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The activity of proteases may be lost during microquantification because of their nonspecific binding to tubes and pipette tips. In this study, the effects of blocking reagents (blockers) in dilutions of digestive proteases of yellowtail, Seriola quinqueradiata, were compared. EzBlock Chemi (EBC; protein free), Western BLoT Blocking Buffer (WBB; protein free), casein-based blocker, and bovine serum albumin were evaluated by the measurement of trypsin, chymotrypsin, and aminopeptidase activities using fluorogenic substrates. Protease activities were linear in extracts of pyloric caeca at dilution rates of up to at least 1/6400 with the four blockers. However, the activities of pancreatic enzymes differed, and the highest levels were observed with EBC and WBB. Extracts diluted with EBC and WBB were incubated for up to 24 h at 25 °C. Pancreatic enzyme activities increased with time in extracts diluted with EBC, but for those diluted with WBB there was no clear trend. The activities of chymotrypsin and aminopeptidase in whole-body extracts of individual yellowtail larvae extracted with EBC were significantly higher than in those extracted with 150 mM NaCl, suggesting that EBC inhibits the loss of enzyme activity that may otherwise occur during extraction. Thus, EBC is considered to be the most effective diluent for the microquantification of proteases amongst the four blockers tested.

The insulin family consists of insulin, IGFs, relaxins, and insulin-like peptides, and is one of the most phylogenetically ancient groups of hormones in animals. Mature peptides are the heterodimers comprising the A- and B-chains. The cysteine motif (Cys–Cys–3X–Cys–8X–Cys) in the A-chain has been termed the insulin signature. The insulin family has a broad spectrum of functions, including roles in nutrient metabolism, cell and organism growth, and reproduction. The receptors are classified into three structurally different types, the heterotetramer type with a tyrosine kinase domain, the GPCR type, and the cation-independent mannose-6-phosphate receptor, indicating that the receptors are phylogenetically polyphyletic. The various functions of the insulin family members are results of co-evolution of ligands and receptors encountered at several different times throughout evolution.

Increased risk of fracture associated with type 2 diabetes has been a topic of recent concern. Fracture risk is related to a decrease in bone strength, which can be affected by bone metabolism and the quality of the bone. To investigate the cause of the increased fracture rate in patients with diabetes through analyses of bone metabolism and bone matrix protein properties, we used goldfish scales as a bone model for hyperglycemia. Using the scales of seven alloxan-treated and seven vehicle-treated control goldfish, we assessed bone metabolism by analyzing the activity of marker enzymes and mRNA expression of marker genes, and we measured the change in molecular weight of scale matrix proteins with SDS-PAGE. After only a 2-week exposure to hyperglycemia, the molecular weight of α- and β-fractions of bone matrix collagen proteins changed incrementally in the regenerating scales of hyperglycemic goldfish compared with those of euglycemic goldfish. In addition, the relative ratio of the γ-fraction significantly increased, and a δ-fraction appeared after adding glyceraldehyde-a candidate for the formation of advanced glycation end products in diabetes-to isolated type 1 collagen in vitro. The enzymatic activity and mRNA expression of osteoblast and osteoclast markers were not significantly different between hyperglycemic and euglycemic goldfish scales. These results indicate that hyperglycemia is likely to affect bone quality through glycation of matrix collagen from an early stage of hyperglycemia. Therefore, non-enzymatic glycation of collagen fibers in bone matrix may lead to the deterioration of bone quality from the onset of diabetes.

We have previously shown that the somatic growth of barfin flounder, Verasper moseri, was promoted by green light. The present study was undertaken to elucidate whether growth-promoting effect of green light can be observed in other flatfishes and to understand the roles of endocrine systems in green light-induced growth. Herein, we demonstrated facilitation of growth by green light in the spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus. Blue and blue-green light showed potencies that were similar to that of green light, while the potencies of red and white light were equivalent to that of ambient light (control). We also examined the effects of green light on growth and endocrine systems of V. variegatus at various water temperatures. Growth of the fish was facilitated by green light at four different water temperatures examined; the fish were reared for 31 days at 12 and 21 °C, and 30 days at 15 and 18 °C. Increase in condition factor was observed at 15 and 18 °C. Among the genes encoding hypothalamic hormones, expression levels of melanin-concentrating hormone 1 (mch1) were enhanced by green light at the four water temperatures. Expression levels of other genes including mch2 increased at certain water temperatures. No difference was observed in the expression levels of pituitary hormone genes, including those of growth hormone and members of proopiomelanocortin family, and in plasma levels of members of the insulin family. The results suggest that green light may generally stimulate growth of flatfishes. Moreover, it is conceivable that MCH, production of which is stimulated by green light, is a key hormone; it augments food intake, which is intimately coupled with somatic growth.

