Evaluation of the in vivo and in vitro interleukin-12 p40 and p35 subunit response in yellowtail (Seriola quinqueradiata) to heat-killed Lactobacillus plantarum strain L-137 (HK L-137) supplementation, and immersion challenge with Lactococcus garvieae.

Dietary supplementation of immunostimulants might be effective to reduce the economic losses due to infectious diseases and the use of antibiotics in aquaculture. To investigate the immune response of interleukin-12 (IL-12) in yellowtail Seriola quinqueradiata to heat-killed Lactobacillus plantarum strain L-137 (HK L-137), we performed a leukocyte culture, feeding trial with diets containing L-137 and an immersion challenge with Lactococcus garvieae. IL-12 (IL-12p70) is a heterodimeric cytokine composed of IL-12p35 and IL-12p40 subunits. In the yellowtail-leukocyte culture, HK L-137 treatment stimulated the mRNA expression of one IL12p35 subunit (p35a) and all IL12p40 subunits (p40a, p40b, and p40c) in a dose-dependent manner. Furthermore, mRNA expression of type-I helper (Th-1) cytokine (tumor necrosis factor α, TNF-α, and interferon γ, IFN-γ) was also stimulated by HK L-137. After 6 weeks of feeding yellowtails with diets containing 0, 20, and 100 ppm of HK L-137, the mRNA expression of p35a and p40b in the spleen leukocytes increased with the dietary concentration of HK L-137, and that of p40b, p40c, and ifng in the head kidney leukocytes were the highest in the 20 ppm HK L-137 group. Survival rates in the 20 ppm HK L-137 group after immersion challenge with L. garvieae were significantly higher than the control (0 ppm of HK L-137). The 100 ppm HK L-137 group did not significantly suppress mortality. HK L-137 showed immunostimulant activity by increasing the expression of il-12, tnfa, and ifng mRNA in both in vitro and in vivo tests in yellowtail. Our results suggest that dietary supplementation with 20 ppm HK L-137 is the most efficient dose for improving immunity in yellowtail. Furthermore, a high dose of HK L-137 and/or long-term feeding of a diet containing HK L-137 might suppress the immune response, which probably decreases the survival rate of fish. To maintain a high immune response in yellowtail, the optimal dietary concentration of HK L-137 and/or feeding regime should be investigated further.

Corrigendum to 'Evaluation of the in vivo and in vitro interleukin-12 p40 and p35 subunit response in yellowtail (Seriola quinqueradiata) to heat-killed Lactobacillus plantarum strain L-137 (HK L-137) supplementation, and immersion challenge with Lactococcus garvieae' [Fish and Shellfish Immunology Reports 4 (2023) 100095].

[This corrects the article DOI: 10.1016/j.fsirep.2023.100095.].

Removal behaviors of sulfamonomethoxine and its degradation intermediates in fresh aquaculture wastewater using zeolite/TiO2 composites.

Removal efficiencies of sulfamonomethoxine (SMM) and its degradation intermediates formed by treatment with zeolite/TiO2 composites through adsorption and photocatalysis were investigated in fresh aquaculture wastewater (FAWW). Coexistent substances in the FAWW showed no inhibitory effects against SMM adsorption. Although coexistent substances in the FAWW inhibited the photocatalytic decomposition of SMM, the composites mitigated the inhibition, possibly because of concentration of SMM on their surface by adsorption. LC/MS/MS analyses revealed that hydroxylation of amino phenyl and pyrimidinyl portions, transformation of the amino group in the amino phenyl portion into a nitroso group, and substitution of the methoxy group with a hydroxyl group occurring in the initial reaction resulted in the formation of various intermediates during the photocatalysis of SMM. All detected intermediates had a ring structure, and almost all intermediates disappeared at the same time SMM was completely decomposed. Ph-OH formed by hydroxylation of the phenyl portion was detected upon decomposition of SMM during photocatalysis. The removal of Ph-OH by the composites proceeded more rapidly than that by TiO2 alone under ultraviolet irradiation. The SMM and Ph-OH were completely degraded by the composites within 30min, showing that the zeolite/TiO2 composites were effective in removing SMM and its intermediates from FAWW.

