Spawning induces a shift in energy metabolism from glucose to lipid in rainbow trout white muscle

Enzymatic changes that occur in the white somatic muscle of rainbow trout (Oncorhynchus mykiss) in response to spawning were investigated, and the evenness of their distribution across the ventral-dorsal plane of this muscle was assessed. Four enzymes that are involved in energy metabolism were measured (phosphofructokinase: glycolytic capacity, 3-hydroxyacyl-CoA dehydrogenase: -oxidation, citrate synthase: citric acid cycle, cytochrome oxidase: oxidative capacity). The enzyme activities were followed in different parts of the white muscle of non-spawning female rainbow trout from May, four months after their first spawning, until December, at second spawning. Samples were taken from white epaxial muscle along the lateral line, on the dorsum, and in between. Samples were also taken from red muscle of non-spawning fish. The isoforms of myosin heavy chains (MyHC) were electrophoretically identified on 6% SDS-PAGE gel to study possible changes in contractile properties of the muscle.Transformation from the non-spawning to spawning phase was associated with dramatic changes in the activity of the enzymes studied in white muscle: glycolytic capacity decreased to less than half, whereas oxidative metabolism increased about two- to four-fold in all areas. Significant quantitative differences in enzyme activities were found between the three epaxial muscle areas: in the non-spawning fish lateral line samples differed from those taken in the other two areas, whereas in spawning fish the dorsal sample difered from the other two. No difference in the expression of MyHC-isoforms was found between spawning and non-spawning fish. Co-expression of both slow and fast isoforms was found in single fibres isolated from red muscle.The results show that the energy metabolism in white muscle of domestic rainbow trout is altered during spawning; i.e., the metabolism becomes increasingly aerobic, with an increased capacity for fatty acid utilization, concomitant with phenotypic changes associated with sexual maturation. These changes are especially pronounced in ventral, superficially located fibres.     

Myosin,parvalbumin and myofibril expression in barbel (Barbus barbus L.) lateral white muscle during development

Histo- and immunohistochemical techniques have recently been used to study the fibre type and myosin expression in fish muscle during development. In the present work, embryonic, larval and adult myosin isozymes (heavy and light chains) and parvalbumin isotypes were analyzed, from fertization to the adult stage, by polyacrylamide gel electrophoresis of barbel (Barbus barbus L.) trunk muscle extracts. The examined myosins display the sequential transitions from embryonic to larval and adult forms characteristic of higher vertebrates. They are characterized by specific heavy chains but their light chains differ only by the LC₁/LC₃ stoichiometry with LC₃ exceeding LC₁ after 10 days. Sarcoplasmic parvalbumins show considerable and unforeseen developmental transitions in their isotype distribution: the PA II isotype first appears after hatching and becomes the predominant form until the length reaches about 6 cm. One month after hatching, the amount of PA II then decreases and the synthesis of PA III and IV further increases to reach the typical adult pattern at a size of 18 cm. These observations show that the distribution of parvalbumin isotypes reflects the stage of development. It suggests a specific role for each isotype in relation to muscle activity. Microscopy illustrates the progressive development of somites, muscles cells, and myofibrils, which accelerates at hatching when movements increase.     

Biochemical differences in lateral muscle of wild and farmed gilthead sea bream (series Sparus aurata L.)

Red and white muscle from specimens of wild and farmed gilthead sea bream (Sparus aurata) were analyzed for histochemical ATPase activity, total protein content, fatty acids, trace element concentrations and myosin isoforms. The fibre type composition of muscle samples was confirmed histochemically by the ATPase reaction, which did not show any differences between the two groups of animals. Myosin ATPase activities, myosin and protein yields were significantly higher in white muscle than in the red muscle and for the red muscle the latter two parameters were higher in wild fish. Fatty acid profiles revealed differences between the two groups of animals, probably because of the fatty acid composition of the diets. Zinc, copper and iron concentrations were higher in red muscle than in white muscle; muscles from wild fish were significantly richer in trace elements. No separation of fast and slow heavy chains of myosin could be obtained on SDS-gel electrophoresis, but two dimensional electrophoresis revealed the presence of three light chains in white muscle (LC1F, LC2F, LC3F), and two main types in red muscle (LC1S, LC2S). Small, variable percentages of LC3F were found in the red muscle samples, especially in the wild fish. It is concluded that the different environmental conditions, experienced by wild and farmed fish, have significantly influenced the biochemical composition of their lateral muscle.     

