Myogenesis and muscle metabolism in juvenile Atlantic salmon (Salmo salar) made transgenic for growth hormone.

Atlantic salmon (Salmo salar) made transgenic for growth hormone (GH) and non-transgenic salmon were sampled at 4 and 7 months of age to estimate myogenic factors, satellite cell proliferation and metabolic enzyme activities. The growth rate of 4 month old transgenic salmon was higher than that of non-transgenic salmon. Myosatellite cell (MC) proliferation rates were higher in cells isolated from GH-transgenic salmon compared with cells from non-transgenic salmon of the same mass. Moreover, MCs extracted from non-transgenic salmon demonstrated a higher proliferation capacity when exposed in vitro to salmon GH. White muscle MyoD I mRNA content was higher in transgenic and non-transgenic salmon at 7 months compared with that at 4 months, indicating an effect of age on MyoD I mRNA expression. White muscle myogenin mRNA content varied with fish age and presence of the transgene, and was higher in transgenic fish at 7 months, suggesting a higher differentiation capacity. MyoD I, MyoD II and myogenin mRNA content was higher in red muscle of GH-transgenic fish at 7 months compared with non-transgenic salmon at 7 months. However, red muscle myogenic factor expression was not different between transgenic and non-transgenic fish of the same weight. Enzyme activities in white muscle and liver were highly affected by the presence of the transgene, although this effect was generally dependent on the age of the fish. Glycolytic and oxidative enzyme activities were increased in transgenic salmon liver, indicating a higher metabolic rate in transgenics. This study demonstrates that (1) the higher growth rate of transgenic salmon particularly at 4 months of age could be explained at least in part by higher numbers and proliferation rates of MCs, (2) GH can directly stimulate the proliferation of myosatellite cells extracted from salmon, indicating that GH is one possible factor involved in the higher myosatellite cell proliferation rates in transgenic salmon, (3) MyoD and myogenin mRNA expression are affected by fish age, and (4) metabolic enzyme activities are affected by the age of the fish at least in liver and white muscle, and any transgene effect is dependent upon the age of the fish.

Cardiorespiratory modifications, and limitations, in post-smolt growth hormone transgenic Atlantic salmon Salmo salar.

In recent years, there has been a great deal of interest in how growth hormone (GH) transgenesis affects fish physiology. However, the results of these studies are often difficult to interpret because the transgenic and non-transgenic fish had very different environmental/rearing histories. This study used a stable line of size-matched GH Atlantic salmon (Salmo salar) that were reared in a shared tank with controls (at 10 degrees C, for approximately 9 months) to perform a comprehensive examination of the cardiorespiratory physiology of GH transgenic salmon, and serves as a novel test of the theory of symmorphosis. The GH transgenic salmon had a 3.6x faster growth rate, and 21 and 25% higher values for mass-specific routine and standard oxygen consumption (M(O(2))), respectively. However, there was no concurrent increase in their maximum M(O(2)), which resulted in them having an 18% lower metabolic scope and a 9% reduction in critical swimming speed. This decreased metabolic capacity/performance was surprising given that the transgenics had a 29% larger heart with an 18% greater mass-specific maximum in situ cardiac output, a 14% greater post-stress blood haemoglobin concentration, 5-10% higher red muscle and heart aerobic enzyme (citrate synthase or cytochrome oxidase) activities, and twofold higher resting and 1.7x higher post-stress, catecholamine levels. However, gill surface area was the only cardiorespiratory parameter that was not enhanced, and our data suggest that gill oxygen transfer may have been limiting. Overall, this research: (1) shows that there are significant metabolic costs associated with GH transgenesis in this line of Atlantic salmon; (2) provides the first direct evidence that cardiac function is enhanced by GH transgenesis; (3) shows that a universal upregulation of post-smolt (adult) GH transgenic salmon cardiorespiratory physiology, as suggested by symmorphosis, does not occur; and (4) supports the idea that whereas differences in arterial oxygen transport (i.e. cardiac output and blood oxygen carrying capacity) are important determinants of inter-specific differences in aerobicity, diffusion-limited processes must be enhanced to achieve substantial intra-specific improvements in metabolic and swimming performance.

Vitrification assays with embryos from a cold tolerant sub-arctic fish species.

