Munetaka Shimizu

Bovine growth hormone treatment of channel catfish : strain and temperature effects on growth, plasma IGF-I levels, feed intake and efficiency and body composition

Channel catfish from two strains, USDA-103 and Norris, were reared in circular 800-l tanks and injected once weekly with 2.5 μg recombinant bovine growth hormone (rbGH) per gram body weight, or the saline vehicle. In addition to the rbGH, and strain treatments, half the tanks were supplied with well water of 26.0°C and the other half received water of 21.7°C. Growth rate, plasma insulin-like growth factor I (IGF-I) levels, feed consumption and body fatness of the fish injected with rbGH were higher than in saline-injected controls. Strain and temperature effects were also significant. Feed consumption was significantly greater with growth hormone treatment, higher temperature, and in the USDA-103 strain of catfish. Feed efficiency was significantly better in rbGH-injected fish. The effect of temperature on feed efficiency was also significant, higher temperature treatments performed better. Growth hormone enabled channel catfish to grow better at lower and higher temperatures than saline-treated counterparts.

Postprandial changes in plasma growth hormone, insulin, insulin-like growth factor (IGF)-I, and IGF-binding proteins in coho salmon fasted for varying periods

We examined postprandial changes in circulating growth hormone (GH), insulin, insulin-like growth factor (IGF)-I, and IGF-binding proteins (IGFBPs) in yearling coho salmon under different feeding regimes. Fish were initially fasted for 1 day, 1 wk, or 3 wk. Fasted fish were then fed, and blood was collected at 4-h intervals over 26 h. After the various periods of fasting, basal levels of insulin were relatively constant, whereas those of IGF-I, IGFBPs and GH changed in proportion to the duration of the fast. A single meal caused a rapid, large increase in the circulating insulin levels, but the degree of the increase was influenced by the fasting period. IGF-I showed a moderate increase 2 h after the meal but only in the regularly fed fish. Plasma levels of 41-kDa IGFBP were increased in all groups within 6 h after the single meal. The fasting period did not influence the response of 41-kDa IGFBP to the meal. IGFBP-1 and GH decreased after the meal to the same extent among groups regardless of the fasting period. The present study shows that insulin and IGF-I respond differently to long (weeks)- and short (hours)-term nutritional changes in salmon; insulin maintains its basal level but changes acutely in response to food intake, whereas IGF-I adjusts its basal levels to the long-term nutritional status and is less responsive to acute nutritional input. IGFBPs maintain their sensitivity to food intake, even after prolonged fasting, suggesting their critical role in the nutritional regulation of salmon growth.

Purification of a 41-kDa insulin-like growth factor binding protein from serum of chinook salmon, Oncorhynchus tshawytscha

In salmon, at least three insulin-like growth factor binding proteins (IGFBPs) with molecular masses of 41, 28, and 22kDa 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 R(3)IGF-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.

Circulating salmon 28- and 22-kDa insulin-like growth factor binding proteins (IGFBPs) are co-orthologs of IGFBP-1.

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.

Circulating salmon 41-kDa insulin-like growth factor binding protein (IGFBP) is not IGFBP-3 but an IGFBP-2 subtype.

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.

Transition of serum vitellogenin cycle in Sakhalin taimen (Hucho perryi)

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) and a single radial immunodiffusion (SRID) were developed for measurement of serum vitellogenin (Vg) levels in Sakhalin taimen (Hucho perryi). Regarding specificity for serum Vg, an antiserum raised against lipovitellin of taimen (a-Lv) was adequate for both assays. ELISA and SRID could detect Vg in serum at concentrations as low as 10 ng/ml and 25 micrograms/ml, respectively. In estrogen administration experiments, the level of serum Vg began clearly increasing within 12 to 24 hr after injection of immature females with estradiol-17 beta (E2). The appearance and levels of Vg in males treated with E2 were delayed and smaller, respectively, than for females. Vg levels varied throughout natural vitellogenesis from 0-4 micrograms/ml (3 years old) to approximately 30 mg/ml (5-6 years old). We observed an early transitory peak of serum Vg levels (primary reaction) at the time of early vitellogenesis and chronic high Vg levels (for 6-7 months) in winter period before ovulation. Changes of serum E2 levels were correlated with Vg levels. However, E2 levels decreased a month earlier than Vg levels near ovulation. It appears that the duration of vitellogenesis in taimen is considerably longer than that in other salmonids, lasting more than 2 years.