Molecular cloning and tissue distribution of cholecystokinin-1 receptor (CCK-1R) in yellowtail Seriola quinqueradiata and its response to feeding and in vitro CCK treatment.

In vertebrates, the peptide cholecystokinin (CCK) is one of the most important neuroregulatory digestive hormones. CCK acts via CCK receptors that are classified into two subtypes, CCK-1 receptor (CCK-1R; formally CCK-A) and CCK-2 receptor (formally CCK-B). In particular, the CCK-1R is involved in digestion and is regulated by CCK. However, very little information is known about CCK-1R in fish. Therefore, we performed molecular cloning of CCK-1R cDNA from the digestive tract of yellowtail Seriola quinqueradiata. Phylogenetic tree analysis showed a high sequence identity between the cloned yellowtail CCK receptor cDNA and CCK-1R, which belongs to the CCK-1R cluster. Furthermore, the expression of yellowtail CCK receptor mRNA was observed in gallbladder, pyloric caeca, and intestines, similarly to CCK-1R mRNA expression in mammals, suggesting that the cloned cDNA is of CCK-1R from yellowtail. In in vivo experiments, the CCK-1R mRNA levels increased in the gallbladder and pyloric caeca after feeding, whereas in vitro, mRNA levels of CCK-1R and digestive enzymes in cultured pyloric caeca increased by the addition of CCK. These results suggest that CCK-1R plays an important role in digestion stimulated by CCK in yellowtail.

Gonads directly regulate growth in teleosts.

In general, there is a relationship between growth and reproduction, and gonads are known to be important organs for growth, but direct evidence for their role is lacking. Here, using a fish model, we report direct evidence that gonads are endocrine organs equal to the pituitary in controlling body growth. Gonadal loss of function, gain of function, and rescue of growth were investigated in tilapia. Gonadectomy experiments were carried out in juvenile males and females. Gonadectomy significantly retarded growth compared with controls; however, this retardation was rescued by the implantation of extirpated gonads. Because gonads express growth hormone, it is possible that gonads control body growth through the secretion of growth hormone and/or other endocrine factors. We propose that gonads are integral players in the dynamic regulation of growth in teleosts.

Molecular characterization, tissue distribution patterns and nutritional regulation of IGFBP-1, -2, -3 and -5 in yellowtail, Seriola quinqueradiata.

Insulin-like growth factor-binding proteins (IGFBPs) play a vital role in regulating the biological activities of IGFs. In this study, we cloned and determined full-length cDNA sequences of yellowtail IGFBP-1, -2, -3 and -5. Their tissue distribution was determined by real-time quantitative RT-PCR, which revealed that IGFBP-1, -2, -3 and -5 are widely distributed in yellowtail tissues. In yellowtail, both IGFBP-1 and -2 are highly expressed in the liver, IGFBP-3 is predominantly expressed in the heart and skin, with the lowest expression in the liver, and IGFBP-5 is highly expressed in the liver and kidneys. The widespread tissue expression of the yellowtail IGFBPs suggests that they may act in an autocrine and/or paracrine manner in the regulation of IGF activity. The effects of nutritional deprivation on yellowtail IGFBPs and IGF-I were also examined. During a 15-day starvation period, significant elevation was observed in hepatic yellowtail IGFBP-1. Refeeding restored its level to that of the control. No significant change was observed in the hepatic yellowtail IGFBP-2 mRNA levels in starved fish compared with control fish during the starvation period. Interestingly, during the early period of food deprivation, a significant increase was observed in hepatic yellowtail IGFBP-3 and -5 mRNA levels, concomitant to the significant elevation in hepatic IGF-I mRNA from day 3 to day 9. The unexpected increase in growth stimulatory IGFBPs and IGF-I during nutritional deprivation may represent a species-specific response to changes in nutritional condition.