Green light irradiation facilitates the somatic growth of barfin flounder ( Verasper moseri ). However, the V. moseri visual system, which may be associated with somatic growth by acting on the endocrine system upon exposure to this particular wavelength, remains largely unexplored. Herein, we characterized the visual opsin repertoire of V. moseri to understand the molecular basis underlying this effect. The five types of visual opsins that are found in vertebrates were cloned from RNA that was extracted from the eyes of V. moseri . Notably, V. moseri possessed one pseudogene ( RH2-A ) and two intact ( RH2-B and RH2-C ) copies of “green-sensitive” opsin genes. The wavelengths of maximum absorption spectra (λ max ) for each of the reconstituted photopigments were 552 nm for “red-sensitive” LWS , 506 nm for RH2-B , 490 nm for RH2-C , 482 nm and 416 nm for “blue-sensitive” SWS2A and SWS2B , respectively, 367 nm for “ultraviolet-sensitive” SWS1 , and 494 nm for “dim-light sensitive rhodopsin” RH1 . The λ max of SWS2A was longer than that of any other reported vertebrate SWS2 opsin. By measuring the expression level of these opsin genes with quantitative RT-PCR in 3-, 15-, and 27-month-old fish, we found that RH2-B and SWS2A were expressed at a constant level, whereas the expression of LWS , RH2-C , SWS2B , and SWS1 opsin genes decreased, and that of RH1 increased with age. Barfin flounders inhabit inshore waters at a young age and expand their habitat to deep sea areas as they age, and green light is relatively abundant in deep water compared to the lights of other wavelengths in shallow water. Our results indicate that gene repertoire and expression profile of the opsin genes of barfin flounder are adaptive to their habitat shift that occurs during development, with some opsins acquiring a distinct λ max .

We evaluated the effect of stress and adrenaline injection on insulin release in barfin flounder Verasper moseri. Flounders were fed to satiation and then exposed to 30 min of stress (chasing using a blunt stick), beginning 15 min after feeding. Plasma insulin concentrations were measured using a time-resolved fluoroimmunoassay for barfin flounder insulin. Peak insulin concentrations in the stressed flounders (3.4 ± 1.0 ng/ml, mean ± SEM) were lower than in the control group (8.3 ± 1.7 ng/ml). Similarly, in flounders injected with l-arginine (Arg; 3.5 mmol/kg body weight), the most potent insulinotropic factor for flounders, insulin levels were lower for up to 5 h in individuals exposed to chasing stress. Furthermore, plasma insulin levels did not increase after injection with Arg followed by exposure to 10 min of stress. This was also true for flounders co-injected with Arg (20 nmol/kg body weight) and adrenalin instead of the chasing stress. We conclude that stress induces a powerful and extended inhibition of insulin release in flounders, even when the stress is acute. We hypothesize that this effect is mediated partly by adrenaline release. Our results provide insight into the deleterious effects of stress on growth.

Insulin is a heterodimeric peptide linked by three disulfide bonds, and was first discovered in 1921. Insulin plays important roles not only in carbohydrate metabolism but also in anabolic regulation of proteins and lipids, somatic growth, and cell proliferation during development. Glucose stimulates the synthesis and release of insulin in mammals. In non-mammalian vertebrates the insulinotropic effect of glucose is weak but that of amino acids is strong, suggesting that the major role of insulin in blood glucose regulation is a relatively derivative function in vertebrate evolution. The actions of insulin are mediated differentially via two receptors (INSR-A and -B) that are alternatively spliced from a single mRNA in mammals. Moreover, hybrid receptors, which are composed of subunits of INSRs and IGF-IR, are present. Different substrate adaptors such as IRS proteins are involved in the signal transduction pathways of insulin action.

The insulinotropic effects of eighteen L-amino acids, two D-amino acids, and glucose were investigated to evaluate the priority of those as stimulators of insulin secretion in barfin flounder (Verasper moseri). This is also the first step in characterizing the insulinotropin-sensing molecule. After intramuscular injection of amino acids or glucose at doses of 3.50 and 1.75 mmol/kg body weight, plasma was collected periodically to determine plasma insulin level. Twelve amino acids and glucose showed insulinotropic effects. Four L-amino acids (Arg, Ala, Met, Ser) produced significantly higher integrated levels of plasma insulin (12.4-34.8 ng/ml) than glucose (average: 4.7 ng/ml) during 3h after injection. D-Amino acids (Arg, Ala) showed no activity. This indicates that many amino acids have strong insulinotropic activities and supports a classic idea, which is well known but has not been confirmed, that amino acids rather than glucose are the important insulinotropins in fish. This study also indicates that the insulinotropic activity of amino acids is restricted to L-amino acids and establishes which amino acids are the strongest stimulators of the insulinotropin sensor in barfin flounder. Co-injection of insulin and L-Thr, L-Ala, or glucose produced a hypoglycemic and hypoaminoacidemic state, indicating that insulin can lower blood amino acid level as well as blood sugar level. This study suggests that insulin plays a more important role than glucose in the regulation of blood L-amino acid metabolism, at least in flounder.

The reef-building (or hermatypic) coral Galaxea fascicularis (Anthozoa, Hexacorallia, Scleractinia) has an annual reproductive cycle. Females of G. fascicularis release packages (or ;bundles') of eggs for external fertilization, whereas male individuals form bundles consisting of sperm and infertile ;pseudo-eggs' that are thought to confer buoyancy to the male bundle. In the egg of G. fascicularis, four proteins (GfEP-1 to 4) were found to be stored in high abundance, and three of them (GfEP-1, 2 and 3) are generated by processing of a vitellogenin (Vg)-like precursor. In the present study, a cDNA encoding GfEP-4 was cloned and its sequence determined (GenBank/EMBL/DDBJ accession no. AB259859). The amino acid sequence of this protein does not exhibit similarity to known proteins, including Vgs or other yolk proteins found in some invertebrates. The expression of GfEP-4 mRNA was observed in females, and also in the majority of males examined, although expression levels were lower than in females. The GfEP-4 protein was detected in pseudo-eggs, where its concentration was 20-100 times lower than in eggs. In contrast, GfEP-1, 2 and 3 were not detected in pseudo-eggs. A protein (28 kDa) which cross-reacted with anti-GfEP-4 antibodies was detected in eggs of the coral Montipora digitata, suggesting the possibility that homologs of this protein are present in the eggs of other scleractinian corals.

