yolk protein, β′-component (β′-c), is the major allergen in chum salmon roe. The effect of proteolysis on the allergenicity of β′-c was estimated. Changes in the IgE-binding ability of β′-c upon pepsin and trypsin digestion were investigated by monitoring the proteolytic cleavage. In the pepsin–trypsin digestion of chum salmon yolk protein, the β′-c contained therein was degraded in a manner similar to that of other yolk proteins, but digestion fragments with a molecular mass of >10kDa remained throughout the digestion process. Specifically, the peptide sequence between 31-Y and 119-Q (10kDa) was stable to pepsin–trypsin digestion and the portion showed high IgE-binding ability. As a result, pepsin–trypsin digestion had little effect on the IgE-binding ability of β′-c. These results suggest that β′-c reaches the small intestine in the form of high-molecular-mass components with IgE-binding ability in vivo. [Copyright &y& Elsevier]

Abstract: Circulating insulin-like growth factor binding proteins (IGFBPs) play pivotal roles in stabilizing IGFs and regulating their availability to target tissues. In the teleost circulation, three major IGFBPs are typically detected by ligand blotting with molecular masses around 20–25, 28–32 and 40–45kDa. However, their identity is poorly established and often confused. We previously identified salmon 22- and 41-kDa forms as IGFBP-1 and -2b, respectively. In the present study, we cloned the cDNA of 28-kDa IGFBP from Chinook salmon (Oncorhynchus tshawytscha) as well as rainbow trout (Oncorhynchus mykiss) based on the partial N-terminal amino acid sequence of purified protein and identified it as an ortholog of IGFBP-1. Structural and phylogenetic analyses revealed that the 28-kDa IGFBP is more closely related to human IGFBP-1 and zebrafish IGFBP-1a than the previously identified salmon IGFBP-1 (i.e. 22-kDa IGFBP). We thus named salmon 28- and 22-kDa forms as IGFBP-1a and -1b, respectively. Salmon IGFBP-1a contains a potential PEST region involved in rapid protein turnover and phosphorylation sites typically found in mammalian IGFBP-1, although the PEST and phosphorylation scores are not as high as those of human IGFBP-1. There was a striking difference in tissue distribution patterns between subtypes; Salmon igfbp-1a was expressed in a variety of tissues while igfbp-1b was almost exclusively expressed in the liver, suggesting that IGFBP-1a has more local actions. Direct seawater exposure (osmotic stress) of Chinook salmon parr caused increases in both IGFBP-1s in plasma, while IGFBP-1b appeared to be more sensitive. The presence of two co-orthologs of IGFBP-1 in the circulation in salmon, and most likely in other teleosts, provides a good opportunity to investigate subfunction partitioning of duplicated IGFBP-1 during postnatal growth. [Copyright &y& Elsevier]

Abstract: In vertebrates, most circulating insulin-like growth factor (IGF) is bound to multiple forms of IGF-binding proteins (IGFBPs) that differ both structurally and functionally. In mammals, the largest reservoir of IGF in the circulation comes from a large (150kDa) ternary complex comprised of IGF bound to IGFBP-3, which is bound to an acid label subunit (ALS), and this variant of IGFBP is regulated by growth hormone (GH) and feed intake. Although multiple variants of IGFBPs ranging from 20 to 50kDa have been found in fishes, no ternary complex is present and it has been assumed that the majority of circulating IGF is bound to fish IGFBP-3. Consistent with this assumption is previous work in salmon showing the presence of a 41-kDa IGFBP that is stimulated by GH, decreases with fasting and increases with feeding. However, the hypothesis that the salmon 41-kDa IGFBP is structurally homologous to mammalian IGFBP-3 has not been directly tested. To address this issue, we cloned cDNAs for several Chinook salmon IGFBPs, and found that the cDNA sequence of the 41-kDa IGFBP is most similar to that of mammalian IGFBP-2 and dissimilar to IGFBP-3. We found an additional IGFBP (termed IGFBP-2a) with high homology to mammalian IGFBP-2. These results demonstrate that salmon 41-kDa IGFBP is not IGFBP-3, but a paralog of IGFBP-2 (termed IGFBP-2b). Salmon IGFBP-2s are also unique in terms of having potential N-glycosylation sites and splice variants. Additional research on non-mammalian IGFBPs is needed to fully understand the molecular/functional evolution of the IGFBP family and the significance of the ternary complex in vertebrates. [Copyright &y& Elsevier]