Nutrient control of release of pancreatic enzymes in yellowtail (Seriola quinqueradiata): involvement of CCK and PY in the regulatory loop.

Cholecystokinin (CCK) and neuropeptide Y (NPY)-related peptides are key regulators of pancreatic enzyme secretion in vertebrates. CCK stimulates enzyme secretion whereas peptide Y (PY), a NPY-related peptide, plays an antagonistic role to that of CCK. In fish, very little is known about how different nutrients affect the synthesis of CCK and PY in the digestive tract, and the mechanism by which CCK and PY actually regulate digestive enzyme secretion is not well understood. In order to determine how different nutrients stimulate the synthesis of CCK and PY in yellowtail (Seriola quinqueradiata), CCK and PY mRNA levels in the digestive tract were measured after oral administration of a single bolus of either phosphate-buffered saline (PBS: control), starch (carbohydrate), casein (protein), oleic acid (fatty acid) or tri-olein (triglyceride). In addition, in order to confirm the synthesis and secretion of digestive enzymes, the mRNA levels and enzymatic activities of three digestive enzymes (lipase, trypsin and amylase) were also analyzed. Casein, oleic acid and tri-olein increased the synthesis of lipase, trypsin and amylase, while starch and PBS did not affect the activity of any of these enzymes. CCK mRNA levels rose, while PY mRNA levels were reduced in fish administered casein, oleic acid and tri-olein. These results suggest that in yellowtail, CCK and PY maintain antagonistic control of pancreatic enzyme secretion after intake of protein and/or fat.

Molecular cloning and characterization of three distinct choriogenins in masu salmon, Oncorhynchus masou.

Three cDNAs, each encoding a different choriogenin (Chg), were isolated from a female masu salmon (Oncorhynchus masou) liver cDNA library. Two of the cDNA clones, Chg Halpha and Chg Hbeta, showed a close relationship and contained the typical domains of zona pellucida (ZP) B genes in fish, namely proline and glutamine rich repeats, a trefoil factor family domain, and a ZP domain. Specific antibodies against recombinant Chg H products (rmHalpha and rmHbeta) were generated to elucidate the relationship between the Chg H cDNAs and two types of serum Chg H protein, which were previously purified and characterized, and designated as very-high-molecular-weight vitelline envelope-related protein (vhVERP) and Chg H of masu salmon. The immunobiochemical analyses revealed that the Chg Halpha and Chg Hbeta clones encoded vhVERP and Chg H proteins, respectively. The third cDNA clone (Chg L) appeared to be a ZPC gene and, by mapping the N-terminal sequence of purified Chg L, was shown to encode serum Chg L protein. Various types of heteromultimer of the three Chgs were identified immunologically as high molecular weight chorion components, indicating the involvement of complex heterodimerization of multiple Chgs in the construction of chorion architecture in masu salmon.

Carp (Cyprinus carpio) vitellogenin: characterization of yolk proteins, development of immunoassays and use as biomarker of exposure to environmental estrogens.

The precursor protein of egg yolk, vitellogenin (Vg), is cleaved into three major components (lipovitellin, phosvitin and beta'-component) at the time of incorporation by growing oocytes. We purified three yolk proteins (YP1, YP2 and YP3) from ovaries of the common carp (Cyprinus carpio) by a combined method of ammonium sulfate precipitation and column chromatography. Biochemical analyses of the purified proteins of this species suggest that YP1, YP2 and YP3 are lipovitellin, beta'-component and phosvitin, respectively. A specific antiserum against purified carp YP1 (lipovitellin) was used to develop a single radial immunodiffusion (SRID) technique and an enzyme-linked immunosorbent assay (ELISA) for carp Vg. By SRID and ELISA, we measured the circulating carp Vg level to be in the ranges of 12.5-400 microg/ml and 2.0-1000 ng/ml, respectively, which cover a wide range of Vg levels. From 1997-1998, male and female carp were captured at points of effluent discharge from a sewage treatment plant connected to the Tama River, where estrogenic compounds were later detected, and the presence of Vg in their circulation was examined. Vg was detected in both male and female carp at the mg/ml level, suggesting that estrogens such as estrone and estradiol were sufficiently high to induce Vg in male carp inhabiting this area. The result of this study supports the use of carp Vg as a biomarker of fish exposure to environmental estrogens.

