Myosatellite cells of Cyprinus carpio (Teleostei) in vitro: isolation,recognition and differentiation

Summary We have developed a method for the dissociation and purification of myosatellite cells from white epaxial muscle of carp. The dissociated myosatellite cells were identified by their morphology, their ultrastructure, the formation of multinucleated myotubes containing myofibrils and the immunocytochemical demonstration of desmin. Desmin and 5-bromo-2-deoxyuridine (BrdU) were used to identify terminally differentiated and proliferating myosatellite cells, respectively. The in vitro behavior of myosatellite cells dissociated from carp of 5 cm standard length differed from that described for myosatellite cells of mammals and birds. No substantial proliferation of the myosatellite cells could be observed. Most cells were differentiated (desmin-positive, BrdU-negative) 17 h after plating, regardless of the medium used. This indicates that the investigated white epaxial muscle of carp of 5 cm standard length contains subpopulations of myosatellite cells, arrested at various stages of differentiation.     

Uptake of tritiated thymidine in muscle of juvenile carp

In juvenile carp (4.5–6 cm s.l .) labelled myosatellite cell nuclei are found 24 h after injection of tritiated thymidine, but labelled myonuclei after 48 h, indicating that fish myonuclei do not proliferate but originate from undifferentiated myogenic cells. The time pattern accords with the estimated cell-cycle time of adult myogenic cells of carp.     

Numbers of myosatellite cells in white axial muscle of growing fish: Cyprinus carpio L. (Teleostei).

By means of transmission electron microscopy (TEM), the percentage of myosatellite cells was shown to decrease from about 6% in carp of 5 cm standard length (SL) to less than 1% in carp larger than 18 cm SL. The ratio between muscle nuclei and non muscle nuclei remained constant. These TEM data, combined with data on the amount of DNA per gram of muscle tissue and per nucleus, were used to calculate the numbers of myosatellite cells per gram of tissue (TEM-DNA method). Total numbers of myosatellite cells could be calculated from the TEM-DNA data and the calculated amounts of muscle tissue per fish. After a slight initial increase, the total number of myosatellite cells in the white axial muscle of a carp appears to be rather constant during the growth phase of the fish. But the myosatellite cells become more and more diluted over an increasing number of myonuclei with age. In addition, the reliability of two new light microscopic methods for the determination of numbers of myosatellite cells was examined. The percentages of myosatellite cells were determined by counting the numbers of total nuclei and of heterochromatic nuclei situated inside the muscle fibers' basal laminae which were stained using an antibody against laminin. These percentages were not significantly different from those determined with TEM. The yield of myosatellite cells per gram of muscle tissue, isolated with a previously developed dissociation method, showed a direct relation to the number of myosatellite cells calculated to be present in the tissue (TEM-DNA method), but at a 1% level. Both methods are alternative ways to determine numbers of myosatellite cells when it is impossible or difficult to use TEM (e.g., large sample sizes, combination with immunohistochemical methods).     

Growth of carp (Cyprinus carpio) white axial muscle; hyperplasia and hypertrophy in relation to the myonucleus/sarcoplasm ratio and the occurrence of different subclasses of myogenic cells

In white axial muscle of carp addition of new fibres to the muscle mass (hyperplasia) decreased with increasing length of the fish. This was deducted from the decrease in the amount of small fibres. In carp larger than about 40 cm standard length (s.l.) hyperplasia no longer occurred (small fibres were absent) and muscle growth only occurred by means of hypertrophy (growth of existing fibres). The stage of growth in which many new fibres were added showed a relatively fast increase in trunk weight, as calculated from growth curves. During the stage of fast growth with a high occurrence of hyperplasia, the DNA/protein ratio decreased. The high percentage of postmitotic myosatellite cells isolated from carp of 5 cm s.l. suggests that in hyperplasia a subpopulation of already differentiated myosatellite cells formed in an earlier stage of development is incorporated in new muscle fibres. The increase of the relative importance of hypertrophy appears to be correlated to an increase in the percentage of proliferating myosatellite cells 17 h after isolation in vitro . This suggests that in hyperplasia and in hypertrophy different subpopulations of myosatellite cells are involved.     