Ovarian follicle cells are the site of yolk protein synthesis in the Ezo abalone Haliotis discus hannai. In this study, histological observations of ovarian follicle cells were conducted in H. discus hannai by immunocytochemical and in situ hybridization methods focusing on their function of yolk protein synthesis. An antibody raised against purified yolk protein (vitellin, Vn) recognized yolk granules in oocytes, and ovarian follicle cells adjacent to the oocytes under yolk accumulation were stained positively with both anti-Vn antibody and an antisense probe for vitellogenin (the precursor for vitellin, Vtg) mRNA. These results indicate that the abalone Vtg gene is transcribed and translated in the ovarian follicle cells. In oocytes in the early phase of yolk accumulation, positive reactions with the antibody appeared first in the stalk part, and the follicle cells adjacent to the stalk were also stained positively. These observations imply local transport of yolk protein from the follicle cells to the oocyte through an extracellular space around the oocyte stalk. Ovarian follicle cells changed their morphological characteristics and reactivity to the antibody in close relation to the stages of the adjacent oocyte, which suggests the presence of functional interactions between follicle cells and oocytes in the course of yolk protein synthesis and accumulation. [ABSTRACT FROM AUTHOR]

Abstract: Sex-steroid hormones are essential for normal reproductive activity in both sexes in all vertebrates. Estrogens are required for ovarian differentiation during a critical developmental stage and promote the growth and differentiation of the female reproductive system following puberty. Recent studies have shown that environmental estrogens influence the developing reproductive system as well as gametogenesis, especially in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor–ligand interactions in Elasmobranchii, we cloned a single estrogen receptor (ESR) from two shark species, the cloudy catshark (Scyliorhinus torazame) and whale shark (Rhincodon typus) and used an ERE-luciferase reporter assay system to characterize the interaction of these receptors with steroidal and other environmental estrogens. In the transient transfection ERE-luciferase reporter assay system, both shark ESR proteins displayed estrogen-dependent activation of transcription, and shark ESRs were more sensitive to 17β-estradiol compared with other natural and synthetic estrogens. Further, the environmental chemicals, bisphenol A, nonylphenol, octylphenol and DDT could activate both shark ESRs. The assay system provides a tool for future studies examining the receptor–ligand interactions and estrogen disrupting mechanisms in Elasmobranchii. [ABSTRACT FROM AUTHOR]

Abstract: A novel, incomplete-type vitellogenin (VgC) and its derived yolk lipovitellin (LvC) were immunologically detected in female serum and egg extracts, respectively, of Sakhalin taimen (Hucho perryi) using a subtype-specific antiserum against LvC of grey mullet (Mugil cephalus). The taimen VgC was purified from the sera of vitellogenic females by a combination of gel filtration, anion exchange, and immunoadsorbent column chromatography. Gel filtration of the purified VgC revealed that it had an apparent native mass of ∼380kDa, while the mass of the VgC polypeptide that appeared following SDS-PAGE was estimated to be ∼140kDa. An antiserum was raised against the purified VgC and utilized for the development of a subtype-specific immunoassay for VgC. Levels of VgC in the serum of female taimen increased from 25μg/mL to ∼1mg/mL, with an increase of GSI. Levels of complete-type Vg and estradiol-17β (E2) in the serum of E2-administered juvenile taimen increased and reached peak levels similar to those found in vitellogenic females. Although VgC could be induced in the serum of E2-administered taimen, it stayed at levels (35.5–73μg/mL) lower than those obtained in females. This is the first report on the presence of serum VgC and yolk LvC in a salmonid species; these findings indicate that for Sakhalin taimen, like other highly-evolved teleost species, this minor subtype of Vg is significant in the formation of egg yolk. [Copyright &y& Elsevier]

Abstract: We monitored the contamination by environmental estrogens (EEs) of coastal areas in Korea and Japan using the wild grey mullet. The grey mullet were collected from Ansan, Jeju, Yeosu, Tongyeong, and Busan in Korea and Nagasaki, Omuta, and Fukuoka in Japan. Contamination by EEs was determined by measuring vitellogenin (VTG) levels in serum and identifying gonadal abnormalities histologically (i.e., testis-ova). In four sites in Korea (Ansan, Yeosu, Tongyeong, and Busan) and two sites in Japan (Nagasaki and Fukuoka), serum VTG in immature and male grey mullet was detected at levels greater than 1.0μg/ml, which is considered to be an abnormal level. Although, testis-ova were observed in some individuals collected in Ansan, Tongyeong, and Busan in Korea and Omuta in Japan, there was no correlation between individuals with testis-ova and individuals with abnormal levels of VTG. Furthermore, in Japan, serum VTG levels of fish collected from Nagasaki and Fukuoka were also greater than 1.0μg/ml. Although individuals with testis-ova were found in Omuta, these fish expressed normal levels of serum VTG. Our results suggest that the grey mullets living in these coastal areas are influenced by EEs in the environment. Furthermore, it appears that the production of VTG and the occurrence of testis-ova are caused by different mechanisms. [Copyright &y& Elsevier]