Expression of four muscle proteins at different growth stages of Günther's walking catfish Clarias macrocephalus

The complete cDNA sequences of four contractile muscle genes of walking catfish Clarias macrocephalus were characterized by assembling partial EST sequences from a skeletal muscle cDNA library. The four genes were parvalbumin 4 (PV4) (670 bp), troponin C (TnC) (1065 bp), troponin I (TnI) (843 bp) and myosin light chain 3 (MLC3) (953 bp), leading to deduced amino acid sequences of 109, 160, 176 and 150 residues respectively. During the larval stage, TnC mRNA showed the highest levels of expression with a 1.4‐fold increase from day 1 to day 30 post hatch. At 90 days, the relative expression levels of PV4, TnC and MLC3 were the highest, with similar proportions in the skeletal muscle, corresponding to the highest relative growth rate of walking catfish. Expression of the three calcium‐binding proteins remained high in 6‐month‐old fish, with higher levels of PV4. The different proportions of muscle proteins expressed suggested the significance of their contributions to fish growth and appeared to be correlated with the functional properties of muscle cells, which were observed from changes in the swimming activity of the fish.     

C-protein (MyBP-C) isoforms from carp ordinary and dark muscles and muscle type-specific binding to myosin

ABSTRACT:   C-protein is a myosin-associated protein of vertebrate striated muscle, and its function and properties have been extensively examined. However, there has been no report of C-protein of fish skeletal muscle so far. C-protein was identified in carp skeletal muscle by immunoassay using antibody against chicken C-protein, and the muscle-type specific C-protein was purified from carp ordinary and dark muscles for the first time. Although C-protein could be prepared from crude myosin by the reported procedure, C-protein degraded appreciably during the purification steps. Accordingly, C-protein was selectively extracted from the muscle with 0.15 M K-phosphate buffer (pH 5.8), and purified by ammonium sulfate fractionation, followed by AF-blue chromatography. Myosin free from the accessory proteins was obtained by diethylaminoethyl (DEAE) chromatography and used to assay the binding of C-protein with myosin. Ordinary muscle C-protein bound to ordinary muscle myosin in a saturable manner, but its maximum amount of binding was approximately twice that of dark muscle myosin. Similarly, dark muscle C-protein bound to dark muscle myosin much more than to ordinary muscle myosin. These results suggest that C-protein isoforms specifically bound with myosin isoforms originated from the same type of muscle.     

鲢肌球蛋白重链球状结构域的cDNA克隆及结构解析

根据已经获得的两种鲢肌球蛋白重链同工型基因(低温型sc-w和高温型sc-s)在3′端展现的明显差异,设计了2个特异性的反向引物,以鲤科鱼类肌球蛋白重链5′端的保守序列为正向引物,通过Long-PCR对编码鲢两种肌球蛋白重链同工型的球状结构域(Subfragment-1,S1)的全长基因进行了克隆和测序,并推断出它们一级结构的氨基酸序列。研究结果表明,sc-w与sc-s在S1的初级结构上显示80.5%的同源性、与已经报道的草鱼低温型(gc10)有97.2%的高同源性;sc-s则与草鱼中间型(gcI)和高温型(gc30)显示了分别为98.4%和97.1%的高同源性。低温型的sc-w和gc10在S1初级结构上展现的特有变异主要发生在43个氨基酸残基位点,其中15个属保守性残基。对S1区域中两个功能性的表面环loop1(与ATP结合位点有关)和loop2(与肌动蛋白结合位点有关)的结构解析发现,sc-w和gc10在两个表面环的长度、残基电荷分布和氨基酸组成等方面与其它同工型之间存在明显差异,揭示了这两个表面环的结构差异可能影响了栖息于不同环境温度下的淡水鱼的肌球蛋白分子马达功能。分子系统树的分析结果进一步证明,鱼类栖...     

