A molecular mechanism for variations in muscle function in rainbow trout

Salmonids undergo a developmental transition from parr to smoltthat involves a number of physiological and morphological changes.In recent years, my laboratory has studied shifts in red musclefunction at this parr-smolt transformation (PST) in rainbowtrout, Oncorhynchus mykiss. Parr red muscle has faster contractionkinetics than smolts, including faster rates of activation andrelaxation and a faster maximum shortening velocity. At PST,a transition in swimming behavior is also observed, with lowertailbeat frequencies and longer EMG duty cycles in the oldersmolts. Lastly, there is molecular correlate to changes in kineticsand behavior. During PST, there is a developmental reductionin the number of myosin heavy chain (MHC) isoforms in the redmuscle of rainbow trout. Since MHC composition of muscle candetermine contractile properties, these molecular results suggesta mechanism for the transition in red muscle kinetics and steadyswimming. The red muscle of parr is more likely to contain thefast-twitch or white isoform of MHC, resulting in faster contractileproperties of that muscle and higher tailbeat frequencies duringsteady swimming. Lastly, experimental work supports the conclusionthat the shift in kinetics causes the observed shift in swimmingbehavior.     

Thyroxine induces transitions in red muscle kinetics and steady swimming kinematics in rainbow trout (Oncorhynchus mykiss)

During normal development, rainbow trout undergo a shift in red muscle contraction kinetics and swimming kinematics. Young trout parr have faster muscle kinetics and faster tailbeat frequency during swimming than older, larger juvenile trout. In this study, the thyroid hormone thyroxine (T(4)) was used to induce these changes in trout parr. This allowed a comparison of swimming kinematics, through the use of video analysis and electromyography, and red muscle contractile properties, through the use of in vitro muscle preparations, between natural parr and same-sized induced juveniles. The red muscle of natural parr has faster contractile properties than induced juveniles, including faster twitch time and a faster maximum shortening velocity (V(max)). Further, natural parr swim with faster tailbeat frequencies than induced juveniles. The results suggest that the natural shift in red muscle contraction kinetics observed during parr-smolt transfomation in trout directly affects swimming behavior in these fish. Also, thyroid hormones appear to induce a shift towards slower isoforms of the muscle protein myosin heavy chain (MHC), a result distinct from work on rats where thyroid hormones induce shifts towards faster forms of MHC. J. Exp. Zool. 290:115-124, 2001.     

Influence of growth rate on white muscle dynamics in rainbow trout and brook trout

In trout, fast growth stimulated white muscle fibre hypertrophy ( P 0·001) and hyperplasia ( P <0·01) in outer fibres but not in deep fibres. Glycogen was most prevalent in outer fibres ( P <0·01) and in brook trout ( P <0·01) that on average had three to four times larger fibres than rainbow trout.     

Dietary phosphorus-responsive genes in the intestine, pyloric ceca, and kidney of rainbow trout

Identification of phosphorus (P)-responsive genes is important in diagnosing the adequacy of dietary P intake well before clinical symptoms arise. The mRNA abundance of selected genes was determined in the intestine, pyloric ceca, and kidney of rainbow trout fed low-P (LP) or sufficient-P (SP) diet for 2, 5, and 20 days. The LP-to-SP ratio (LP/SP) of mRNA abundance was used to evaluate the difference in gene expression between LP and SP fish, and to compare the response with bone and serum P, which are conventional indicators of P status. The LP/SP of intestinal, cecal, and renal type II sodium-phosphate cotransporter (NaPi-II) mRNA abundance changed from approximately 1-2 (day 2) to approximately 1.4-4 (day 5) and to approximately 2-10 (day 20). The LP/SP of renal NaPi-II, vitamin D 24-hydroxylase, and vitamin D receptor mRNA abundance correlated inversely with serum P on day 5 but not on day 2 and day 20. In another study, differentially expressed genes between LP and SP fish were examined by subtractive hybridization, confirmed by Northern blot, and evaluated by t-test and correlation with serum and bone P concentrations. About 30 genes were identified as dietary P responsive at day 20, including intestinal meprin and cysteinesulfinic acid decarboxylase, renal S100 calcium-binding protein and mitochondrial P(i) carrier, and cecal apolipoprotein E, somatomedin B-related protein, and NaPi-II. The LP/SP of mRNA abundance of renal mitochondrial P(i) carrier and intestinal cysteinesulfinic acid decarboxylase changed significantly by day 2, and intestinal meprin by day 5. Hence, these genes and NaPi-II are among the earliest steady-response genes capable of predicting P deficiency well before the onset of clinical deficiency.     

