Gregory M. Weber

Effects of Insulin-Like Growth Factor-I on In Vitro Final Oocyte Maturation and Ovarian Steroidogenesis in Striped Bass, Morone saxatilis

Abstract Recombinant human (rh) insulin-like growth factor-I (IGF-I) was more potent than rhIGF-II at inducing in vitro germinal vesicle breakdown (GVBD), a marker for resumption of meiosis, in oocytes of striped bass. Treatment of ovarian fragments containing oocytes in intact follicles with rhIGF-I increased concentrations of estradiol-17β and maturation-inducing steroid (MIS) 17,20β,21-trihydoxy-4-pregnen-3-one (20β-S) in the culture medium and decreased testosterone levels. The follicles were too immature for oocytes to complete GVBD in response to 20β-S (MIS incompetent) or hCG. Addition of 20β-S to cultures did not increase the percentage of oocytes completing GVBD in response to rhIGF-I or rhIGF-II. Bovine insulin was without effect on GVBD or steroid production. Incubation of MIS-competent follicles with actinomycin D, cyanoketone, trilostane, 1-heptanol, or 1-octanol had no effect on rhIGF-I-induced GVBD, but attenuated hCG-induced GVBD and 20β-S production. Cycloheximide inhibited rhIGF-I-induced GVBD. Collectively, these observations indicate that IGF-I can induce GVBD via MIS- and transcription-independent pathways without coupled gap junctions between oocytes and granulosa cells or among granulosa cells, but requires protein synthesis to do so. An rhIGF-I analogue that does not bind IGF-binding proteins, des(1,3)IGF-I, was more potent than rhIGF-I in inducing GVBD, suggesting ovarian IGF-binding proteins may inhibit IGF-I action.

Isolation and characterization of myostatin complementary deoxyribonucleic acid clones from two commercially important fish: Oreochromis mossambicus and Morone chrysops.

In mammals, skeletal muscle mass is negatively regulated by a muscle-derived growth/differentiating factor named myostatin (MSTN) that belongs to the transforming growth factor-β superfamily. Although putative MSTN homologs have been identified from several vertebrates, nonmammalian orthologs remained poorly defined. Thus, we isolated and characterized MSTN complementary DNA clones from the skeletal muscle of the tilapia Oreochromis mossambicus and the white bass Morone chrysops. The nucleic and amino acid sequences from both fish species are highly homologous to the previously identified mammalian and avian orthologs, and both possess conserved cysteine residues and putative RXXR proteolytic processing sites that are common to all transforming growth factor-β family members. Western blotting of conditioned medium from human embryonal kidney (HEK293) cells overexpressing a His-tagged tilapia MSTN indicates that the secreted fish protein is processed in a manner similar to mouse MSTN. However, in contrast ...

Vitellogenin-Derived Yolk Proteins of White Perch, Morone americana: Purification, Characterization, and Vitellogenin-Receptor Binding1

Abstract The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a ∼20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, β′-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the β′-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.

Effects of insulin-like growth factor-I, insulin, and leucine on protein turnover and ubiquitin ligase expression in rainbow trout primary myocytes

The effects of insulin-like growth factor-I (IGF-I), insulin, and leucine on protein turnover and pathways that regulate proteolytic gene expression and protein polyubiquitination were investigated in primary cultures of 4-day-old rainbow trout myocytes. Supplementing media with 100 nM IGF-I increased protein synthesis by 13% (P < 0.05) and decreased protein degradation by 14% (P < 0.05). Treatment with 1 microM insulin increased protein synthesis by 13% (P < 0.05) and decreased protein degradation by 17% (P < 0.05). Supplementing media containing 0.6 mM leucine with an additional 2.5 mM leucine did not increase protein synthesis rates but reduced rates of protein degradation by 8% (P < 0.05). IGF-I (1 nM-100 nM) and insulin (1 nM-1 microM) independently reduced the abundance of ubiquitin ligase mRNA in a dose-dependent manner, with maximal reductions of approximately 70% for muscle atrophy F-box (Fbx) 32, 40% for Fbx25, and 25% for muscle RING finger-1 (MuRF1, P < 0.05). IGF-I and insulin stimulated phosphorylation of FOXO1 and FOXO4 (P < 0.05), which was inhibited by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, and decreased the abundance of polyubiquitinated proteins by 10-20% (P < 0.05). Supplementing media with leucine reduced Fbx32 expression by 25% (P < 0.05) but did not affect Fbx25 nor MuRF1 transcript abundance. Serum deprivation decreased rates of protein synthesis by 60% (P < 0.05), increased protein degradation by 40% (P < 0.05), and increased expression of all ubiquitin ligases. These data suggest that, similar to mammals, the inhibitory effects of IGF-I and insulin on proteolysis occur via P I3-kinase/protein kinase B signaling and are partially responsible for the ability of these compounds to promote protein accretion.