Changes in cholecystokinin and peptide Y gene expression with feeding in yellowtail (Seriola quinqueradiata): relation to pancreatic exocrine regulation.

In fish, the regulation of digestive enzyme secretion by hormonal control such as cholecystokinin (CCK) and neuropeptide Y (NPY)-related peptide is not well understood. To investigate the roles of fish CCK and peptide Y (PY) in digestive enzyme secretion, mRNA levels of CCK and PY, pyloric caeca enzyme activities and mRNA levels of pancreatic digestive enzymes (lipase, trypsin and amylase) were measured at pre- and post-prandial stages in yellowtail. Pyloric caeca were sampled at 0, 0.5, 1.5, 3, 6, 12, 24 and 48 h after feeding. The mRNA levels of trypsin and amylase increased after feeding, suggesting that transcription was induced by feed ingestion. Digestive enzyme activities decreased in exocrine pancreas after feeding, suggesting the stored enzyme was secreted from pancreas post-prandially. mRNA levels for CCK displayed a time-dependent increase, peaking between 1.5 and 3 h after-feeding followed by a rapid decrease 3 to 6 h after feeding. The mRNA expression pattern of PY was inverse to the pattern of CCK, decreasing until 1.5 h after feeding and then rising to initial levels by 12 h after feeding. These results suggest that CCK and PY work antagonistically in the exocrine pancreas of yellowtail.

Expression of growth hormone family and growth hormone receptor during early development in the Japanese eel (Anguilla japonica).

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.

Molecular cloning and characterization of growth hormone receptor and its homologue in the Japanese eel (Anguilla japonica).

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.

Cholecystokinin and peptide Y in yellowtail (Seriola quinqueradiata): molecular cloning, real-time quantitative RT-PCR, and response to feeding and fasting.

In fish, the peptide hormones cholecystokinin (CCK) and peptide Y (PY) may be involved in pancreatic exocrine secretion, as found with mammalian CCK and peptide YY (PYY); CCK stimulates, whereas PYY inhibits, pancreatic exocrine secretion in mammals. However, there is very little information on these hormones in fish; in particular, the function of PY is still unknown. Therefore, as a first step for understanding the role of CCK and PY in regulating pancreatic exocrine in fish, the cDNAs of CCK and PY were cloned from the digestive tract of yellowtail (Seriola quinqueradiata). The peptide sequence of yellowtail CCK-8, DYLGWMDF, is identical to sequences found in several teleosts. The mature form of yellowtail PY consists of 36 amino acids and has high identity to other fish PYs (88.9-97.2%). Real-time quantitative RT-PCR assays were developed to measure yellowtail CCK and PY mRNA levels. CCK mRNA levels were extremely high in the brain and, among the digestive organs, high concentrations were found in the pyloric caeca and anterior intestine. PY mRNA levels were low in the brain and highest in the anterior intestine. In fasting experiments, mRNA levels of CCK and PY in the anterior intestine showed an antagonistic change after fasting; CCK decreased whereas PY increased. These data suggest that CCK and PY in yellowtail may relate to digestion including, enzyme secretion.

Annual changes in serum levels of two choriogenins and vitellogenin in masu salmon, Oncorhynchus masou.

Annual changes in serum levels of two chorion precursors, choriogenin H (Chg H) and choriogenin L (Chg L), vitellogenin (Vg) and estradiol-17beta (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 ( approximately 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.

Purification of two lipovitellins and development of immunoassays for two forms of their precursors (vitellogenins) in medaka (Oryzias latipes).