The non-aromatizable androgen, dihydrotestosterone, induces antiestrogenic responses in the rainbow trout

In order to satisfy government mandates, numerous studies have been performed categorizing potential endocrine disrupting chemicals as (anti)estrogens or (anti)androgens. We report here that dihydrotestosterone (DHT), a potent, non-aromatizable androgen receptor agonist, induces antiestrogenic responses through direct and/or indirect modulation of vitellogenin (Vg), steroid hormone and total cytochrome P450 levels. DHT and two weak, aromatizable androgens, DHEA and androstenedione (0.05-50 mg/kg per day), were fed to juvenile trout for 2 weeks. DHEA and androstenedione significantly increased blood plasma Vg by up to 30- and 45-fold, respectively (P<0.05, t-test). 17beta-Estradiol (E2) increases were also observed with both androgens, albeit with lower sensitivity. DHT markedly decreased Vg and E2 levels, suggesting that DHEA and androstenedione increased Vg and E2 via conversion to E2 and not by estrogen receptor agonism. DHEA and androstenedione had no effect on total cytochrome P450 content, while DHT significantly decreased P450 content in a dose dependent fashion. These results indicate that alterations in metabolism mediated by androgen receptor binding may be responsible for the Vg and E2 decreases by DHT. In an attempt to decipher between receptor and non-receptor androgenic mechanisms of the observed DHT effects, DHT (0, 50 or 100 mg/kg per day) and flutamide (0-1250 mg/kg per day), an androgen receptor antagonist, were fed to juvenile rainbow trout for 2 weeks. Flutamide alone was as effective as DHT in decreasing E2 and Vg levels in males but did not significantly reverse DHT induced Vg decreases in either sex (P>0.05, F-test). DHT decreases in total P450 content were partially attenuated in males by flutamide co-treatment, but not females, suggesting a partial androgenic mechanism to the P450 decreases as well as a fundamental sex difference responding to androgen receptor binding. Moreover, flutamide alone decreased P450 content by up to 30% in males and 40% in females. These effects may be mediated through direct androgen receptor binding irrespective of whether the binding is agonistic or antagonistic. This study indicates that androgen receptor agonists/antagonists can elicit significant antiestrogenic effects that may not necessarily be mediated through classic receptor binding mechanisms and signal transduction pathways.     

Flesh qualities and muscle fiber characteristics in triploid and diploid rainbow trout

Analyzed with regard to their slaughter weights and flesh quality 78‐month‐old diploid and triploid rainbow trout (full sibs) were reared together in a pond after tagging at an age of 12 months. Triploids had higher body weights and carcass percentages than diploids (6 kg vs 4 kg and 66% vs 52%). Triploid fish also displayed lower electrical conductivity values and darker (L* value) and redder (a* value) flesh color. The fillets of the triploid trout contained more crude fat and less moisture than the diploids (6% vs 3% and 68% vs 74%, respectively). No effect of ploidy was found with regard to the protein contents. Triploid rainbow trout had larger mean white and intermediate muscle fiber areas than diploid fish in the dorsal and pelvic fin regions. In the pelvic fin part, the white muscle fiber areas were larger than in the dorsal fin part. In conclusion, adult triploid rainbow trout grow faster especially by fiber hypertrophy and have better flesh quality parameters than diploid fish.     