Abstract: Two vitelline envelope precursors (choriogenin H: Chg H; choriogenin L: Chg L) and an egg yolk precursor (vitellogenin B: VgB) were purified from red lip mullet. The mass of intact Chg H and Chg L were estimated to be ~215 kDa and ~69 kDa, respectively. In SDS-PAGE, Chg H and Chg L separated to positions corresponding to ~51 kDa and ~44 kDa, respectively. The mass of intact VgB was ~530 kDa and resolved into a polypeptide of ~185 kDa in SDS-PAGE. Specific antisera were raised against each purified protein and specific immunoassays were developed. When Chg H, Chg L and VgB were induced in the serum of immature mullet by injection with various doses of estradiol-17β (E2), VgB exhibited the most sensitive response exhibiting high variation in its induced levels. The variation in induced levels of Chg H and L was relatively minimal although induction required higher doses of E2 than with VgB. Serum samples obtained from immature mullet populations collected from their natural habitat exhibited similar profiles in the levels of these proteins. The present study suggests that the utilization of multiple biomarkers holds great importance for the reliable and accurate evaluation of estrogenic activity in aquatic environments. [Copyright &y& Elsevier]

• Choriogenin, vitellogenin and estrogen receptor-1a genes were estrogen-responsive. • These estrogen responsive genes responded differentially to estradiol-17β (E2). • E2 transactivated these gene promoters in the presence of four estrogen receptors. Estradiol-17β (E2) regulates transcription of estrogen-responsive genes via estrogen receptors (Esr). In many teleost species, choriogenin (chg), vitellogenin (vtg) and esr genes are transactivated by E2 in the liver. This study aimed i) to compare expression properties of all subtypes of these genes (chg : chgHα , chgHβ , chgL ; vtg : vtgAs , vtgC ; esr : esr1a , esr1b , esr2a , esr2b) in response to estrogen stimulation, and ii) to confirm how each of four Esr subtypes is involved in the transcriptional regulation of these estrogen-responsive genes in cutthroat trout hepatocytes. In hepatocytes in primary culture, all chg and vtg subtype mRNA levels, and those of esr1a , were increased by E2 treatment (10−6 M) at 24 and 72 h post initiation (hpi), but esr1b , esr2a and esr2b mRNA levels were not. Treatment of hepatocytes with various concentrations of E2 (10−11–10−6 M) induced dose-dependent increases in the levels of all chg and vtg subtype mRNAs at 24 and 72 hpi. At both time points, the lowest dose that induced a significant increase in the expression levels of mRNAs (LOEC) for E2 differed among the genes; LOECs were estimated as 10−11 M for chgHα at 24 hpi, as 10−9 M for vtgC at 72 hpi, and as 10−10 M for other mRNAs at both 24 and 72 hpi. Meanwhile, the levels of esr1a mRNA exhibited a dose-dependent increase at 24 and 72 hpi, but the LOEC shifted from 10−9 M at 24 hpi to 10−7 M at 72 hpi because of a decrease in mRNA levels at treatment groups exposed to high concentrations of E2. All Esr subtypes transactivated chg , vtg and esr1a promoters in the presence of E2 in vitro. The activation levels indicated that promoter activity of chgHα ≥ vtgAs > chgHβ > chgL ≥ vtgC ≥ esr1a when mediated by Esr1a, chgHβ > chgHα > chgHL > vtgAs ≥ vtgC ≥ esr1a by Esr1b, chgHβ ≥ chgL > chgHα ≥ vtgAs > vtgC > esr1a by Esr2a, and chgHβ ≥ chgHα ≥ vtgAs > chgL ≥ vtgC > esr1a by Esr2b. Collectively, different Esr subtypes were distinctly different in their ability to transactivate estrogen-responsive target genes, resulting in differential expression of chg , vtg and esr1a genes in the estrogen-exposed hepatocytes. [ABSTRACT FROM AUTHOR]