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 380 kDa, 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-17beta (E2). The measurable range of both CLIAs was from 0.975 to 1000 ng/ml. The cross-reactivity to the alternative Vg in each CLIA was extremely low (

Identification of the salmon somatolactin receptor, a new member of the cytokine receptor family.

Somatolactin (SL) is a pituitary hormone of the GH/prolactin (PRL) family that so far has been found only in fish. Compared with GH and PRL, the primary structure of SL is highly conserved among divergent fish species, suggesting it has an important function and a discriminating receptor that constrains structural change. However, SL functions are poorly understood, and receptors for SL have not yet been identified. During cloning of GH receptor cDNA from salmon, we found a variant with relatively high (38-58%) sequence identity to vertebrate GH receptors and low (28-33%) identity to PRL receptors; however, the recombinant protein encoding the extracellular domain showed only weak binding of GH. Ligand binding of the recombinant extracellular domain for this receptor confirmed that the cDNA encoded a specific receptor for SL. The SL receptor (SLR) has common features of a GH receptor including FGEFS motif, six cysteine residues in the extracellular domain, a single transmembrane region, and Box 1 and 2 regions in the intracellular domain. These structural characteristics place the SLR in the cytokine receptor type I homodimeric group, which includes receptors for GH, PRL, erythropoietin, thrombopoietin, granulocyte-colony stimulating factor, and leptin. Transcripts for SLR were found in 11 tissues with highest levels in liver and fat, supporting the notion that a major function of SL is regulation of lipid metabolism. Cloning SLR cDNA opens the way for discovery of new SL functions and target tissues in fish, and perhaps novel members of this receptor family in other vertebrates.

Salmon growth hormone receptor: molecular cloning, ligand specificity, and response to fasting.

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.

Quantification of serum levels of precursors to vitelline envelope proteins (choriogenins) and vitellogenin in estrogen treated masu salmon, Oncorhynchus masou.

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-17beta (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 microg/kg BW of E2 injection. A similar trend was seen in the response time to exogenous E2. Serum Chgs were induced from 8h after E2 injection and reached a peak of about 5 microg/ml at 24h. Although Vg was not detected until 8h after E2 injection, its levels remained considerably low at around 0.03 microg/ml, even after 24 h. Chg H was more sensitive than was Chg L to 1 microg/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.

Carp (Cyprinus carpio) vitellogenin: purification and development of a simultaneous chemiluminescent immunoassay.

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.

Purification and identification of a second form of vitellogenin from ascites of medaka (Oryzias latipes) treated with estrogen.

Estrogen treatment of medaka leads to accumulation of ascites, in which vitellogenin (Vg) and choriogenins (precursors to vitelline envelope) are abundant. Besides those female-specific proteins, we detected a new component in ascites that cross-reacts with antiserum against egg yolk proteins. We tentatively named it egg yolk-related protein (YRP). YRP was purified from ascites by hydroxylapatite chromatography followed by gel filtration. Purified YRP had a molecular mass of 460 kDa in intact state while 570 kDa for Vg. The molecular weight of purified YRP on SDS-PAGE under both reducing and nonreducing conditions was 130 kDa. YRP was confirmed to be a lipoglycophosphoprotein by staining with Sudan black, periodic acid-Schiff (PAS) and methyl green. Amino acid composition of YRP resembled that of Vg except for a relatively low content of serine. A specific antiserum against YRP was raised in a rabbit. Antiserum against YRP specifically immunostained its antigen but not Vg or choriogenins. YRP was detected as a female-specific protein in serum of breeding medaka. The antiserum also cross-reacted with a band at 29 kDa in egg extracts, which is not immunoreactive to antiserum against Vg. These data show that YRP is a precursor to some egg yolk proteins with differing antigenicity from Vg (Hamazaki et al. '87). We thus conclude that YRP is a second form of medaka Vg and rename YRP as Vg 2 while formerly reported Vg as Vg 1.