Amino acids are important insulinotropins in fish, and their effects vary between amino acids and fish species. Insulin levels are indicative of growth efficiency and stress levels in fish; however, interspecies comparisons of insulin levels are hampered by the difficulty of measuring insulin concentration in each fish. We developed a widely applicable competitive immunoassay using biotinylated yellowtail (Seriola quinqueradiata) insulin for measuring insulin in marine teleosts, including yellowtail and red seabream (Pagrus major), which are the most common species raised by aquaculture in Japan. Amino acid sequence substitution was limited at the ninth residue of the A-chain (A9) between these two species, and analysis of the primary structures of insulins from six phylogenetically far teleosts suggested that the sequences of yellowtail and red seabream insulins are identical to those of many teleosts, except the A9 residue. However, A9 is known to be an epitope that confers cross-reactive differences on insulin. We solved this problem through immunoreactive invalidation of this residue by biotinylation. The binding-inhibition curves of yellowtail and red seabream insulins were identical following the use of this technique. However, yellowtail and red seabream plasma was found to contain components that interfere with immunoassays. This problem was solved by the extraction of plasma using equal volume of acid-ethanol in yellowtail and by cooling at 0°C during the cross-reaction between the ligand and antibody in red seabream. Serially diluted plasma samples from both species exhibited linearity after these treatments. In a recovery test using plasma with added yellowtail insulin, the average recovery varied from 96.2% to 109.4%. A post-feeding rise in insulin was confirmed by this immunoassay in yellowtail, and peak of the rise was 39.8±7ng/ml at 1h postfeeding from 3.9±1.1ng/ml at 0h. This indicates that this assay is sufficient for measuring the baseline concentration of plasma insulin after starvation and is a useful indicator of nutritional status in yellowtail, as in other teleosts. This immunoassay demonstrated high performance and resisted interference from plasma components; consequently, it constitutes a useful tool for the interspecies evaluation of insulinotropins and represents a widely applicable insulin immunoassay for many teleosts.

We investigated the effects of specific wavelengths of light on the growth of barfin flounder. The fish, reared in white tanks in a dark room, were irradiated with light from light-emitting diodes (LEDs) with peak wavelengths of 464nm (blue), 518nm (green), and 635nm (red) under a controlled photoperiod (10.5:13.5, light-dark cycle; 06:00-16:30, light). Fish were reared for four weeks in three independent experiments at three different water temperatures (averages of 14.9°C, 8.6°C, and 6.6°C). The fish irradiated with blue and green light had higher specific growth rates (% body weight⋅day(-1)) than fish irradiated with red light. Notably, green light had the greatest effect on growth among the three light wavelengths at 6.6°C. In the brains of fish reared at 6.6°C, the amounts of melanin-concentrating hormone 1 mRNA under green light were lower than those under red light, and amounts of proopiomelanocortin-C mRNA under blue and green light were higher than those under red light. No differences were observed for other neuropeptides tested. In the pituitary, no difference was observed in growth hormone mRNA content. In plasma, higher levels of insulin and insulin-like growth factor-I were observed in fish under green light than those of fish under red light. These results suggest that the endocrine systems of barfin flounder are modulated by a specific wavelength of light that stimulates somatic growth.

In the egg of the reef coral Galaxea fascicularis, four proteins (named GfEP-1 to -4) are stored in high abundance. In the present study, a cDNA containing a full-length open reading frame for GfEP-1 was cloned, and the translated protein sequence was compared to the N-terminal sequences of GfEP-2, -3, and -4. GfEP-1 and -2 were shown to be generated by processing of a precursor of 1439 amino acids, and GfEP-3 turned out to be a partial fragment of GfEP-2. The precursor protein contained regions which exhibited similarities to vitellogenins (Vgs) in bilaterian animals (oviparous vertebrates and invertebrates including nematodes, arthropods, and molluscs). This study reports the first cloning and characterization of a full-length cDNA encoding a Vg in a non-bilaterian animal, and argues that the emergence of Vg as a precursor of egg yolk proteins predated the divergence of the cnidarian and bilaterian lineages.

A time-resolved fluoroimmunoassay system (TR-FIA) for measuring flounder insulin-like growth factor-I (IGF-I) was developed using biotinylated flounder IGF-I, anti-fish IGF-I antiserum and europium-avidin conjugate. The detection limit per well was5 pg/well corresponding to0.5 ng/ml in a basic procedure for sample of 10 microl/well and to0.08 ng/ml in a procedure modified for high volume samples (up to 70 microl/well). Specificity of the assay was validated using various IGF-Is and insulins. All IGFs except seabream IGF-I showed very low or no crossreactivity. Binding inhibition curves for flounder and seabream IGF-Is were completely identical to each other. Intra- and interassay variations ranged from coefficients of variations of 3.9% to 7.2%. Recovery tests using barfin flounder plasma varied from 82.7 to 101.6% in the added range from 20 to 160 ng/ml. This assay system was applied for measuring total plasma IGF-I in barfin flounder injected porcine growth hormone (GH). A group injected with GH at the dose of 0.05 IU/gBW showed a significant increase of total plasma IGF-I compared with those of albumin-injected (control) and initial groups. In addition, I was able to substitute time-resolved fluorometric detection in this assay system with enzymatic fluorometric detection (FIA). Binding inhibition curve for flounder IGF-I in this substituted assay system showed equal performance with that of the TR-FIA system. Correlation of IGF-I levels between TR-FIA and FIA was high (r(2)=0.957) in plasma samples from barfin flounders in various physiological conditions. Thus, the present study shows precision and efficiency of two non-radioisotopic immunoassay systems for measuring flounder IGF-I.