Pseudopleuronectes americanus is a Northern teleost species that produces antifreeze proteins (AFPs) to protect them from freezing during the winter. These AFPs bind to ice crystals to inhibit their growth, and they also protect cell membranes at low temperatures. In this study, vitrification trials were done with fish embryos at three different developmental stages, using two different protocols for incorporating the vitrifying solutions. Toxicity of the cryoprotectants and permeability to dimethyl sulfoxide were analyzed. Embryos were vitrified in 0.5 ml straws by direct immersion in liquid nitrogen, and their morphology and development analyzed following thaw. The embryos responded well to vitrification as evidenced by the high percentage that exhibited good morphology following thaw. Although none of the embryos hatched, a small percentage (0.92%) of them showed active movements within the chorion and continued to develop for a number of days following thaw. This is the first record of post-thaw development of vitrified fish embryos.

Growth enhancement in transgenic Atlantic salmon by the use of an "all fish" chimeric growth hormone gene construct.

We have developed an "all fish" growth hormone (GH) chimeric gene construct by using an antifreeze protein gene (AFP) promoter from ocean pout linked to a chinook salmon GH cDNA clone. After microinjection into fertilized, nonactivated Atlantic salmon eggs via the micropyle, transgenic Atlantic salmon were generated. The presence of the transgene was detected by polymerase chain reaction (PCR) using specific oligonucleotide primers. A number of these transgenic fish showed dramatic increases in their growth rate. At one year old, the average increase of the transgenic fish was 2 to 6 fold and the largest transgenic fish was 13 times that of the average non-transgenic control.

Antifreeze proteins in the urine of marine fish.

Several species of marine teleosts have evolved blood plasma antifreeze polypeptides which enable them to survive in ice-laden seawater. Four distinct antifreeze protein classes differing in carbohydrate content, amino acid composition, protein sequence and secondary structure are currently known. Although all of these antifreezes are relatively small (2.6-33 kd) it was generally thought that they were excluded from the urine by a variety of glomerular mechanisms. In the present study antifreeze polypeptides were found in the bladder urine of winter flounder (Pseudopleuronectes americanus), sea raven (Hemitripterus americanus), ocean pout (Macrozoarces americanus) and Atlantic cod (Gadus morhua). Since the plasma of each of these fish contains a different antifreeze class it would appear that all four classes of antifreeze can enter the urine. The major antifreeze components in the urine of winter flounder were found to be identical to the major plasma components in terms of high performance liquid chromatography retention times and amino acid composition. It is concluded that plasma antifreeze peptides need not be chemically modified before they can enter the urine.

Structural variations in the alanine-rich antifreeze proteins of the pleuronectinae.

The sequence and activity of antifreeze proteins from two right eye flounder species were compared to assess the influence of structural variations on antifreeze capacity. The cDNA encoding the major serum antifreeze protein in the yellowtail flounder (Limanda ferruginea) was cloned from liver tissue. Its DNA sequence shows that the precursor to the antifreeze is a 97-residue preproportion. Edman degradation identified the N-terminus of the 48-amino-acid mature serum antifreeze protein and confirmed the sequence of the first 36 residues. A comparison with the previously determined winter flounder antifreeze protein and mRNA sequences shows strong homology through the 5' and 3' untranslated regions and in the peptide region. The mature protein section has the greatest sequence variation. Specifically, the yellowtail antifreeze protein, in contrast to that of the winter flounder, contains a fourth 11-amino-acid repeat and lacks several of the hydrophilic residues that have been postulated to aid in the binding of the protein to ice crystals. Intramolecular salt bridges are present in the antifreeze proteins from both species but in different registries with respect to the 11-amino-acid repeats. On a mass basis the yellowtail flounder antifreeze, though longer than that of the winter flounder, is only 80% as effective at depressing the freezing temperature of aqueous solutions. This lower activity might be due to the reduced number of hydrophilic ice-binding residues per molecule.

The binding of zinc to the soluble proteins of intestinal mucosa in winter flounder (Pseudopleuronectes americanus).

1. The binding of Zn2+ to soluble proteins of intestinal mucosa of winter flounder was examined using an equilibrium dialysis technique. 2. There appeared to be more than one binding system present for Zn2+ in the mucosal cytosol. 3. It required four times the normal endogenous Zn2+ level found in the mucosal cytosol to saturate the highest affinity (K1 = 2.42 x 10(7] binding system. 4. Of 10 metals tested Cu2+ was the only one which interfered with Zn2+ binding to the mucosal cytosol proteins. 5. It is postulated that binding proteins in the mucosal cytosol of winter flounder may play a role in the transport of Zn2+.