• Choriogenin, vitellogenin and estrogen receptor-1a genes were estrogen-responsive. • These estrogen responsive genes responded differentially to estradiol-17β (E2). • E2 transactivated these gene promoters in the presence of four estrogen receptors. Estradiol-17β (E2) regulates transcription of estrogen-responsive genes via estrogen receptors (Esr). In many teleost species, choriogenin (chg), vitellogenin (vtg) and esr genes are transactivated by E2 in the liver. This study aimed i) to compare expression properties of all subtypes of these genes (chg : chgHα , chgHβ , chgL ; vtg : vtgAs , vtgC ; esr : esr1a , esr1b , esr2a , esr2b) in response to estrogen stimulation, and ii) to confirm how each of four Esr subtypes is involved in the transcriptional regulation of these estrogen-responsive genes in cutthroat trout hepatocytes. In hepatocytes in primary culture, all chg and vtg subtype mRNA levels, and those of esr1a , were increased by E2 treatment (10−6 M) at 24 and 72 h post initiation (hpi), but esr1b , esr2a and esr2b mRNA levels were not. Treatment of hepatocytes with various concentrations of E2 (10−11–10−6 M) induced dose-dependent increases in the levels of all chg and vtg subtype mRNAs at 24 and 72 hpi. At both time points, the lowest dose that induced a significant increase in the expression levels of mRNAs (LOEC) for E2 differed among the genes; LOECs were estimated as 10−11 M for chgHα at 24 hpi, as 10−9 M for vtgC at 72 hpi, and as 10−10 M for other mRNAs at both 24 and 72 hpi. Meanwhile, the levels of esr1a mRNA exhibited a dose-dependent increase at 24 and 72 hpi, but the LOEC shifted from 10−9 M at 24 hpi to 10−7 M at 72 hpi because of a decrease in mRNA levels at treatment groups exposed to high concentrations of E2. All Esr subtypes transactivated chg , vtg and esr1a promoters in the presence of E2 in vitro. The activation levels indicated that promoter activity of chgHα ≥ vtgAs > chgHβ > chgL ≥ vtgC ≥ esr1a when mediated by Esr1a, chgHβ > chgHα > chgHL > vtgAs ≥ vtgC ≥ esr1a by Esr1b, chgHβ ≥ chgL > chgHα ≥ vtgAs > vtgC > esr1a by Esr2a, and chgHβ ≥ chgHα ≥ vtgAs > chgL ≥ vtgC > esr1a by Esr2b. Collectively, different Esr subtypes were distinctly different in their ability to transactivate estrogen-responsive target genes, resulting in differential expression of chg , vtg and esr1a genes in the estrogen-exposed hepatocytes. [ABSTRACT FROM AUTHOR]

Lipovitellin (Lv), the major yolk protein derived from vitellogenin (Vg), was purified from vitellogenic ovaries of Pacific saury Cololabis saira using hydroxylapatite column chromatography followed by gel filtration. The apparent native mass of purified Lv was approximately 420 kDa, while the tertiary structure of Lv revealed by sodium dodecylsulfate–polyacrylamide gel electrophoresis was typical of teleost Lvs, consisting of a heavy chain (∼99 kDa) and a light chain (∼34 kDa). Western blot analysis using rabbit antiserum raised against Pacific saury Lv revealed a specific reaction with a polypeptide (∼194 kDa) that is present in serum from female Pacific saury but not in male serum, suggesting the approximately 194-kDa polypeptide to be the Vg monomer. This study describes the first step toward the development of specific immunoassays for Pacific saury Vg, which will be an effective tool for monitoring the reproductive development of this species. [ABSTRACT FROM AUTHOR]

A glycerolipid acyl-hydrolase was purified 19-fold with a yield of 11% from the prostaglandin-producing red alga Gracilaria vermiculophylla by ammonium sulfate precipitation, anion-exchange chromatoraphy and gel filtration chromatography. Sodium dodecylsulfate– polyacrylamide gel electrophoresis of the final preparation showed a single band corresponding to a molecular mass of 20 kDa, but Superdex 200 fast protein liquid chromatography exhibited a molecular mass of 40 kDa. Accordingly, it was suggested that the purified enzyme was a homodimer of a 20 kDa subunit. The optimal temperature and pH were 37°C and 7–8, respectively. The purified enzyme catalyzed hydrolysis of the acyl groups of both glycoglycerolipids and phospholipids, especially monogalactosyldiacylglycerol and phosphatidylcholine. These results suggest that the enzyme hydrolyze the membrane lipids of the alga to release various saturated and unsaturated fatty acids, including arachidonic acid as substrate for prostaglandin synthesis. [ABSTRACT FROM AUTHOR]