Vitellogenin (Vg) is the precursor to egg yolk proteins of teleost fishes, including lipovitellin (Lv), phosvitin (Pv), and β′-component (β′-c). Complete Vg molecules contain the yolk protein domains arranged in linear fashion: NH2-Lv heavy chain, Pv, Lv light chain, β′-c, C-terminal coding region (C-t)-COOH. Western blots employing an antiserum raised against a recombinant C-t polypeptide revealed that the C-t domain of Vg gives rise to a fourth yolk protein in barfin flounder oocytes. Three classes of Vg appear to exist in teleosts, including two types of complete Vg (A-type and B-type) and a smaller, incomplete Vg lacking a Pv domain (Pv-less type). By sequencing cDNA amplified from distinctive regions of each type of Vg transcript from several teleosts, we discovered that members of higher taxa (e.g. Paracanthopterygii and Acanthopterygii) express both Vg A and Vg B, and that Pv-less Vg is widely present among teleosts. Vitellogenins A and B play distinct roles in regulation of egg buoyancy in barfin flounder through selective proteolysis of their product yolk proteins in oocytes undergoing final maturation. Protease specification procedures confirmed involvement of a cathepsin B-like enzyme in this maturation-associated proteolysis. Measurement of changing pH revealed that drastic acidification of the ooplasm accompanies hydrolysis of the yolk proteins. Thus, with respect to yolk protein hydrolysis and oocyte hydration, cytoplasmic maturation appears to be controlled by changing pH in maturing oocytes.

Our previous study shows that seminal plasma of a teleost, the Nile tilapia, contains a glycoprotein Mr = 120,000 named as SPP (Seminal plasma glycoprotein)120 which forms a homopolymer that has sperm immobilizing activity. In order to elucidate the mechanisms of the formation of the homopolymer and the immobilization of sperm, molecular cloning of SPP120 was conducted. The cDNA for SPP120 contains a complete open reading frame encoding 797 amino acid residues with 14 potential N-glycosylation sites. The predicted amino acid sequence of SPP120 contains a partial von Willebrand factor type D domain and a zona pellucida domain, that are involved in protein-protein adhesion that form filamentous structures in various kinds of cells. This result suggests that SPP120 forms a homopolymer via these domains in seminal plasma and probably interacts with spermatozoa. Northern blotting reveals that the gene is also expressed in ovary, even in ovulated eggs. The results of in situ hybridization indicate that in testis the gene is expressed in Sertoli cells and epithelial cells of sperm ducts, and the localization corresponds to that of the protein analyzed by immunohistochemistry. In the ovary, the gene is expressed at the perinucleolus stage of oocytes; however, the protein is not detected in any cells other than oocytes.

We evaluated whether transcription or translation of the preproinsulin gene or insulin release into plasma is the primary regulator of plasma insulin level in barfin flounder. Three experimental groups were used: one tested 2h after feeding (Fed), one tested after fasting for 5 days (Fasted), and one tested 2 h after feeding following 5 days of fasting (Refed). No significant differences in insulin transcription, insulin concentrations in the principal islets (PI), or plasma total insulin-like growth factor-I (IGF-I) levels were observed between the three groups. In contrast, plasma insulin level in the Fasted group was significantly lower (P0.002) than that in the other groups. These results suggest that insulin release is the primary regulator of plasma insulin level and is more sensitive to short-term changes in nutritional conditions than IGF-I level. Furthermore, we estimated the capacity for insulin release. Based on various individual measures, the average insulin stored in the PI was 82.8 μg/kg body weight (BW), and the maximum plasma content of insulin was estimated to be1.7 μg/kg BW. The half-life of plasma insulin in diabetogenic chemically (alloxan) treated flounder injected with insulin was estimated to be 2.79 h, which is much longer than that in mammals, assuming a two-compartment model for the β phase. These results suggest that the capacity for insulin release in fish is ensured by at least two systems, such as the ability to store excess insulin in Brockman bodies, and enhanced efficiency of insulin storage by elongating its half-life.

To understand the developmental process of the endocrine system, which controls the pancreatic exocrine function in Japanese flounder, Paralichthys olivaceus, the expression patterns of cholecystokinin (CCK) and pancreatic polypeptide (PP) during the larval stage were analyzed by immunohistochemistry and in situ hybridization. Expression of CCK in the intestinal epithelia started at 2 days posthatching (dph), 1 day prior to the first feeding. Endocrine pancreatic cells containing insulin were present in the pancreas primodium at hatching, and these endocrine cells formed an islet at 3 dph and developed into the principle islet neighboring gallbladder at 20 dph. However, PP cells were not contained in the principle islet even after metamorphosis. PP cells were contained in the accessory islets differentiated around the proximal part of the pyloric appendages at 30 dph (early metamorphosis stage). Therefore, we infer that the stimulative regulation of pancreatic enzyme secretion by CCK starts to function at the first feeding, whereas the restrictive regulation by PP develops about 1 month later in flounder larvae. In addition, we observed that CCK immunoreactive cells appeared in the accessory islets at 30 dph, similar to PP cells, even though CCK mRNA expression could not be detected in cells from the islets. This indicates the possibility that a peptide that is cross-reacted with the CCK antibody, i.e., gastrin, is synthesized in the flounder islets.