Responses of insulin-like growth factor (IGF)-I and two IGF-binding protein-1 subtypes to fasting and re-feeding, and their relationships with individual growth rates in yearling masu salmon (Oncorhynchus masou).

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 5 weeks 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.

PURIFICATION OF THE PRECURSORS TO VITELLINE ENVELOPE PROTEINS FROM SERUM OF SAKHALIN TAIMEN, HUCHO PERRYI

High and low molecular weight vitelline envelope-related proteins (hVERP and lVERP) were purified from serum of vitellogenic female Sakhalin taimen (Hucho perryi) by a combination of ion-exchange, hydroxylapatite and gel filtration chromatography. The molecular weight of hVERP was estimated to be 83 kDa by gel filtration, and 48 kDa and 54 kDa in SDS-PAGE under non-reduced and reduced conditions, respectively. The molecular weight of lVERP was 56 kDa by gel filtration, and 42 kDa and 46 kDa on SDS-PAGE (non-reduced and reduced, respectively). Amino acid composition of hVERP was characterized by high content of proline (15.9%) and glutamic acid (13.8%). The lVERP had high contents of glutamic acid (10.8%) and aspartic acid (10.5%). Specific antibodies against hVERP and against lVERP were prepared by immunizing rabbits. The antiserum to hVERP stained bands corresponding to 98 kDa and 48 kDa of vitelline envelope (VE) in SDS-PAGE without reduction, whereas the antiserum to lVERP immunostained 98 kDa and 42 kDa bands. Both specific antibodies recognized the vitelline envelope of vitellogenic oocytes immunocytochemically. Thus, hVERP and lVERP are precursors to vitelline envelope proteins in this species.

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 Hα and Chg Hβ, 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 (rmHα and rmHβ) 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 Hα and Chg Hβ 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. Mol. Reprod. Dev. 75: 1217–1228, 2008.

Circulating insulin-like growth factor binding proteins in fish: Their identities and physiological regulation

Insulin-like growth factor binding proteins (IGFBPs) play crucial roles in regulating the availability of IGFs to receptors and prolong the half-lives of IGFs. There are six IGFBPs present in the mammalian circulation with IGFBP-3 being most abundant. In mammals IGFBP-3 is the major carrier of circulating IGFs, facilitated by forming a ternary complex with IGF and an acid-labile subunit (ALS). IGFBP-1 is generally inhibitory to IGF action by preventing it from interacting with its receptors. In teleosts, the third-round of vertebrate whole genome duplication created paralogs of each IGFBP, except IGFBP-4. In the fish circulation, three major IGFBPs are typically detected at molecular ranges of 20-25, 28-32 and 40-50kDa. However, their identities are not well established. Three major circulating IGFBPs in Chinook salmon have been identified through protein purification and cDNA cloning. Salmon 28- and 22-kDa IGFBPs are co-orthologs of IGFBP-1, termed IGFBP-1a and -1b, respectively. They are induced under catabolic conditions such as stress and fasting but their responses are somewhat different, with IGFBP-1b being the most sensitive of the two. Cortisol stimulates production and secretion of these IGFBP-1 subtypes while, unlike in mammals, insulin may not be a primary suppressor. Salmon 41-kDa IGFBP, a major carrier of IGF-I, is not IGFBP-3, as might be expected extrapolating from mammals, but is in fact IGFBP-2b. Salmon IGFBP-2b levels in plasma are high when fish are fed, and GH treatment increases its circulating levels similar to mammalian IGFBP-3. These findings suggest that salmon IGFBP-2b acquired the role and regulation similar to mammalian IGFBP-3. Multiple replications of fish IGFBPs offer a unique opportunity to investigate molecular evolution of IGFBPs.