Abstract: Only a few biochemical and molecular studies on yolk proteins (vitellins) have been carried out in mollusks, mainly in bivalves, while information on prosobranch vitellogenesis is still limited. In this study, we cloned a full-length cDNA encoding vitellogenin (Vg) in the Pacific abalone Haliotis discus hannai. The complete Vg cDNA consists of 7753 nucleotides with a long open reading frame encoding 2391 amino acid residues. The deduced primary structure contains the N-terminal amino acid sequences of the 95 kDa and 150 kDa subunits of vitellin of the abalone and shows similarities to Vgs of other mollusk, fish, nematode and coral species. In common with bivalve Vgs, the abalone Vg gene was expressed only in the ovary. In situ hybridization analysis further localized Vg mRNA to the follicle cells in the ovary. We conclude that the follicle cells are the site of Vg synthesis in H. discus hannai. [Copyright &y& Elsevier]

Three female specific serum proteins were detected immunologically in the sera of grey mullet ( Mugil cephalus) which were named vitellogenin A (VgA), VgB, and VgC, based upon their distinct antigenicity against specific antisera raised against three types of mullet lipovitellins (Lvs). These Vgs were subsequently purified from the serum of estradiol-treated mullet by combining several types of chromatography columns (anion exchanger, hydroxylapatite, immunoadsorbent column, and gel filtration). Purified native VgA, VgB, and VgC exhibited molecular masses of 570, 580, and 335 kDa, respectively. Following, SDS-PAGE, the estimated mass of polypeptide bands evident for VgA and VgB were ∼179 and ∼175 kDa, respectively; VgC appeared to be ∼132 kDa. The two larger Vgs (VgA and VgB) appeared to be phosphorylated, suggesting that these Vgs contain a highly phosphorylated, serine-rich phosvitin (Pv) domain. Furthermore, two discrete Vg-type specific antisera, anti-VgA and anti-VgB, were developed and each generated two precipitin lines against ovary extracts in immunoelectrophoresis, indicating that these Vgs contain additional antigenic yolk protein domains: Lv and β′-component. The small Vg (VgC) appeared to lack a Pv domain because of its low serine content (5.35%) and failure to show positive results in phospho-staining experiments. In conjunction with N-terminal amino acid sequencing analyses of the purified Vgs, our present results have conclusively identified the purified Vg products in grey mullet as typical A-type (VgA), B-type (VgB), and C-type (VgC) Vgs. [ABSTRACT FROM AUTHOR]

Abstract: In a previous study, we identified cDNAs encoding the growth hormone receptor (eGHR1) and eGHR1 homologue (eGHR2) in Japanese eel (Anguilla japonica). In the present study, changes in the developmental expression of growth hormone (GH), eGHR1 and eGHR2 were investigated in the Japanese eel eggs and preleptocephali by RT-PCR and immunohistochemical methods in an attempt to examine the involvement of these proteins in larval growth. The GH transcripts and the production of GH protein were not detected in the newly hatched larvae and preleptocephali at day 3 post-hatch, however, these were detected at day 6 post-hatch, and also detected at higher levels at day 10 post-hatch. In contrast, prolactin and somatolactin transcripts could not be detected in all preleptocephalus specimens (newly hatched larvae and preleptocephali at day 3, 6 and 10 post-hatch). eGHR1 and eGHR2 transcripts were detected in all preleptocephalus specimens. Therefore, it is plausible that the actions of GH during the preleptocephalus stage are mediated through the eGHRs. The present data suggest that GHR-mediated actions of GH begin at the same time as the initiation of GH production, and that GH plays important roles in larval growth and survival to the leptocephalus stage. eGHR1 mRNA, which is thought to be of maternal origin, was also detected in ovulated eggs. However, the role of eGHR1 mRNA in eggs is not clear. [Copyright &y& Elsevier]

Vitellogenin (Vg), a major estrogen-inducible yolk precursor protein, has become an important biomarker for assessing the estrogenic potency of chemicals and the exposure of animals to estrogenic contaminants present in aquatic environments. These contaminants, which can disrupt functioning of the vertebrate neuroendocrine system, are known as endocrine disrupting chemicals (EDCs). In general, investigations of the significance of estrogenic EDCs have failed to keep pace with recent developments in our understanding of vitellogenesis in fishes. Recent gene cloning and immunobiochemical analyses have verified the general multiplicity of piscine Vg and led to exploration of the unique roles of yolk proteins derived from different forms of Vg in the processes of oogenesis and embryogenesis. The levels of circulating Vg proteins (or Vg gene transcripts) during oogenesis and their degree of induction by estrogens appear to vary among species and among different types of Vg within species. The kinetics of induction of distinct types of Vg by estrogens in fishes appears to depend on environmental factors (e.g., water temperature and photoperiod), life history stage, and the concentration and type of estrogenic compound. Consideration of these findings will contribute to development of Vg-based bioassays superior to those currently based on the outdated “single Vg” model. [ABSTRACT FROM AUTHOR]