Insulin-I and -II were purified from stone flounder (Kareius bicoloratus), and their primary structures were determined. The amino acid sequences of insulin-I and -II from stone flounder were identical with those of barfin flounder (Verasper moseri) except for position 2 of both B-chains. Insulin-II of stone flounder had an extension of the two amino acid residues (Pyr-Ala), at the N-terminus of the B-chain. These structural characteristics of insulins from stone flounder support the idea (Andoh and Nagasawa, Zool. Sci. 15: 901, 1998) that insulin-I and -II of flounders arise from a single preproinsulin in each species by proteolytic cleavage at different sites of the signal peptide region, and suggest that this generation system of two molecular forms of insulin is not specific for barfin flounder. In the course of the purification of insulins, somatostatin-14 and two glucagons (glucagon-I and -II) were also purified from the extract of the Brockmann body. The amino acid sequence of somatostatin...

We have purified and characterized two molecular forms of insulin from the Brockmann bodies of barfin flounder, Verasper moseri: a normal type of insulin (insulin-I), which consisted of 21 amino acid residues for the A-chain and 30 residues for the B-chain, and a novel type of insulin (insulin-II), which had an extension of two amino acid residues at the N-terminus of the B-chain. The additional two residues at the N-terminus of B-chain of insulin-II were Pyr-Ala which had not yet been reported in vertebrate insulins. Except for these two residues, the amino acid sequence of insulin-II was completely identical with that of insulin-I. Each Brockmann body extract from five individuals contained both insulins, indicating that insulin-I and -II were the products of non-allelic expression. By polymerase chain reaction, only one nucleotide sequence of preproinsulin gene encoding insulin-I and -II was obtained, and the amino acid sequence of A-and B-chains deduced from the nucleotide sequence was identi...

Ghrelin is a peptide hormone that is acylated with a fatty acid, usually n-octanoic acid, at the third amino acid residue (usually a serine or threonine), and this acylation is known to be essential for ghrelin activity not only in mammals but also in non-mammals, such as fish. However, the modification mechanisms of ghrelin modification in fish are not known. In this study, we elucidated the structure of ghrelin in a teleost, the barfin flounder (Verasper moseri), and determined whether ingested free fatty acids of various chain lengths participated in ghrelin acylation. Complementary DNA cloning revealed the barfin flounder prepro-ghrelin to be a 106-amino acid (aa) peptide and the mature ghrelin to be a 20-aa peptide (GSSFLSPSHKPPNKGKPPRA). However, purification of ghrelin peptides from stomach extracts demonstrated that the major form of the hormone was a 19-aa decanoylated peptide (GSS[C10:0]FLSPSHKPPNKGKPPR) missing the last alanine of the 20-aa peptide. Ingestion of feed enriched with n-heptanoic acid (C7), n-octanoic acid (C8), or n-nonanoic acid (C9) changed the modification status of the peptide: ingestion of C8 or C9 increased the amount of C8:0 or C9:0 19-aa ghrelin, respectively, but no C7:0 ghrelin was isolated after ingestion of C7. These results indicate that ingested free fatty acids are substrates for ghrelin acylation in the barfin flounder, but the types of free fatty acids utilized as substrates may be limited.

In some animals, such as fish, insects, and cephalopods, the thick egg coat has a narrow canal—a micropyle—through which spermatozoa enter the eggs. In fish, there is no indication that spermatozoa are attracted by eggs from a distance, but once spermatozoa come near the outer opening of the micropyle, they exhibit directed movement toward it, suggesting that a substance exists in this defined region to attract spermatozoa. Since Coomassie Blue (CB) binds preferentially to the micropyle region in flounder, herring, steelhead, and other fish, it probably stains this sperm guidance substance. This substance—a glycoprotein based on lectin staining—is bound tightly to the surface of the chorion, but can be removed readily by protease treatment. Although fertilization in fish (flounder) is possible after removal of this substance, its absence makes fertilization inefficient, as reflected by a drastic reduction in fertilization rate. The sperm “attraction” to the micropyle opening is species specific a...

In some animals, such as fish, insects, and cephalopods, the thick egg coat has a narrow canal-a micropyle-through which spermatozoa enter the eggs. In fish, there is no indication that spermatozoa are attracted by eggs from a distance, but once spermatozoa come near the outer opening of the micropyle, they exhibit directed movement toward it, suggesting that a substance exists in this defined region to attract spermatozoa. Since Coomassie Blue (CB) binds preferentially to the micropyle region in flounder, herring, steelhead, and other fish, it probably stains this sperm guidance substance. This substance-a glycoprotein based on lectin staining-is bound tightly to the surface of the chorion, but can be removed readily by protease treatment. Although fertilization in fish (flounder) is possible after removal of this substance, its absence makes fertilization inefficient, as reflected by a drastic reduction in fertilization rate. The sperm "attraction" to the micropyle opening is species specific and is dependent on extracellular Ca(2+). Eggs of some insects, including Drosophila, have distinct micropyle caps with CB affinity, which also may prove to assist sperm entry. Our attempts to fertilize fly eggs in vitro were not successful.

