Long-term culture of muscle explants from Sparus aurata.
Although there are mammalian myoblast cell lines, no fish myoblast cell line has been developed so far. The aim of this study was to develop a culture system of muscle explants for fish, as explants provide an approximation of the in vivo conditions for cell proliferation and differentiation, and enable a close comparison with events in muscle regenerating in vivo. Here we describe the main features of a long-term in vitro culture system for muscle explants from Sparus aurata fry. At the time of sampling, the original fibres were damaged and subsequently degenerated as shown by the loss of parvalbumin (PV) and presence of apoptotic nuclei. This mechanical damage provoked a myogenic response by activation of myogenic precursor cells. After a few days, new mononucleate cells aligned with the original fibres were seen in the explants, some with proliferating cell nuclear antigen (PCNA-) and Myf-5-positive nuclei, indicating proliferation and their myogenic fate. By 1 week, multinucleate cells with desmin immunoreactivity but PCNA- and Myf5-negative nuclei were present, equivalent to differentiated, postmitotic myotubes. Some of these myotubes were also immunoreactive for PV and insulin-like growth factors (IGFs). By 11 days, many of the myotubes were also immunoreactive for myostatin (MSTN). By 23 days, many of the myotubes had increased in diameter, were packed with myofibrils, and were strongly PV-positive and immunoreactive for MSTN, IGF-I and IGF-I receptor. This study shows that a proliferative process occurs in the explants despite the death of the original muscle fibres, and new muscle fibres expressing growth regulators are formed by regeneration from myogenic precursors present in the explants at the time of sampling.
Funkenstein
B
B
Department of Marine Biology & Biotechnology, Israel Oceanographic & Limnological Research, PO Box 8030, Tel-Shikmona, Haifa 31080, Israel. bruria@ocean.org.il
Balas
V
V
Skopal
T
T
Radaelli
G
G
Rowlerson
A
A
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D007150:Immunohistochemistry; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D009210:Myofibrils / Q000378:metabolism*; D051787:Myogenic Regulatory Factor 5 / Q000378:metabolism; D055435:Myostatin; D010320:Parvalbumins / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021541:Sea Bream / Q000378:metabolism*; D046509:Tissue Culture Techniques; D016212:Transforming Growth Factor beta / Q000378:metabolism
D015415:Biomarkers; D051787:Myogenic Regulatory Factor 5; D055435:Myostatin; D010320:Parvalbumins; D018809:Proliferating Cell Nuclear Antigen; D016212:Transforming Growth Factor beta; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1