Abstract: Two cDNAs encoding growth hormone receptor (GHR)-like genes, eGHR1 and eGHR2, were isolated from Japanese eel (Anguilla japonica) liver tissue. The putative eel GHR proteins showed conserved structural features of vertebrate GHRs, including six cysteine residues and a YGEFS motif in the extracellular domain, a single transmembrane region, and proline-rich box 1 and box 2 domains. Northern blot analysis showed a single eGHR1 transcript in liver, while two sizes of eGHR2 transcripts, thought to be produced by alternative splicing, were present. RT–PCR revealed that eGHR1 and eGHR2 transcripts were widely distributed throughout the whole body of the Japanese eel. Moreover, the results of binding assays showed the specific binding of growth hormone to recombinant eGHR1. Since these putative eGHR proteins show all characteristics of the GHR family, we conclude that eGHR1 and eGHR2 cDNA encode two different GHRs in Japanese eel. We confirmed the ligand specificity of eGHR1 by binding assay, and further research is needed to allow characterization of the binding capability of eGHR2. [Copyright &y& Elsevier]

Abstract: Two distinct yolk proteins (YP1 and YP2) were purified from the ovary of medaka, and specific antisera against YPs were generated to characterize YPs and reveal their relation to two vitellogenins (Vg1 and Vg2). The molecular masses of purified YP1 and YP2 on gel filtration were 270 and 380kDa, respectively. YPs were confirmed to be lipoproteins by staining with Sudan black. Amino acid compositions of YP1 and YP2 were similar to those of Vg1 and Vg2, respectively. In double immunodiffusion using anti-Vg1, a precipitin line of YP1 formed a spur against the Vg1 line. YP2 and Vg2 were reacted with anti-Vg2, and a precipitin line of YP2 formed a fuse against the Vg2 line. These biochemical and immunological analyses of purified YPs revealed that YP1 is lipovitellin 1 (Lv1) derived from Vg1 and YP2 is lipovitellin 2 (Lv2) derived from Vg2. Using specific antibodies against Lvs and Vgs, specific, high sensitivity chemiluminescent immunoassays (CLIAs) for two Vgs were developed to reveal basal Vg levels and response to exogenous estradiol-17β (E2). The measurable range of both CLIAs was from 0.975 to 1000ng/ml. The cross-reactivity to the alternative Vg in each CLIA was extremely low (≦0.57%). When immature fish were immersed in water containing E2 for 1h, both Vgs were induced by 0.5μg/L of E2 at 24h after treatment. Vg1 increased in a concentration-dependant manner up to 100μg/L E2, while Vg2 reached a plateau at 10μg/L of E2. The ratio of Vg1:Vg2 in E2-treated fish changed in a concentration-dependent manner from 1.5:1 to 8.5:1. The results from E2-treatment suggest that differential regulation may control the expression of medaka Vgs. [Copyright &y& Elsevier]

Congenital diaphragmatic hernia (CDH) occurs in ∼1:2,500 human births and has high morbidity and mortality rates, primarily due to pulmonary hypoplasia and pulmonary hypertension. Tracheal occlusion (TO), in experimental animals, distends lungs and increases lung growth and alveolar type I cell maturation but decreases surfactant components and reduces alveolar type II cell density. We examined effects of CDH and CDH + TO on lung growth and maturation in fetal rats. To induce CDH, we administered nitrofen (100 mg) to dams at 9.5 days of gestation. We compared lungs from fetuses with CDH, CDH+TO, and those exposed to nitrofen without CDH. CDH decreased lung wet weight bilaterally (P < 0.0001) and DNA content in lung ipsilateral to CDH (P < 0.05). CDH+TO significantly increased lung wet weights bilaterally; DNA content was intermediate between CDH and NC. To evaluate effects on the distal pulmonary epithelium, we examined surfactant mRNA and protein levels, type I and II cellspecific markers (RTI40 and RTII70, respectively), and transcriptional regulator thyroid transcription factor-1 (TTF-1). Decreased lung distension (due to CDH) increased SP-C mRNA and TTF-1 protein expression and reduced RTI40 (P < 0.05 for all). Increased lung distension (due to CDH+TO) reduced expression of SP mRNAs and pro-SP-C and TTF-1 proteins and enhanced expression of RTI40 (mRNA and protein; P < 0.05 for all). We conclude that CDH+TO partially reverses effects of CDH; it corrects the pulmonary hypoplasia and restores type I cell differentiation but adversely affects SP expression in type II cells. These effects may be mediated through changes in TTF-1 expression. [ABSTRACT FROM AUTHOR]