We constructed ammonia monooxygenase alpha subunit (amoA) gene clone libraries of ammonia-oxidizing archaea (AOA) and bacteria (AOB) from three biofiltration tanks used for closed marine fish culture systems. The number of operational taxonomic units (OTUs) found in any one place was 76%–80% of the total OTUs in each tank for AOA and 100% for AOB when OUTs were defined on the basis of a 5% nucleotide difference. In a phylogenetic tree, all of the AOA amoA sequences fell into a cluster, which contained Candidatus Nitrosopumilus maritimus. All of the AOB amoA sequences were related to the Nitrosospira lineage. These results indicated that different ammonia oxidizers were present in different tanks, but that the dominant phylogenetic types were stable. In a biofiltration tank to which a high concentration of ammonium chloride was added periodically to condition the biofilter materials, most of the AOA amoA sequences were different from the dominant one observed in the fish culture tanks. The AOB amoA sequences were also different, and were similar to those of Nitrosomonas aestuarii. These findings suggest that high concentration ammonia loads have a considerable affect on ammonia-oxidizer community composition.

The fish egg is surrounded by a thick envelope called the chorion. The fertilizing spermatozoon enters the egg through a canal-like structure in the chorion, the micropyle. Examination of micropyle at fertilization is difficult if eggs are large and have no distinct landmarks surrounding the micropyle, or if they are positively buoyant in water. Eggs of many commercially important fishes (e.g., flounder, sea bream and eel) are buoyant in water or only slightly adhere to solid objects (e.g., sands, rock and water plants), which makes observation of spermatozoa at fertilization difficult. Here, we report that such eggs can be firmly attached to plastic and glass dishes that have been previously coated with poly-L-lysine. These adhering eggs can be fertilized and develop normally on the dishes. Observations of micropyles of three fish species, before and after sperm entry are presented.

The presence of endothelin (ET)-like immunoreactivity and the cardiovascular effects of mammalian ET-1 in fish have been reported. To identify ET-related peptides in fish, we screened the cDNA library of the salmon (Oncorhynchus keta) stomach by means of rapid amplification of cDNA ends, and we cloned cDNAs encoding an ET-related peptide. The salmon ET-related sequence of 21 amino acids is identical to the trout ET-1 peptide recently purified from kidney specimens of Oncorhynchus mykiss. The deduced amino acid sequence of salmon pre-proET-1 (PPET-1) comprises 244 amino acids, including a putative signal sequence and mature ET-1, as well as big ET-1 and ET-1-like sequences. This precursor, the first reported PPET-1 sequence for Salmoniformes, Teleostei, has low homology with the sequences of human, mouse, frog (Xenopus laevis), and zebrafish (Danio rerio) PPET-1 (26%, 29%, 24%, and 39%, respectively).

The objectives of this study were to characterize multiple forms of vitellogenin (Vg) in mosquitofish (Gambusia affinis) and to discover the fate of each Vg during its processing into product yolk proteins. Two Vg preparations, with apparent masses of 600 kDa (600 Vg) and 400 kDa (400 Vg), were isolated from the plasma of fish treated with estradiol-17beta (E(2)) by various chromatographic procedures. Immunological analyses verified the presence of two different Vg proteins (600 VgA and 600 VgB) in the 600 Vg preparation and of a single protein in the 400 Vg preparation. Three major yolk proteins (Yps) with apparent masses of 560, 400, and 28 kDa were observed in extracts of ovarian follicles from vitellogenic females. Immunological analyses demonstrated that the 400 Vg underwent no change in native mass after being incorporated into oocytes. The 600 Vgs gave rise to a 28 kDa beta'-component and a native 560 kDa Yp, which was heterodimeric in structure, consisting of two types of complexes between phosvitin (Pv) and lipovitellin (Lv) heavy- and light-chains. Full-length cDNAs encoding the 600 VgA, 600 VgB, and 400 Vg were isolated from a liver cDNA library of E(2) treated fish. Similar to the zebrafish vg3 gene, the 400 Vg cDNA lacked a Pv domain and was classified as an incomplete or phosvitinless (C-type) Vg. The deduced primary structures of 600 VgA and 600 VgB were complete, and these were categorized as type A and type B Vgs, respectively, according to our recent classification scheme. This is the first report on the characterization of three functional Vg genes and their circulating and yolk protein products in any vertebrate species.

We report herein a rare case of esophageal carcinoma producing alpha-fetoprotein (AFP). A 69-year-old man presenting elevated AFP was admitted in order to investigate its origin, which several liver examinations done before admission had not revealed. At admission, his serum AFP was 76.9 ng/ml whereas other tumor markers were within normal range. As the patient complained of mild swallowing disturbance, gastrointestinal examinations were performed, and an esophageal carcinoma was found at the esophagogastric junction. The patient underwent subtotal esophagectomy and the esophagus was reconstructed by gastric tube. The postoperative course was uneventful and the serum AFP level normalized immediately after the operation. Histopathological examination demonstrated the tumor to be poorly-differentiated adenosquamous carcinoma, which contained scattered adenocarcinoma composed of clear cells positive to AFP by an immunohistochemical stain. The patient has been well for six months after the surgery without any sign of recurrence.