Numbers of muscle nuclei and myosatellite cell nuclei in red and white axial muscle during growth of the carp (Cyprinus carpio)

We determined the percentages of muscle fibie nuclei and satellite nuclei over a growth range of carp ( Cyprinus carpio ), as the increase in the number of muscle fibre nuclei is an important aspect of the increase in muscle mass, and myosatellite cells are believed to be the source of new muscle fibre nuclei. In white as well as in red axial muscle the percentage of the nuclei present in muscle that are muscle nuclei (muscle fibre nuclei+myosatellite nuclei) remained constant during growth (54 and 32% respectively). The difference in the percentage of non-muscle nuclei between white and red axial muscle is mainly caused by the higher content of endothelial nuclei in red axial muscle.
In white axial muscle the DNA/protein ratio (nucleus/sarcoplasm ratio) decreased between 3 and 15 cm S.l. In red axial muscle we found a continuous decrease in DNA/protein ratio over the entire investigated size range (3–50 cm s.l.). This may be related to a longer occurrence of hyperplasia in red than in white axial muscle.
In both fibre types the percentage of muscle nuclei being myosatellite nuclei decreased with increasing length, In white axial muscle it decreased from about 5% in carp of 5 cm s.l. to less than 1% in carp of 20 cm S.L.; for red muscle these values were 11 and 3% respectively.
For white axial muscle we calculated that, especially in larger fish, the myosatellite ceils alone cannot account for the increase in the number of muscle fibre nuclei during growth. The percentage of proliferating nuclei in muscle tissue, measured by the uptake of 5-bromo-2'-deoxy-uridine, is high enough to account for the total increase in nuclei. So indirect evidence is available that another cell type present in the muscle tissue may also be involved in the formation of additional muscle fibre nuclei.     

Primary rat muscle progenitor cells have decreased proliferation and myotube formation during passages

Abstract.  Adult skeletal muscle contains populations of satellite cells and muscle-derived stem cells that are capable of forming multinucleate myotubes. The purpose of this study was to determine the phenotype of cells isolated from a common satellite cell isolation and passaging procedure from whole skeletal muscle. To ascertain the characteristics of the cellular phenotype, the myogenic markers MyoD and desmin, the satellite-cell-specific marker Pax7, and the haemopoietic stem cell markers CD34 and CD45 were examined by immunohistochemical analysis. Immediately after isolation, > 90% myogenic marker-positive cells were positive for desmin, MyoD and Pax7. In contrast, ∼10% of the isolated cells expressed only CD34 or CD45. After three passages, the percentage of cells that were positive for the myogenic markers desmin, MyoD and Pax7 was reduced to ∼55%, while the population of CD34- or CD45-positive cells increased to ∼30% after the third passage. Immunohistochemical detection of bromodeoxyuridine demonstrated that the number of proliferating cells decreased progressively after each passaging. Finally, after the third passage the percentage of nuclei in myotubes decreased from 46.7% to 12.5%. Since passaging of muscle progenitor cells is common practice, the results of the current report suggest that characterization of cell heterogeneity needs to be made frequently.     

Estimates of daily food intake by an inshore population of Pleuronectes platessa L. off eastern Anglesey, North Wales

A sequence of trawl surveys showed that the diet of plaice off eastern Anglesey was dominated by Abra alba in spring and summer, and by Pectinaria koreni in early spring and autumn. Plaice ate little food in winter. Despite the relatively small stomach in this species, plaice were able to achieve high rates of daily food intake during bouts of heavy feeding by rapidly transferring newly-ingested prey items into the anterior intestine without full gastric digestion. Seasonal daily feeding rates (percentage body weight) varied between 5 and 10% for small fish (15–20 cm total length), 3 and 12% for medium fish (20–25 cm) and 2.8 and 4.4% for large fish (30–35 cm).     

Differential Location of Different Types of Intermediate-Sized Filaments in Various Tissues of the Chicken Embryo

The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11–20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to – and specific for – epithelial cells; vimentin filaments are seen – at this stage of embryogenesis – only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.e., desmin) are significant only in muscle cells; and intermediate filament protein of brain, most probably neurofilament protein, is present only in nerve cells. We conclude that for most tissues the expression of filaments of cytokeratin, vimentin, desmin, and neurofilament protein is mutually exclusive, and that these protein structures provide useful markers for histochemical and cytochemical differentiation of cells of epithelial, mesenchymal, myogenic, and neurogenic differentiation.     