Abstract: Annual changes in serum levels of two chorion precursors, choriogenin H (Chg H) and choriogenin L (Chg L), vitellogenin (Vg) and estradiol-17β (E2) were quantified in masu salmon, Oncorhynchus masou, using specific immunoassays. Serum Chg levels were higher than Vg during the previtellogenic growth phase when circulating E2 levels were low (∼0.1 ng/mL), suggesting higher sensitivity of Chg to E2. When oocyte growth shifted to the vitellogenic phase, Vg levels increased and became the most abundant in serum coincident with elevations of E2 and GSI. Chg H, Chg L and Vg peaked 1 month prior to ovulation at 0.61±0.08, 0.98±0.18 and 10.93±3.24 mg/mL, respectively. These results suggest that chorion formation by Chgs occurs prior to vitellogenesis and that the sensitivity of Chgs to low circulating E2 is closely related to the sequential events of oocyte growth. [Copyright &y& Elsevier]

In order to identify the dominant contributors to estrogenic activity in environmental waters, a comprehensive fractionation method using silica gel column chromatography, combined with recombinant yeast assay for detecting estrogenic activity and with gas chromatography-mass spectrometry for quantifying endocrine disruptors and natural estrogens, was developed. The method was applied to the municipal sewage treatment plant (STP) secondary effluent discharged to the Tamagawa River in Tokyo, Japan, where endocrine disruption was observed in wild carp. The instrumental analysis demonstrated that averaged concentrations of nonylphenol, bisphenol A, estrone (E1), and 17β-estradiol (E2) were 564 ± 127, 27 ± 19, 33 ± 11, and 4.6 ± 3.0 ng/L, respectively. Based on the concentration and relative potency of these compounds, the natural estrogens E1 and E2 represented more than 98% of the total estrogen equivalent concentration (EEQ) in the STP effluent, while the contribution of phenolic compounds to total EEQ was less than 2%. Estrogenic activities associated with the dissolved phase of the effluent samples were detected by a recombinant yeast assay. By using silica gel column chromatography, the dissolved phase was separated into several fractions that were subjected to the bioassay. The polar fractions exhibited estrogenic activity. The greatest estrogenic activity was found in a polar fraction containing E1 and E2 and represented 66 to 88% of the total estrogenic activities estimated from the bioassay data. These results lead to the conclusion that E1 and E2 were the dominant environmental estrogens in the STP effluent, but a significant contribution to estrogenic activities stems from unidentified components in the effluents. [ABSTRACT FROM AUTHOR]

To better understand the role of growth hormone in regulating fish growth, the cDNA of growth hormone receptor (GHR) was cloned from the liver of masu salmon (Oncorhynchus masou) and characterized. The masu salmon GHR (msGHR) sequence revealed common features of a GHR, including a (Y/F)GEFS motif in the extracellular domain, a single transmembrane region, and Box 1 and Box 2 in the intracellular domain. However, the amino acid sequence identity was low (⩽49%) compared to GHRs of other vertebrates including seven teleosts, and the putative msGHR protein lacked one pair of cysteine residues in the extracellular domain. To verify the identity of the msGHR, the recombinant protein of the extracellular domain was expressed with a histidine tag protein (His-msGHR-ECD), refolded and purified for analysis of its ligand specificity. In competition experiments, the specific binding between His-msGHR-ECD and radioiodine-labeled salmon GH was displaced completely by only salmon GH, and not by salmon prolactin or somatolactin. A real-time RT-PCR assay was used to measure salmon GHR mRNA in the liver of fed and fasted coho salmon (Oncorhynchus kisutch). The levels of hepatic GHR mRNA were lower in fasted fish compared to fed fish after 3 weeks, suggesting that GHR gene expression is reduced following a long-term fast. These results confirm the identity of the salmon GHR based on ligand specificity and response to fasting. [Copyright &y& Elsevier]