Relationship between heat-shock protein synthesis and thermotolerance in rainbow trout fibroblasts

Summary The role of heat-shock protein synthesis in the development of thermotolerance by rainbow trout fibroblasts was examined. During the first 6 h after being shifted from 22°C to 28°C, cells of the rainbow trout fibroblast line, RTG-2, rapidly synthesized the major heat-shock proteins (hsps), hsps 87, 70 and 27, and developed tolerance to 32°C. After 24 h at 28°C hsp synthesis was drastically reduced but thermotolerance was maintained. If these thermotolerant cells were shifted to 32°C, hsp synthesis continued at a very low level, but if they were subsequently returned to 22°C, synthesis of hsps 70 and 27 was induced again. The addition of actinomycin D during the first 6 h at 28°C prevented hsp synthesis and the development of thermotolerance. The presence of actinomycin D during the incubation of thermotolerant cultures at 32°C blocked the reinitiation of hsps synthesis at 22°C but had no effect on survival. Therefore, the hsps that accumulated at 28°C were sufficient to allow cells to survive a subsequent thermal stress at 32°C.     

Tissue damage in organic rainbow trout muscle investigated by proteomics and bioinformatics

The response to tissue damage is a complex process, which involves the coordinated regulation of multiple proteins to ensure tissue repair. In order to investigate the effect of tissue damage in a lower vertebrate, samples were taken from rainbow trout (Oncorhynchus mykiss) at day 7 after damage and proteins were separated using 2DE. The experimental design included two groups of rainbow trout, which were fed organic feed either with or without astaxanthin. In total, 96 proteins were found to be affected by tissue damage, clearly demonstrating in this lower vertebrate the complexity and magnitude of the cellular response, in the context of a regenerative process. Using a bioinformatics approach, the main biological function of these proteins were assigned, showing the regulation of proteins involved in processes such as apoptosis, iron homeostasis, and regulation of muscular structure. Interestingly, it was established that exclusively within the astaxanthin feed group, three members of the annexin protein family (annexin IV, V, and VI) were regulated in response to tissue damage.     

Calcitonin induces hypercalcemia in grey mullet and immature freshwater and sea-water adapted rainbow trout

1. Changes in plasma calcium levels, in response to salmon calcitonin injections, were studied in freshwater and sea-water adapted trout (Salmo gairdnerii) and in grey mullet (Chelon labrosus). 2. Low doses (0.1 ng sCT/100 g body weight) elicited hypercalcemia in the two species studied. 3. High doses (0.5 microgram) provoked hypocalcemia only in freshwater and sea-water adapted trout. 4. An hypercalcemic response appears as the primordial effects of CT injections, higher doses of CT leading to hypocalcemic effects.     

Temperature and oxygen tension influence the development of muscle cellularity in embryonic rainbow trout

Muscle cellularity at a developmental stage around the time of hatching was examined in rainbow trout which had been reared from the eyed stage at three different temperature regimes (5, 10 and 15° C) and different O₂ tensions [70% of air saturation value (ASV) at 5° C, 100% of ASV at all temperatures, and 150% of ASV at 10 and 15° C]. It was found that, as has been shown for other species, there was a difference in muscle fibre numbers and fibre cross-sectional areas between some of the regimes. There was a decrease in fibre number at the intermediate and higher temperature, and a decrease in fibre size at the high temperature. The temperature effects observed were modified by the applied changes in O₂ tension. An increased O₂ tension at 10° C led to an increase in fibre size whereas a decrease in O₂ tension at the low temperature resulted in a decrease in fibre number. The largest total white muscle cross-sectional area was achieved at 10° C under high O₂ conditions. Temperature and O₂ tension therefore had a clear effect on muscle cellularity and there was a significant interaction between the two parameters.     

