Gilles Paboeuf

A gene with homology to myogenin is expressed in developing myotomal musculature of the rainbow trout and in vitro during the conversion of myosatellite cells to myotubes

We report the cloning of a new trout myogenic cDNA which encodes helix‐loop‐helix protein homologous to the myogenic factor myogenin. Northern analyses indicate that trout myogenin (Tmyogenin) transcripts accumulate in large amounts in the myotomal masculature of embryos and frys. In adults, transcripts concentrate within the thin lateral layer of red (slow oxydative) muscle fibres. They are present only in low amounts in white (fast glycolytic) muscle fibres which constitute the major part of the trunk musculature. Using an in vitro myogenesis system, we observed that the trout myogenin encoding gene is not activated until myosatellite cells fuse to generate multinucleated myotubes, indicating that Tmyogenin lies downstream of muscle determination factors. All these observations show that in a major taxinomic group like teleosts, a gene with homology to myogenin exists. Its activation during myogenesis suggests that it acts as a major developmental regulator of muscle differentiation.

Effect of fasting and refeeding on in vitro muscle cell proliferation in rainbow trout (Oncorhynchus mykiss).

Abstract. The effects of short-term fasting and refeeding were studied on satellite cells extracted from white epaxial muscle of juvenile rainbow trout (1–3xa0g body weight). In vitro changes in the proliferation of satellite cells were analyzed using bromodeoxyuridine (BrdU) incorporation over a 24-h period. Proliferation in fed control fish was characterized by an initial basal proliferation rate of 5–10% BrdU-labeled nuclei·day–1, followed by an exponential increase at a rate of +18–20%·day–1, up to a maximum of 60–70% BrdU-labeled nuclei·day–1. Characteristics of satellite cells extracted from starved fish, namely extraction yield, morphology, and proliferation, were different from those of fed fish. Fasting (8–10xa0days) completely suppressed initial proliferation of satellite cells in vitro over a period of 4xa0days. After this delay, proliferation resumed and changes in proliferation rates over time were similar to those of the control group. In fish fed for 4xa0days after an 8-day fast, the initial proliferation rate and the changes in proliferation rates over time were completely restored. These findings demonstrate that satellite cells express different behavior depending on feeding status, which could be due to the presence of different satellite cell populations.

Changes induced by dietary energy intake and divergent selection for muscle fat content in rainbow trout (Oncorhynchus mykiss), assessed by transcriptome and proteome analysis of the liver

BackgroundGrowing interest is turned to fat storage levels and allocation within body compartments, due to their impact on human health and quality properties of farm animals. Energy intake and genetic background are major determinants of fattening in most animals, including humans. Previous studies have evidenced that fat deposition depends upon balance between various metabolic pathways. Using divergent selection, we obtained rainbow trout with differences in fat allocation between visceral adipose tissue and muscle, and no change in overall body fat content. Transcriptome and proteome analysis were applied to characterize the molecular changes occurring between these two lines when fed a low or a high energy diet. We focused on the liver, center of intermediary metabolism and the main site for lipogenesis in fish, as in humans and most avian species.ResultsThe proteome and transcriptome analyses provided concordant results. The main changes induced by the dietary treatment were observed in lipid metabolism. The level of transcripts and proteins involved in intracellular lipid transport, fatty acid biosynthesis and anti-oxidant metabolism were lower with the lipid rich diet. In addition, genes and proteins involved in amino-acid catabolism and proteolysis were also under expressed with this diet. The major changes related to the selection effect were observed in levels of transcripts and proteins involved in amino-acid catabolism and proteolysis that were higher in the fat muscle line than in the lean muscle line.ConclusionThe present study led to the identification of novel genes and proteins that responded to long term feeding with a high energy/high fat diet. Although muscle was the direct target, the selection procedure applied significantly affected hepatic metabolism, particularly protein and amino acid derivative metabolism. Interestingly, the selection procedure and the dietary treatment used to increase muscle fat content exerted opposite effects on the expression of the liver genes and proteins, with little interaction between the two factors. Some of the molecules we identified could be used as markers to prevent excess muscle fat accumulation.

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.