Complete nucleotide sequences of two forms of vitellogenin (Vg) cDNA in Japanese common goby were determined from a liver cDNA library of E(2)-treated male fish. These two Vg cDNAs contained complete open reading frames encoding 1664 and 1238 amino acid residues including signal peptides, respectively. From comparison of the deduced amino acid sequences of both Vgs and the partial amino acid sequences of the yolk proteins, the longer sequence was concluded to be cDNA of the Vg-530 and the shorter one was that of the Vg-320 of the Japanese common goby which were reported in our previous paper. The deduced sequence of Vg-530 without signal peptide was arranged by lipovitellin heavy-chain (LvH), phosvitin (Pv), lipovitellin light-chain (LvL), and beta'-component beta'-c) domains from the N-terminus, and showed a range of 40-45% sequence identity to those of other fish. Furthermore, the deduced sequence of Vg-320 showed no obvious Pv domain, has a shortened C-terminal coding region after the LvH domain, and showed a close similarity to the phosvitin-less Vg of zebrafish. Moreover, biochemical analysis of the yolk proteins verified that Vg-530 cleaves into the Lv-Pv complex (molecular mass: 470 kDa) and beta'-c (33 kDa), while Vg-320 showed no change when incorporated into oocytes. The present study demonstrated the existence of the two different forms of Vgs at both the cDNA and protein level, and showed molecular alteration of the two Vgs during vitellogenesis. Two Vg sequence data will aid in designing nucleotide probes for detecting Vg gene expressions as a biomarker of environmental estrogens.

We previously produced four monoclonal antibodies to testicular proteins of a teleost, the Nile tilapia. One of the monoclonal antibodies, TAT(Testicular Antigen of Tilapia)-10, recognizes a Mr=27,000 protein (27 kD protein), which is present in A and early B type spermatogonia, spermatids, and spermatozoa in testis. In order to clarify the function of this protein, molecular cloning was conducted. The cDNA for the 27 kD protein contains a complete open reading frame encoding 220 amino acid residues. The predicted amino acid sequence of the 27 kD protein was homologous to those of the ubiquitin carboxy-terminal hydrolases (UCH) reported in mammals. The measurement of the ubiquitin-releasing activity of the recombinant 27 kD protein revealed that the protein is the active form of UCH. Northern blot analysis showed that the UCH mRNA was expressed in ovary and brain in addition to the testis. Immunohistochemical study showed that, in brain, UCH was localized especially on the olfactory organ including the olfactory bulb and olfactory epithelium in olfactory rosetta, suggesting the involvement of the protein in chemoreceptive function. In the Tilapia ovary, UCH localized especially in pre-vitellogenic oocytes, suggesting that the enzyme activity could be important in oocyte growth. This is the first report for the cDNA cloning and cellular localization of UCH in fish. J. Exp. Zool. 293:368-383, 2002.

A time-resolved fluoroimmunoassay (TR-FIA) system was developed to quantify insulin levels in the barfin flounder. This TR-FIA system is a solid-phase assay based on competition of unlabeled insulins and biotinylated barfin flounder insulin-II against an anti-barfin flounder insulin-II antibody. The minimum detectable level of barfin flounder insulin-I and -II in this TR-FIA was 10 pg/well which corresponded to 1.0 ng/ml, and insulin-II showed slightly higher crossreactivity than insulin-I. The accuracy of this TR-FIA was assured by specificity test, validation test, and recovery test using plasma added insulin-II. The results indicated the high specificity and sufficient accuracy of this assay system for insulin level measurement. This system was applied to the measurement of plasma insulin levels of fed and fasted barfin flounders. Plasma insulin levels (average +/- SEM) in fed flounders reached a maximum 2 h (9.3 +/- 1.7 ng/ml) and decreased gradually thereafter, while those in fasted flounders remained at low levels (1.1 +/- 0.1-2.0 +/- 0.2 ng/ml) during the experiment. After removing proteins by acidification and subsequent gel filtration, plasma samples taken from fed and fasted flounders at 2 h after feeding were fractionated separately by reversed-phase HPLC. In fed flounders, insulin immunoreactivity was detected in fractions corresponding to those of insulin-I or -II. The ratio of integrated insulin immunoreactivities of each peak was 0.378 +/- 0.044 (average +/- SD). This value was in good agreement with those (0.355 +/- 0.019) of absorbance areas of each insulin from Brockmann body extracts of the barfin flounder on reversed-phase HPLC. In fasted flounders, very weak insulin immunoreactivities were observed at retention times corresponding to those of insulin-I and -II. These results indicated that both insulin-I and -II were secreted into the blood being induced by feeding stimulation with approximately the same ratio as that of the quantities harbored in the Brockmann body.

Five molecular forms of glucagon and two molecular forms of insulin were characterized from the kaluga sturgeon. Substitutions occurred at two to thirteen internal amino acid residues among the five molecular forms of glucagons, indicating that these glucagons were encoded by five distinct genes. The amino acid sequences of two insulins from the kaluga sturgeon were identical to those of paddlefish insulin-II and Russian sturgeon insulin except that kaluga sturgeon insulin-I had an extension of five residues at the B-chain N-terminus. This is the first demonstration that more than two molecular forms of glucagon have been characterized from a single animal species.