Previously two precursors to vitelline envelope proteins, choriogenin H (Chg H) and choriogenin L (Chg L), were identified in masu salmon, Oncorhynchus masou, and specific antisera against these two proteins were generated in rabbits. In this study, two methods of immunoassay have been developed using these specific antibodies: single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA). Non-competitive sandwich ELISAs for Chg H and Chg L were designed using digoxigenin-labeled antibodies and purified Chgs as assay components. The working range of the ELISAs was 1–128 and 2–256 ng/ml for Chg H and Chg L, respectively. Using these immunoassays and a chemiluminescent immunoassay for vitellogenin (Vg), the changes in these three estrogen-responsive proteins were measured in the serum of masu salmon after treatment with various doses of estradiol-17β (E2). The changes in serum levels of Chgs and Vg in male fish differed according to the E2 dose. When fish were given a 5 mg/kg body weight (BW) of E2, Vg was induced to a greater extent than Chgs. By contrast, Chg levels were higher than that of Vg after a 10 μg/kg BW of E2 injection. A similar trend was seen in the response time to exogenous E2. Serum Chgs were induced from 8 h after E2 injection and reached a peak of about 5 μg/ml at 24 h. Although Vg was not detected until 8 h after E2 injection, its levels remained considerably low at around 0.03 μg/ml, even after 24 h. Chg H was more sensitive than was Chg L to 1 μg/kg BW of estrogen: the long-term exposure of fish to E2 showed that Chg H could be induced from a lower dose of E2 than could Chg L. Taken together, these results suggest that the serum levels of Chg H, Chg L, and Vg in masu salmon are regulated by circulating levels of E2. [Copyright &y& Elsevier]

Surveys of the estrogenic activity in coastal areas of Japan were performed by using two serum vitellogenins (Vg-530 and Vg-320) of male Japanese common goby Acanthogobius flavimanus as biomarkers. To evaluate the relationship between the Vg concentrations in the serum of male fish and the concentrations of environmental estrogens in seawater, 3 week exposure experiments were performed using estradiol (E2), nonylphenol (NP) and bisphenol A (BPA). From the results of the E2 exposure, mean concentrations of the Vg and the ratio of Vg-positive fish increased from actual concentration at 10.5 ng/L E2 treatment. Exposure of NP and BPA showed that the 19.0 µg/L NP treatment induced the Vg but 133.7 µg/L BPA did not. As for the survey of the estrogenic activity in coastal areas, the mean Vg concentrations and ratio of Vg-positive fish in some urban areas were similar to values of the 10.5 ng/L E2 treatment. Although E2 and NP were detected in the seawater from urban areas, the concentrations were concluded as not sufficient to independently induce the Vg. Considering these results, it is concluded that the total estrogenic activity in some urban areas is dependent not only on E2 but synergistically with other environmental estrogens. [ABSTRACT FROM AUTHOR]

Two vitellogenins (Vgs) were detected in serum from estradiol-17β (E2)-injected Japanese common goby (Acanthogobius flavimanus). Vitellogenins with molecular masses of 530 kDa (Vg-530) and 320 kDa (Vg-320) were purified, and used to raise specific antisera in rabbits. Sandwich enzyme-linked immunosorbent assays (ELISAs) for Vg-530 and Vg-320 were developed using the antisera and the isolated Vgs. The sensitivity ranges of these ELISAs were 1.25–160 ng/ml for Vg-530 and 0.26–66 ng/ml for Vg-320, and very low cross-reactivity was found with the alternate Vg in each assay. Treatment of male gobys with E2 by injection and immersion induced both Vgs in sera in a dose-dependent manner. The mean concentrations of the Vgs increased from 10 ng/L E2 exposure for three weeks. Serum concentrations of the two Vgs in field-collected maturing females increased in accordance with increment of E2 level and ovarian development, and the mean concentrations of Vg-530 were higher than those of Vg-320 in maturing female. These results indicate that the sandwich ELISAs for Vg-530 and Vg-320 developed in the present study is useful as an assay system for surveys of estrogenic activity in coastal areas of Japan. [Copyright &y& Elsevier]

Vitellogenin (Vg) was purified from the serum of vitellogenic female carp (Cyprinus carpio) by hydroxylapatite column chromatography and gel filtration. Vg had an apparent molecular mass of 490 kDa and appeared as two bands corresponding to 190 and 156 kDa after SDS-PAGE under reducing conditions. These bands were immunoreacted in Western blotting using antiserum against carp lipovitellin (anti-Lv) which is an egg yolk protein derived from Vg. The amino acid composition of carp Vg was similar to previous reports of cyprinids. The chemiluminescent immunoassay (CLIA) for carp Vg was developed to quantify serum Vg using purified carp Vg and anti-Lv. Its measurable range was from 1.95 to 1000 ng/ml. The dilution curve in the CLIA of vitellogenic female serum was parallel to the standard curve of purified Vg. The coefficient variations of intra- and inter-assay were less than 5%, respectively. Furthermore, the assay had cross-reactivity with the sera of other female cyprinids (crucian carp and Japanese dace). In fish diets-experiments, Vg was detected in all fish in the fish meal containing soybean (20%) group, but was not detected in almost all of the fish in the fish meal-group. This suggests that a soybean based-diet may induce Vg production in the serum of cultivated carp. [Copyright &y& Elsevier]