Protamine messenger RNA: evidence for early synthesis and accumulation during spermatogenesis in rainbow trout

Trout testis cells were separated into various developmental classes by velocity sedimentation in bovine serum albumin gradients and were identified morphologically with particular stages of the process of spermatogenesis. The stage of testis cell differentiation at which protamine mRNA appears in the cell cytoplasm for the first time was determined by hybridization of RNA populations extracted from the separated cells to radioactively labeled protamine cDNA. Primary spermatocytes represent the earliest stage of differentiation at which protamine mRNA can be detected in large quantities in the cell cytoplasm, establishing that the synthesis of this class of mRNA occurs at a much earlier stage than the time of its translation at the spermatid stage. Protamine mRNA sequences were found in both the polysomes and postribosomal supernatant of the spermatid cells which are involved in the synthesis of protamine, while primary and secondary spermatocytes contained the mRNA sequences only in their postribosomal supernatant fractions. These findings strongly suggest that protamine mRNA is synthesized, accumulated, and stored in the cell sap of primary and secondary spermatocytes in the form of “inactive” messenger ribonucleoprotein particles, which are “activated” and translated at the spermatid stage.     

Control of skeletal muscle fibres and adipose cells size in the flesh of rainbow trout

The distributions of the diameters of skeletal muscle fibres and adipocytes were studied in rainbow trout. The cellularity of perivisceral adipose tissues and subcutaneous ventral and dorsal adipose tissues were characterized more specifically. In these tissues, a population of small adipocytes was distinguishable from larger adipocytes. The same was observed in white muscle. The effects of extrinsic factors (dietary lipid in two different thermal conditions) and intrinsic factors (strains in two different saline conditions, growth hormone) on the long-term response of the cellularity of both muscle and adipose tissues were studied. The effects of thermal environment were tested on fish fed the same ration and the effects of saline environment on fish fed ad libitum. The mean size of white muscle fibres was relatively unaffected by the different treatments tested: genetic origin and dietary lipid in different environmental conditions. There were significant differences in growth rate due to genetic origin and saline environment. The possible involvement of hyperplasia in response to these different factors is discussed. Growth hormone supplementation enhanced the percentage of small diameter fibres indicating a role of this hormone in the control of muscle hyperplastic growth. The mean size of adipose cells was affected only slightly by the different treatments tested. An increase in adipose cell size with aging and lipid content was observed. The percentage of small adipocytes also increased with aging. Thus, it is proposed that the development of adipose tissues, and thus fat retention, both result from the recruitment of new adipocytes and from the increase in size of existing adipocytes. The hyperplastic process contributed significantly to the differences in fat retention due to different treatments tested (strains, thermal and saline environments). When partially substituting fish oils for corn oils in the diet, a large increase in the ventral adipose cell size was seen indicating a potential negative effect of n-6 fatty acids on cell proliferation. Growth hormone treatment, on the contrary, induced a decrease in the size of perivisceral adipocytes. Thus, diet and hormonal status affect adipose cells size through two different metabolic pathways: lipogenesis and lipolysis respectively.     

Zinc uptake across the apical membrane of freshwater rainbow trout intestine is mediated by high affinity, low affinity, and histidine-facilitated pathways

Zinc is both a vital nutrient and an important toxicant to aquatic biota. In order to understand the interplay between nutrition and toxicity, it will be important to determine the mechanisms and the factors that regulate zinc uptake. The mechanism of apical intestinal Zn(II) uptake in freshwater rainbow trout and its potential modification by the complexing amino acid histidine was investigated using brush-border membrane vesicles (BBMVs). Following characterisation of the BBMV preparation, zinc uptake in the absence of histidine was both time- and concentration-dependent and consisted of two components. A saturable phase of uptake was described by an affinity constant of 57±17 μM and a transport capacity of 1867±296 nmol mg membrane protein⁻¹ min⁻¹. At higher zinc levels (>500 μM) a linear, diffusive component of uptake was evident. Zinc transport was also temperature-dependent, with Q₁₀ values suggesting zinc uptake was a carrier-mediated process. Zinc uptake by vesicles in the presence of histidine was correlated to a mono-histidine species (Zn(His)⁺) at all Zn(II) concentrations examined.     