Two forms of vitellogenin (Vg), Vg A and Vg B, were identified in serum from estrogen-treated barfin flounder (Verasper moseri). Structural changes of lipovitellins (Lvs) derived from the two Vgs were examined during vitellogenesis and oocyte maturation. Two Lvs, vLv A and vLv B, were identified electrophoretically and immunologically in postvitellogenic oocytes. Each appeared to be composed of distinct heavy chains (vLvH A, M(r) 107,000, and vLvH B, M(r) 94,000) and light chains (vLvL A, M(r) 30,000, and vLvL B, M(r) 28,000) when analyzed by SDS-PAGE. Results from N-terminal amino acid sequencing and Western blotting using antisera to vLvH A and vLvH B verified that there are two Vg polypeptides in serum from estrogen-treated fish, Vg A (M(r) 168,000) and Vg B (M(r) 175,000), which give rise to vLvH A-vLvL A and vLvH B-vLvL B, respectively. N-terminal sequencing revealed two sequences for both phosvitin and beta'-component, supporting the concept of duality for all three classes of Vg-derived yolk proteins. During oocyte maturation, native dimeric vLv B was dissociated into a native M(r) 170,000 monomer (oLv B). Meanwhile, vLv A was extensively cleaved including complete degradation of vLvH A into free amino acids. We propose that the quantitative ratio of vLv A to vLv B in postvitellogenic oocytes regulates the buoyancy of the spawned pelagic eggs by controlling availability of free amino acids which function as osmotic effectors during oocyte hydration. The vLv A/vLv B ratio likely also controls the proportional availability of different types of nutrients, free amino acids versus Lv, for use during embryonic development.

The profile of fecal bile acids was examined in 13 children with short bowel syndrome; 7 of the 13 did not have diarrhea and the other 6 had intractable diarrhea. In children without diarrhea, no severe fat malabsorption was recognized, and the content of total bile acids in the feces was within the normal range or slightly higher. The ratio of primary to total bile acids showed various patterns. In children with intractable diarrhea, in contrast, fat malabsorption was observed and the fecal content of total bile acids in these patients was more than ten times higher than that of the control group, primary bile acids accounting for more than 95% of the total bile acids and taurine- or glycine-conjugated bile acids for 10%. In the children with intractable diarrhea, the values for the D-xylose absorption test were lower than the normal range. These results suggested that, in children with short bowel syndrome with diarrhea, the loss of bile acids was strongly associated with a decrease in the actual absorptive surface area of the residual small intestine, and the growth of the normal bacterial flora was disturbed in the residual intestine. Some children with or without diarrhea also had hyper bile acidemia. Ursodeoxycholic acid was not effective for the treatment of hyper bile acidemia or fat malabsorption.

Recently defined formula diets are widely used for patients with digestive diseases. Long-term administration of such diets is presumed to change the absorptive characteristics of the small intestine. We investigated the influence of the defined formula diet on the absorptive capacity of growing rats by measuring the potential difference of sugars, amino acid, half-maximum concentration (kt) of these substrates, the activities of disaccharidase and dipeptidase, and the portal amino acid concentrations. There was no significant difference in the body weight of rats fed amino acid or peptide diets and those given the normal chow, but the administration of the defined formula diets reduced the absorption of amino acid and small peptide per serosal area and kt. On the other hand, absorption of sugars was not significantly influenced by the type of the diets. The differences in the absorptions of amino acid and peptide following the administration of the defined formula diet might be associated with the change in the resistance of the unstirred water layer or the alteration in the active transport system of amino acids or peptides in the small intestine. No significant differences were observed between the influences of the amino acid and the peptide diets.

Polypatric speciation from an amphidromous ancestor has been suggested for a fluvial gobyRhinogobius flumineus; a similar history may explain life-history and genetic variation inCottus species. We used starch-gel electrophoresis to examine intra- and interspecific genetic differentiation of two siblingCottus species, amphidromousC. amblystomopsis and fluvialC. nozawae, which are presumed to be in an ancestor-descendant phyletic relationship. Genetic differentiation was markedly less withinC. amblystomopsis, than inC. nozawae. One group ofC. nozawae populations was closer toC. amblystomopsis (by Nei's genetic distance) than it was to other populations ofC. nozawae. This suggests that the differentiation of presently identifiable groups ofC. nozawae populations might have occurred polypatrically from an amphidromous ancestral population at different times. The two groups are presumed to be different species in spite of their quite similar morphological and ecological characteristics: we propose to call this type of speciation ‘Polypatric and parallel speciation’. This model of ‘Polypatric and parallel speciation’ might be generalized to explain evolution of amphidromous or anadromous species to fluvial or lacustrine land-locked species.

An electrophoretic study on the biochemical genetics of two sibling river-sculpinsCottus amblystomopsis andC. nozawae was undertaken with the primary objective of clarifying the reproductive isolation between the sympatric populations in three rivers around Cape Erimo of Hokkaido, where their distributions overlap widely along the river courses. At the 3 lociAcp,Ldh and6Pgd, out of 20 examined loci, evident displacement of alleles were observed between the two species. In addition, no genetical evidences for hybridization between the two species were detected in the three rivers examined. These results strongly suggest that the two species are reproductively isolated from each other even when they are distributed sympatrically and their distributions overlap widely along the course of a river.

ブタ肝移植において移植後のエンドトキシン (Et) の変動について検討した. 3時間 (n=5) および12時間保存 (n=2) の移植モデルで全例において血中Etが検出され, 移植後24時間の間に8～380pg/mlのピ-ク値を示し以後漸減した. このEtは従来の合成基質法では陽性に検出されたがβ-glucan感受性因子を選択的に除去したエンドトキシン特異テストでは全く検出されなかった, また移植前後での血液培養は全例が陰性であった. この現象はヒトの肝不全時にみられる, 矢島らのいうところのnon-septic endotoxemiaと同一のものであると考えられた. 移植肝の状態が悪く, 数時間で死亡した例では血中Etが低値を示す傾向が認められ, この意味で生着予後の1つの指標となりうると考えられたが, この現象は従来のspill-over学説では説明困難であった.