Predation by Nile perch Lates niloticus (L.) on Oreochromis niloticus (L.), Cyprinus carpio (L.), Mugil sp. and its role in controlling tilapia recruitment in Egypt

Vulnerability of three fish groups (tilapia, common carp, mullet) to predation by Nile perch Lates niloticus was evaluated. The study was conducted in aquaria and in cages placed in concrete ponds. Common carp Cyprinus curpio was the more vulnerable to predation by Nile perch followed in order by mullet ( Mugil cephulus and M. cupito ) and Nile tilapia Oreochromis niloricus . Medium-size Nile perch (30–32 cm t.l. ) were able to consume tilapia up to 14 cm t.l. , while tilapia of 15 cm total length were not eaten. Ratios of 1 : 10 to 1 : 15 between Nile perch and ready-to-spawn females of O. niloticus were found to be adequate to control tilapia reproduction.     

Age estimation, growth, maturity and distribution of the roundnose grenadier from the Rockall trough

This paper summarizes the history of commercial exploitation of roundnose grenadier Coryphaenoides rupestris in the North Atlantic. Length frequencies of C. rupestris in 1993, from 400 to 1200 m on the slopes of the Rockall trough indicate a reduction since the 1970s in the modal length of fish found at 700–1000 m. Ages ranged from 2 to 50 years for males and 2 to 60 years for females, with most between 10–38 years. Females attained a greater asymptotic pre-anus length ( L _∞=19.5 cm) than males ( L _∞=15.5 cm) and had a greater weight for a given age (male W _∞=761g, female W _∞=1132g). This species may have a protracted spawning period. Using pre-anus lengths, 50% of male fish were mature at 10 cm (ages 8–10) while 50% of female fish were mature at 12 cm (ages 9–11). At the greatest depths sampled the length frequency of fish was bimodal with a hiatus between 9 and 11 cm (ages 8–12). Highest catch rates occurred on the Donegal slope in September at a depth of 800–1000 m.     

Intensive culture of grass carp and hybrid grass carp larvae

Intensive culture of grass carp and hybrid grass carp (Ctenopharyngodon idella x Aristichthys nobilis ) larvae was conducted under Florida (U.S.A.) conditions. The influence of different rearing facilities (indoor tanks, outdoor tanks and cages) and different methods of fish feeding using live zooplankton and artificial food was tested. High survival (86–100%) and satisfactory grass carp growth (47–56 mg) were obtained in the outdoor tanks and cages during the 10–day experiment. It is believed, that the technique described can be used after some improvements for commercial–scale grass carp larvae rearing. Low survival (1–3%) was obtained in the hybrid culture experiment even though satisfactory growth rates (84–212 mg) were obtained after 13 days. High mortality was attributed to genetic abnormalities caused by hybridization. The hybrid does not appear to be a promising fish for culture.     

Lake Tana large barbs: phenetics, growth and diversification

Among the Lake Tana large barbs of about 10 cm SL only representatives of the' acute'morphotype can be distinguished, in the size range 10–20 cm SL' bigmouth big eye' can be identified also. As for the rest, very few individuals can be confidently affiliated with a particular morphotype most of them looking like' intermedius'. Even within the range of 20–30 cm SL some individuals are still difficult to identify. Principal component analysis of cranial characters revealed discrete groups of morphotypes. Differences in both external and cranial characters of the morphotypes result from divergence which is most pronounced when fish are 4–5 years old and 20–25 cm SL. This divergence cannot be related exclusively with differences in the growth rates of individuals representing different morphotypes. Differences in food composition between the morphotypes probably increase in parallel with their morphological divergence. Differences between the morphotypes in the lateral line (11) and the gill rakers (Sb) counts were revealed using ANOVA. Comparison of the Lake Tana Barbus complex of forms with that previously known from Lake Lanao (Philippines) suggests that in both lakes the different forms arose sympatrically but that sympatric speciation in Lake Lanao has advanced further than in Lake Tana.     