Identification of a muscle factor related to MyoD in a fish species

We have isolated the cDNA encoding a myogenic factor expressed in embryonic trout muscle by hybridization with a Xenopus MyoD cDNA. Nucleotide sequence analysis and amino acid comparison showed that this cDNA called TMyoD encodes a polypeptide of 276 amino acids with 70% identity to the entire Xenopus MyoD protein and 92% identity within the basic and myc-like region. Results from Northern blotting showed that the corresponding transcript is expressed both in adult and embryonic skeletal musculature and in an in vitro myogenesis system, but is undetectable in cardiac and smooth muscles and in non muscle tissues.

4 - Muscle Satellite Cells in Fish

The characteristics of fish satellite cells in situ and in vitro are described. The in situ proliferation of satellite cells appears to be very low and therefore not easy to quantify. Differences in the in vitro proliferation capacities of satellite cells have been observed between species, but a large part of these differences could be related to methodology. Most studies on fish satellite cells have been carried out on cyprinids and salmonids. The relationship between proliferation capacity and muscle growth is discussed in these fish. The differentiation of fish satellite cells is partially similar to that of myoblasts. However, there is a great heterogeneity in satellite cell differentiation that needs to be characterized.

Differentially expressed proteins in rainbow trout adipocytes isolated from visceral and subcutaneous tissues

In rainbow trout, subcutaneous (in dorsal and ventral positions) and visceral fat deposits are known to influence the yield of edible flesh, whilst their respective roles in metabolism, storage and release of fatty acids have not, so far, been directly studied. The present work aimed to identify, by using 2D electrophoresis, proteins differentially expressed in isolated mature adipocytes originating from these various localizations in prepubescent females. A total of nine proteins were estimated to be differentially expressed according to the localisation of the adipocytes. Seven protein spots were considered to be present in the three fat deposits at differing abundances, and among them only six were estimated as being specific to fat tissues. Among these, five were more abundant in subcutaneous adipocytes of both sites compared to perivisceral adipocytes. Four were identified: three as H-FABP, ATP synthase, serum deprivation-response protein, indicating higher metabolic activity in subcutaneous adipocytes, while the latter, annexin, indicative of a higher proportion of less mature adipocytes, as also suggested by their smaller mean diameter. The more abundant protein in visceral isolated adipocytes is actin, known to be involved in cytoskeleton structure and to increase during adipogenesis. This allows us to suggest their more mature stage of development, in relation with their higher mean diameter.

GABA-ergic control of prolactin release in rainbow trout (Oncorhynchus mykiss) pituitaries in vitro

The involvement of γ-aminobutyric acid (GABA) in the control of prolactin (PRL) release was investigated in rainbow trout using both perifused pituitary fragments and pituitary cells in primary culture. In our perifusion system, infusion of GABA (10−6 to 10−4 M) caused an inhibition of PRL release (between 20 and 40%). Administration on perifused pituitary fragments of 3APS, a GABAa agonist, mimicked this inhibitory effect. Moreover, bicuculline, a specific antagonist of GABAa receptors, totally abolished GABA effect. When tested on cultured pituitary cells during 40h exposure, GABA (10−5 M) caused a significant decrease in PRL release (24.5%). Baclofen, a specific agonist for GABAb receptor tested at 10−6 and 10−5 M, also inhibited PRL released from cultured pituitary cells. These results demonstrate that GABA inhibits PRL release by acting directly on pituitary cells and that probably both types of GABA receptor (a and b) are involved in this regulation.RésuméNous avons étudié limplication de lacide γ-aminobutirique (GABA) dans le controle de la sécrétion de prolactine (PRL) chez la truite arc-en-ciel en utilisant à la fois des fragments dhypophyse perifusés et des cellules hypophysaires en culture. Dans notre système de périfusion, le GABA (10−6 à 10−4 M) inhibe la libération de PRL (entre 20 et 40%). Ladministration sur les fragments dhypophyse périfusés de 3APS, un agoniste des récepteurs GABAa, reproduit ces effets inhibiteurs. De plus, la bicuculline, un antagoniste spécifique des récepteurs de type GABAa, abolie complètement les effets du GABA. Lorsquil est testé pendant 40h sur des cellules en culture, le GABA (10−5 M) réduit de manière significative la libération de PRL (24.5%). Le Baclofen, un agoniste spécifique des récepteurs GABAb testé à 10−6 et 10−5 M, inhibe aussi la libération de PRL par les cellules en culture. Ces résultats démontrent que le GABA inhibe la libération de PRL en agissant directement sur les cellules hypophysaires et que les 2 types de récepteurs GABA (a et b) sont impliqués dans cette régulation.