Response to selection for bacterial cold water disease resistance in rainbow trout

A family-based selection program was initiated at the National Center for Cool and Cold Water Aquaculture in 2005 to improve resistance to bacterial cold water disease (BCWD) in rainbow trout. The objective of this study was to estimate response to 2 generations of selection. A total of 14,841 juvenile fish (BW = 3.1 g; SD = 1.1 g) from 230 full-sib families and 3 randomly mated control lines were challenged intraperitoneally with Flavobacterium psychrophilum, the bacterium that causes BCWD, and mortalities were observed for 21 d. Selection was applied to family EBV derived from a proportional-hazards frailty (animal) model while constraining rate of inbreeding to <or=1% per generation. After adjusting for nongenetic effects, survival rate of select-line families increased by 24.6 +/- 6.8 and 44.7 +/- 6.7 (cumulative) percentage points after 1 and 2 generations of selection, respectively (P < 0.01). Genetic trend, estimated from a linear animal model that fit genetic group effects, was 19.0 +/- 4.1 percentage points per generation and approached significance (P = 0.07). Heritability estimates from the proportional-hazards frailty model and linear animal model were similar (0.22 and 0.23, respectively), and family EBV from both models were highly correlated (-0.92). Accuracy of selection, estimated as the correlation between mid-parent EBV and progeny survival rate, was 0.20 (P < 0.01) for the proportional-hazards frailty model and 0.18 (P = 0.01) for the linear animal model. Accuracy estimates were not different (P = 0.81) between the models. This study demonstrates that selective breeding can be effective for improving resistance to experimental BCWD challenge in rainbow trout.

Insulin-like growth factor-I and genetic effects on indexes of protein degradation in response to feed deprivation in rainbow trout (Oncorhynchus mykiss)

This study determined the effect of genetic variation, feed deprivation, and insulin-like growth factor-I (IGF-I) on weight loss, plasma IGF-I and growth hormone, and indexes of protein degradation in eight full-sibling families of rainbow trout. After 2 wk of feed deprivation, fish treated with IGF-I lost 16% less (P < 0.05) wet weight than untreated fish. Feed deprivation increased growth hormone (P < 0.05) and decreased IGF-I (P < 0.05), but hormone levels were not altered by IGF-I. Plasma 3-methylhistidine concentrations were not affected by IGF-I but were decreased after 2 wk (P < 0.05) and increased after 4 wk (P < 0.05) of feed deprivation. In white muscle, transcript abundance of genes in the ubiquitin-proteasome, lysosomal, and calpain- and caspase-dependent pathways were affected by feed deprivation (P < 0.05). IGF-I prevented the feed deprivation-induced upregulation of MAFbx (F-box) and cathepsin transcripts and reduced abundance of proteasomal mRNAs (P < 0.05), suggesting that reduction of protein degradation via these pathways may be partially responsible for the IGF-I-induced reduction of weight loss. Family variations in gene expression, IGF-I concentrations, and weight loss during fasting suggest genetic variation in the fasting response, with considerable impact on regulation of proteolytic pathways. These data indicate that nutrient availability, IGF-I, and genetic variation affect weight loss, in part through alterations of proteolytic pathways in rainbow trout, and that regulation of genes within these pathways is coordinated in a way that supports a similar physiological response.

Generation of a reference transcriptome for evaluating rainbow trout responses to various stressors

BackgroundFish under intensive culture conditions are exposed to a variety of acute and chronic stressors, including high rearing densities, sub-optimal water quality, and severe thermal fluctuations. Such stressors are inherent in aquaculture production and can induce physiological responses with adverse effects on traits important to producers and consumers, including those associated with growth, nutrition, reproduction, immune response, and fillet quality. Understanding and monitoring the biological mechanisms underlying stress responses will facilitate alleviating their negative effects through selective breeding and changes in management practices, resulting in improved animal welfare and production efficiency.ResultsPhysiological responses to five treatments associated with stress were characterized by measuring plasma lysozyme activity, glucose, lactate, chloride, and cortisol concentrations, in addition to stress-associated transcripts by quantitative PCR. Results indicate that the fish had significant stressor-specific changes in their physiological conditions. Sequencing of a pooled normalized transcriptome library created from gill, brain, liver, spleen, kidney and muscle RNA of control and stressed fish produced 3,160,306 expressed sequence tags which were assembled and annotated. SNP discovery resulted in identification of ~58,000 putative single nucleotide polymorphisms including 24,479 which were predicted to fall within exons. Of these, 4907 were predicted to occupy the first position of a codon and 4110 the second, increasing the probability to impact amino acid sequence variation and potentially gene function.ConclusionWe have generated and characterized a reference transcriptome for rainbow trout that represents multiple tissues responding to multiple stressors common to aquaculture production environments. This resource compliments existing public transcriptome data and will facilitate approaches aiming to evaluate gene expression associated with stress in this species.