Diet of otters (Lutra lutra) inhabiting small rivers in the Bialowiea National Park, eastern Poland

The diet of otters Lutra lutra L. inhabiting small rivers in the primeval forests of the Bialowieza National Park was studied in 1988–91 by analysis of 135 spraints. Anurans, mainly Rana temporaria, and fish, mainly Cyprinidae, were two staple prey of otters. In autumn and winter (1 October–31 March), 99% of spraints contained anuran remains and 45% fish remains. This corresponded to 66 and 34% of biomass consumed by otters, respectively. In spring and summer, anurans dropped to 55% occurrence in spraints (38% of biomass eaten by otters) and that of fish increased to 69% occurrence (50% of biomass). Fifty per cent of fish caught by otters were very small specimens (body length 6.5–10 cm) and 35% were small (> 10–15 cm). Pike were the only big fish captured by otters (mean body length 31 cm). Water beetles Dytiscidae were commonly eaten by otters in warm seasons (72% of occurrence in spraints, 9% of biomass taken by otters in spring and summer). Birds, snails, mammals and crayfish were minor supplements to the otters' diet.     

Insulin-like growth factor as a novel stimulator for somatolactin secretion and synthesis in carp pituitary cells via activation of MAPK cascades

Somatolactin (SL), a member of the growth hormone/prolactin family, is a pituitary hormone unique to fish models. Although SL is known to have diverse functions in fish, the mechanisms regulating its secretion and synthesis have not been fully characterized. Using grass carp pituitary cells as a model, here we examined the role of insulin-like growth factor (IGF) in SL regulation at the pituitary level. As a first step, the antisera for the two SL isoforms expressed in the carp pituitary, SLα and SLβ, were produced, and their specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the carp pituitary. Western blot using these antisera revealed that grass carp SLα and SLβ could be N-linked glycosylated and their basal secretion and cell content in carp pituitary cells could be elevated by IGF-I and -II treatment. These stimulatory effects occurred with parallel rises in SLα and SLβ mRNA levels, and these SL gene expression responses were not mimicked by insulin but blocked by IGF-I receptor inactivation. In carp pituitary cells, IGF-I and -II could induce rapid phosphorylation of IGF-I receptor, MEK1/2, ERK1/2, MKK3/6, and p38 MAPK; and SLα and SLβ secretion, protein production, and mRNA expression caused by IGF-I and -II stimulation were negated by inactivating MEK1/2 and p38 MAPK. Parallel inhibition of PI3K and Akt, however, were not effective in these regards. These results, taken together, provide evidence that IGF can upregulate SL secretion and synthesis at the pituitary level via stimulation of MAPK- but not PI3K/Akt-dependent pathways.     

Reproduction in the greenback grey mullet, Liza subviridis (Valenciennes, 1836)

This paper presents data on the reproductive biology of Liza subviridis , a little studied mullet species. The fish is heterosexual, exhibiting external fertilization. Six maturity stages can be macroscopically identified in the testes and seven in the ovaries. The macroscopic changes in the gonads are manifestations of histological changes occurring in the development of sperm cells and oocytes. First sexual maturity is attained in the length ranges of 9.5–11.5 cm and 10.5–11.5 cm in male and female fish respectively. The fecundity for fish measuring 10.3–13.9 cm in standard length ranged from 40 000–145 000 eggs. The relationship between fecundity ( F ) and length ( L ) can be represented as: F = 1.9044 L ^4.2998. The spawning duration in L. subviridis is restricted to a short and definite period, with all ripe ova being released within a single spawning act. A pronounced spawning season can be detected to extend from June to November. However, during off-seasons, some spawning also occurs. The correlations between spawning, rainfall and air temperature is discussed.     

In vitro characterization of proliferation and differentiation of trout satellite cells

