Isolation and Identification of Myotube-forming Stimulating Factor on Skeletal Muscle of Domestic Animals

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A large number of in vitro studies using primary myogenic cells isolated from the skeletal muscle of mouse, rat, chicken and swine have showed that the cell growth and terminal differentiation are controlled by several growrth factors. However, the myogenesis of bovine myoblasts, the transformation from cell proliferation to terminal differentiation, is not always understood. Present study was conducted to isolate and identify a myotube-forming stimulating factor (MTF) in regulating the myogenesis of bovine myoblasts in vitro and to characterize the possible role. Experiments were conducted as fouows ;(1) Establishment of myogenesis by bovine myoblasts in cultures. Bovine myoblast culture was established by the medium which mixed medium 199, Dulbecco's modified Eagle's medium and FCS in 8 : 2 : 1, and supplemented 1.5% swine fetal extract (SFE). Bovine myoblasts were enriched by the combination procedure of differential adhesion method and a selection of myoblasts by cytochalasin B tre … More atment. SFE,Insulin (10^<-6>M) and IGF-1 (10^<-7> -10^<-8>M) caused a significant increase of myotubes by the myoblast fusion.(2) Isolation and identification of MTF.MTF activity was recognized in SFE of fetal muscle, lung and kidney. By gel filtration, the activity was detected in 3-10kD and 10-30kD fraction. MTF was eluted with 0.9-1.2M NaCI solution by heparin-affinity chromatography and was identified as a single protein band which was different from IGF-1.(3) Effects of MTF on AchE and AchR expression of bovine myoblasts. AchE and AchR were expressed on the committed bovine myoblasts which differentiated in G_0 phase. MTF and IGF-1 induced the AchE and AchR expression of bovine myoblasts.(4) Establishment of cloned bovine myoblasts. Cloned bovine myoblasts, Bmb-1 and Bmb-2, were established in the colony cultures of bovine myoblast. They were stained by anti-desmin antibody and formed myotubes. The myotube formation of Bmb-2 cloned cells was stimulated by MTF and IGF-1.(5) Formation of three-dimensional tissues from primary cultured bovine myoblasts. Cell aggregates which bunched into loosely bound masses in the myoblast cultures on the agar plates were transferred to Culstir flask and cultured at rotation speeds around 70 rpm. The mass increased progressively in compactness and formed various size (250-500 mu m in diameter) of multicellular spheroids. The spheroid formation was accelerated by MTF and IGF-1. The results demonstrated that three-dimensional structures by bovine myoblasts call be reconstituted in culture system. Less