Temporal mRNA expression of transforming growth factor-beta superfamily members and inhibitors in the developing rainbow trout ovary ☆

During mammalian ovarian development transforming growth factor-beta (TGFbeta) superfamily members and their inhibitors are critical paracrine regulators, yet the intraovarian functions of these proteins have received less attention in fish. Using quantitative real-time RT-PCR, changes in ovarian mRNA expression of six TGFbeta members and two inhibitors were investigated in rainbow trout across a wide range of fish ovarian stages (i.e., early perinucleous stage through acquisition of maturational competence). Transcript changes for insulin-like growth factor 1 and 2, and five enzymes associated with steroidogenesis, as well as plasma levels of three sex steroids were also measured to provide a framework of established intraovarian regulators in trout. Expression of bone morphogenetic protein 4 (bmp4), bone morphogenetic protein7 (bmp7), and growth differentiation factor 9 (gdf9) peaked during pre-vitellogenic stages and steadily decreased through advancing stages implicating these genes in early ovarian development. A dramatic increase in inhibin beta(A) and decrease in follistatin expression occurred during early to mid-vitellogenic stages, which corresponded with increased 17beta-estradiol plasma levels suggesting a vitellogenic role for ovarian activin A. Follicles that were competent to respond to the maturation-inducing hormone had decreased levels of inhibin beta(B) and increased expression of bambi (bmp and activin membrane-bound inhibitor) suggesting their roles in maturation processes. Furthermore, bmp4, bmp7 and gdf9 are primarily expressed in the oocyte whereas the inhibin subunits, follistatin, and bambi are primarily expressed in the somatic follicle cells. These results support TGFbeta superfamily members and their inhibitors have wide-ranging and disparate roles in regulating ovarian development in fish.

In vitro hormone induction of final oocyte maturation in striped bass (Morone saxatilis) follicles is inhibited by blockers of phosphatidylinositol 3-kinase activity.

Oocyte germinal vesicle breakdown (GVBD) was induced in striped bass ovarian fragments when tissues were incubated with 100-nM recombinant human insulin-like growth factor-I (rhIGF-I), 25-IU human chorionic gonadotropin (hCG) ml(-1), or 290 nM of the maturation-inducing steroid (MIS), 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S). Inhibitors of phosphatidylinositol 3-kinase (PI 3-K), wortmannin (100 nM) and LY 294002 (50 microM), inhibited GVBD induced by these hormones. Furthermore, the inhibitors attenuated hCG-induced steroid hormone synthesis. Previous studies report that gap junction uncouplers inhibit GVBD induced by hCG, but not by rhIGF-I, in striped bass. We show that 20beta-S-induced GVBD is also attenuated by 1 mM 1-heptanol or 1-octanol without being affected by incubation with 3 mM ethanol. Thus, the effects of inhibiting PI 3-K activity on GtH and MIS actions are similar to effects of uncoupling gap junctions. These data suggest that PI 3-K activity is required for GtH- MIS- and IGF-I induction of GVBD in striped bass. Our data are also consistent with the notion that a ligand that regulates PI 3-K activity, possibly an IGF, participates in maintenance of gap junctional communication required for maximal GtH and MIS action.

Associations between Plasma Growth Hormone, Insulin-Like Growth Factor-I, and Cortisol with Stress Responsiveness and Growth Performance in a Selective Breeding Program for Rainbow Trout

Abstract The National Center for Cool and Cold Water Aquaculture is conducting a selective breeding program to improve the germplasm of rainbow trout Oncorhynchus mykiss for aquaculture. Two lines are being selected for improved growth. We previously found a positive correlation between stress responsiveness, indicated by plasma cortisol concentration after a 3-h confinement stressor, and growth performance, indicated by body weight at selection (approximately 300 d posthatching [dph]), in families from the first line. This correlation is confirmed for the second line in the present study. In addition to stressor-induced cortisol levels, resting plasma growth hormone (GH), insulin-like growth factor-I (IGF-I), and cortisol concentrations were measured in the second line as a first step in determining the physiological and genetic bases for growth performance variation and the association between stress responsiveness and growth performance in our germplasm. Hormone levels were compared against two measure...