Fish satellite cells have been extracted from various species, but the myogenic characteristics of these cells in culture remain largely unknown. We show here that 60%-70% of the adherent cells are myogenic based on their immunoreactivity for the myogenic regulatory factor MyoD. In DMEM containing 10% fetal calf serum (FCS), trout myoblasts display rapid expression of myogenin (18% of myogenin-positive cells at day 2) combined with rapid fusion into myotubes (50% of myogenin-positive nuclei and 30% nuclei in myosin heavy chain [MyHC]-positive cells at day 7). These kinetics of differentiation are reminiscent of the behavior of fetal myoblasts in mammals. However, not all the myogenic cells differentiate; this subpopulation of cells might correspond to the previously named “reserve” cells. More than 90% of the BrdU-positive cells are also positive for MyoD, indicating that myogenic cells proliferate in vitro. By contrast, less than 1% of myogenin-positive cells are positive for BrdU suggesting that myogenin expression occurs only in post-mitotic cells. In order to maximize either the proliferation or the differentiation of cells, we have defined new culture conditions based on the use of a proliferation medium (F10+10%FCS) and a differentiation medium (DMEM+2%FCS). Three days after switching the medium, the differentiation index (% MyHC-positive nuclei) is 40-fold higher than that in proliferation medium, whereas the proliferation index (% BrdU-positive nuclei) is three-fold lower. Stimulation of cell proliferation by insulin-like growth factor 1 (IGF1), IGF2, and FGF2 is greater in F10 medium. The characterization of these extracted muscle cells thus validates the use of this in vitro system of myogenesis in further studies of the myogenic activity of growth factors in trout.     

Contribution to the feeding ecology of the predatory wingfin anchovy Pterengraulis atherinoides (L.) in north Brazilian mangrove creeks

Stomach contents were examined from 136 Amazonian wingfin anchovy, Pterengraulis atherinoides (Engraulidae), caught from intertidal mangrove creeks at diurnal neap tides between June and September 1997 (early dry season) near Bragança (northern Brazil). The study found that P. atherinoides are specialized predators of juvenile Natantia and Teleostei (mean: 67 and 28% by dry weight, respectively). On average, 5.2 g ha^?1 day^?1 of Natantia and 2.6 g ha^?1 day^?1 of Teleostei (wet weight) were eaten by P. atherinoides. Diet changed with fish size as well as by month. While smaller sizes still fed on several food items (e.g. the copepod Pseudiaptomus marshii, the brachyuran crab Pachygrapsus gracilis, amphipods), fish >13 cm standard length (SL) fed exclusively on Natantia and Teleostei. Copepods were especially abundant in July and August, dominating the diet of fish <9 cm SL in numbers (92%). Our results suggest a positive relationship between predator size and prey size, both in penaeid and piscine prey. However, the largest predator size class apparently selected fewer but larger Teleostei prey. More than 64% of Natantia were juvenile penaeid shrimps of commercial importance (Fenneropenaeus subtilis, F. schmitti, Xiphopenaeus kroyeri). Comparison with ichthyoplankton samples taken simultaneously showed that Sciaenidae and Mugilidae were positively selected while Gobiidae and Engraulidae were negatively selected. The presence of pranzia larvae in the stomachs of fish <10 cm SL, from July onward, suggests that these sizes fulfil a mutually beneficial ‘cleaning’ function on other fish. Block net sampling at neap tides showed that P. atherinoides were present in intertidal mangrove creeks throughout the submergence period, suggesting temporal optimization of the foraging time in the eulittoral.     

Grass carp somatolactin: I. Evidence for PACAP induction of somatolactin-alpha and -beta gene expression via activation of pituitary PAC-I receptors

Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. At present, SL can be identified only in fish but not in tetrapods and its regulation at the pituitary level has not been fully characterized. Using grass carp as a model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL secretion and synthesis at the pituitary cell level. As a first step, the structural identity of grass carp SL, SLalpha and SLbeta, was established by 5'/3'-rapid amplification of cDNA ends. These two SL isoforms are single-copy genes and are expressed in two separate populations of pituitary cells located in the pars intermedia. In the carp pituitary, PACAP nerve fibers were detected in the nerve tracts of the neurohypophysis and extended into the vicinity of pituitary cells forming the pars intermedia. In primary cultures of grass carp pituitary cells, PACAP was effective in stimulating SL release, cellular SL content, and total SL production. The increase in SL production also occurred with parallel rises in SLalpha and SLbeta mRNA levels. With the use of a combination of molecular and pharmacological approaches, PACAP-induced SL release and SL gene expression were shown to be mediated by pituitary PAC-I receptors. These findings, as a whole, suggest that PACAP may serve as a hypophysiotropic factor in fish stimulating SL secretion and synthesis at the pituitary level. Apparently, PACAP-induced SL production is mediated by upregulation of SLalpha and SLbeta gene expression through activation of PAC-I receptors.