Species-specific thermal denaturation pattern of fish myosin when heated as myofibrils as studied by myosin subfragment-1 and rod denaturation rates

Thermal denaturation of myofibrils from various species of fish was investigated by measuring ATPase inactivation, myosin aggregation, myosin subfragment-1 (S-1) and rod denaturation rates as studied by chymotryptic digestion. Decrease in monomeric myosin (myosin aggregation) was always faster than the ATPase inactivation for all myofibrils tested. The relative denaturation rate of rod to that of S-1 differed from species to species. Preceded denaturation of rod was observed with some species, and the opposite was true with other species. The denaturation pattern was explained by the different magnitude of S-1 stabilization by F-actin in myofibrils at low salt medium. Myofibrils which receive a great stabilization by F-actin as studied by ATPase inactivation showed the preceded rod denaturation pattern, and vice versa. S-1 portion, not F-actin, determined the different stabilization of S-1 by F-actin in myofibrils.     

Expression of the myosin light chains 1, 2 and 3 in the muscle of blackspot seabream (<Emphasis Type="Italic">Pagellus bogaraveo</Emphasis>, Brunnich), during development

Previous studies on the histochemistry and immunoreactivity of fibres in lateral muscle of blackspot seabream indicated that there is a developmental transition in the composition of myofibrillar proteins, which presumably reflects changes in contractile function as the fish grows. We hypothesize that the phenomenon underscores age and spatial differences in the expression of myosin light chains (MLC), not studied yet in this species. In this study, we examined selected stages in the post-hatching development of the muscle of blackspot seabream: hatching (0 days), mouth opening (5 days), weaning (40 days) and juveniles (70 days). The spatial expression of embryonic MLC 1 (MLC1), 2 (MLC2) and 3 (MLC3) was studied by in situ hybridization. Overall, MLC expression patterns were overlapping and restricted to the fast muscle. At hatching and mouth opening, all MLC types were highly expressed throughout the musculature in fast muscle. The expression levels in fast muscle remained high until weaning when germinal zones appeared on the dorsal and ventral areas. The germinal zones were characterized by small-diameter fast fibres with high levels of MLC expression. This pattern persisted up to day 70, when the germinal zones disappeared and expression of MLCs was observed only in the smaller cells of the fast muscle mosaic. These results support our hypothesis and, together with previous imuno- and histochemistry results, allow a better understanding of the mechanism of muscle differentiation and growth in fish beyond larval stages, and form- the basis for further comparative and experimental studies with this economically relevant species.     

MyoD,myogenin and proliferating cell nuclear antigen expression in growing Nile tilapia (Oreochromis niloticus L.)

In the present study, immunohistochemical and morphometric analysis was used to characterize variations in muscle growth performance during muscle fibre recruitment and hypertrophy. As in fisheries, fish were classified into four age stages: alevin 35 days (0.65±0.08 g); juvenile 60 days (13.67±1.35 g); adult 90 days (73.18±4.70 g) and adult 190 days (349.76±34.62 g). The number of nuclei expressing MyoD and myogenin was similar in alevin, juvenile and adult 90 days; however, in adult 190 days, the number of nuclei expressing myogenin was higher than the number expressing MyoD. The number of proliferating cell nuclear antigen‐stained nuclei in each stage was higher than MyoD and myogenin staining with peaks in alevin and adult 90 days. These data suggest that growth per se stimulated cellular proliferation and nuclei accretion of Nile tilapia muscle fibres in alevin, juvenile and adult 90 days. Muscle fibre differentiation was more pronounced in adult 190 days.     

Mechanochemical properties of ordinary and dark muscle myosins from seawater fish

Myosin was isolated from two types of muscle, ordinary and dark muscles, of three species of fish living in sea water. The compositions of light chains were visualized by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the mechanochemical activity was examined by in vitro motility and ATPase assays. Ordinary muscle myosin of either species had three species of light chain, whereas dark muscle myosin had another two species of light chain judged by SDS-PAGE. Sliding velocity of ordinary muscle myosin was in the range of 4.92–6.89 μm/S, whereas that of dark muscle myosin was in the range of 3.07–4.25 μm/s. Therefore, ordinary muscle myosin showed 1.26–1.95 times higher sliding velocity than dark muscle myosin in either species. The ratios of V_max of actin-activated Mg²⁺-ATPase activity of ordinary to dark muscle myosins were correlated quite well to the ratios of sliding velocity. Activity of ordinary muscle myosin was comparable to that of mammalian fast muscle myosin, but that of dark muscle myosin was twice of that of mammalian slow muscle myosin. These results may reflect the essential role of fish dark muscle myosin always used in slow cruising.