Zinc uptake across the apical membrane of freshwater rainbow trout intestine is mediated by high affinity,low affinity,and histidine-facilitated pathways

Zinc is both a vital nutrient and an important toxicant to aquatic biota. In order to understand the interplay between nutrition and toxicity, it will be important to determine the mechanisms and the factors that regulate zinc uptake. The mechanism of apical intestinal Zn(II) uptake in freshwater rainbow trout and its potential modification by the complexing amino acid histidine was investigated using brush-border membrane vesicles (BBMVs). Following characterisation of the BBMV preparation, zinc uptake in the absence of histidine was both time- and concentration-dependent and consisted of two components. A saturable phase of uptake was described by an affinity constant of 57+/-17 microM and a transport capacity of 1867+/-296 nmol mg membrane protein(-1) min(-1). At higher zinc levels (>500 microM) a linear, diffusive component of uptake was evident. Zinc transport was also temperature-dependent, with Q10 values suggesting zinc uptake was a carrier-mediated process. Zinc uptake by vesicles in the presence of histidine was correlated to a mono-histidine species (Zn(His)+) at all Zn(II) concentrations examined.     

Biochemical polymorphisms in muscle and liver extracts and in the serum of the rainbow trout Salmo gairdneri

The frequency of polymorphic phenotypes determined by starch gel electrophoresis from six enzyme systems was investigated on 664 rainbow trout (stock I) originating equally from six full-sib families. The enzyme systems studied were CA, AKP, G-6-PD, SOD, AEP, and 6-PGD.
On material deriving from six parental matings totalling 212 offspring (stock II) the mode of inheritance of the first four enzymes (AEP and 6-PGD were not polymorphic) and the additional systems IDH, PGM, Alb, and Psta, not analysed in stock I, were investigated.
The G-6-PD system showed no polymorphism in the family material. The CA, PGM, Alb, and Psta systems were easily identifiable. Their mode of inheritance with two alleles each can be considered as proven. For SOD three alleles, in four out of six possible progeny types, were found, for which the postulated mode of inheritance was confirmed.
For IDH the mode of inheritance found by Allendorff & Utter (1973) was confirmed. This pattern shows two disomic gene loci, one of which is monomorphic, while the other carries four different alleles.
The number of alleles and their mode of inheritance for the AEP system, which was not clearly identifiable, could not be elucidated.     

Piscirickettsia salmonis induces apoptosis in macrophages and monocyte‐like cells from rainbow trout

Piscirickettsia salmonis is the etiologic agent of the salmonid rickettsial septicemia (SRS) which causes significant losses in salmon production in Chile and other and in other regions in the southern hemisphere. As the killing of phagocytes is an important pathogenic mechanism for other bacteria to establish infections in vertebrates, we investigated whether P. salmonis kills trout macrophages by apoptosis. Apoptosis in infected macrophages was demonstrated by techniques based on morphological changes and host cell DNA fragmentation. Transmission electron microcopy showed classic apoptotic characteristics and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling showed fragmented DNA. Programmed cell death type I was further confirmed by increased binding of annexin V to externalized phosphatidylserine in infected macrophages. Moreover, significant increases of caspase 3 activation were detected in infected cells and treatment with caspase inhibitor caused a decrease in levels of apoptosis. This is the first evidence that P. salmonis induces cell death in trout macrophages. This could lead to bacterial survival and evasion of the host immune response and play an important role in the establishment of infection in the host. J. Cell. Biochem. 110: 468–476, 2010. © 2010 Wiley‐Liss, Inc.     

Developmental events of the pectoral muscle in rainbow trout larvae (Salmo gairdneri)

Skeletal muscles of developing pectoral fins in rainbow trout larvae (Salmo gairdneri) were analyzed by electron microscopy. Large, branched mitochondria were dominant structures in developing myotubes. Mitochondria were associated with the tubular system (T and SR). New mitochondria arose from old ones when the latter extruded whorls of paired membranes surrounding a nonmembranous core. The core was comprised in part of a dense material, presumably, DNA. The developing muscles were characterized by two sets of caveolae which provided the major contributions to the tubular system. Large caveolae gave rise to elements traditionally designated as SR tubules but which later lost their exterior connections. Small caveolae gave rise to small diameter tubules that appear to be analogous to T tubules, which maintained connections with the exterior. Both tubular elements abutted mitochondria. The two elements ran parallel to each other and intersected with each other to form junctions. Each set of elements possessed intratubular junctions.