Dependence of in vitro myogenesis on a trophic protein present in chicken embryo extract.

Abstract

A trophic protein (sciatin) purified from sciatic nerves has been shown to enhance the morphological develop ment and to promote the maintenance of sletal muscle cells in vitro Proc. NatL Acad.Sci. USA 76,2470-24741. We have elicited a specific antiserum against purified sciatin in rabbits. By using this antiserum, we have examined whether sciatin is also required for the initial differentiation of avian myogenic cells in vitro. Sciatin was found to be a component of chicken embryo extract, a constituent of culture medium required for myogenesis in vitro, by an immunodiffusion assay and by NaDodSO4 gel electrophoresis of immunoprecipitates. The removal of sciatin from chicken embryo extracts by immunoprecipitation with antiserum against sciatin completely inhibited myogenesis. When myogenic cells were grown in culture medium from which sciatin had been removed, the cells failed to differentiate beyond the myoblast stage. However, when sciatin (25 pg/ml) was added to the sciatin-absorbed culture medium, normal myogenesis ensued. Furthermore, myogenic cells underwent normal myogenesis in the absence of embryo extract if sciatin (25 ,g/ml) was added to the culture medium. These results demonstrate that sciatin is the component of chicken embryo extract required for myogenesis and that the protein influences the initial differentiation of myogenic cells in vitro. Innervation exerts a "trophic" influence on the morphologic, physiologic, and metabolic properties of skeletal muscle (for reviews, see refs. 1 and 2). To explain the mechanism by which the motor nerve conveys trophic influence to muscle, it has been postulated that such trophic influence is mediated in part by a trophic substance(s) released by the nerve (1-3).The best evidence for the trophic influence of a neurally derived substance has been obtained from in vitro experiments (4-11). By using embryonic muscle culture, Oh (12, 13) demonstrated that extracts of central and peripheral nervous tissue enhanced the morphological development of aneural muscle cells, increased protein synthesis and acetylcholinesterase activity, and maintained matured, cross-striated muscle fibers for several months in the absence of innervation. In the absence of the extracts or innervation, cultured muscle fibers atrophied and degenerated rapidly. The active agent in peripheral nervous tissue was characterized as a protein of rather high molecular weight (13) and was partially purified (14-16).We have purified a biologically active protein from adult chicken sciatic nerves that duplicates many of the trophic effects of innervation on cultured muscle (17). In preliminary publications*t we have referred to this trophic protein as "neurotrophic factor" or NTF. We have now formally adopted the name sciatin in order to avoid confusion with the neuronotrophic factors described by workers in other laboratories.The term sciatin is intended to designate the trophic protein that was isolated and purified from the sciatic nerve. Sciatin is acidic, migrates with a molecular weig...

Transferrin receptor numbers and transferrin and iron uptake in cultured chick muscle cells at different stages of development

“…Chicken transferrin has been found to be necessary for the normal growth and differentiation of chick embryo muscle cells in culture. It was first shown that chick embryo extract was an absolute requirement for myogenesis in vitro (Oh and Markelonis, 1980;Hagiwara, Kimura, and Ozawa, 1981;Ii, Kimura, and Ozawa, 1982) and that its trophic effects could be replaced by a small amount of embryonic or adult chicken serum Ozawa and Kohama, 1978), extracts of adult chicken muscle Matsuda, Spector, and Strohman, 19841, extracts of adult chicken neural tissues (Markelonis et al, 1982;Popiela, Ellis, and Festoff, 19821, and also microgram quantities of purified chicken transferrin (Kimura, Hasegawa, and Ozawa, 1982;Saito et al, 1982). This muscle trophic factor was now been isolated and identified as Tf-Fez .…”

Transferrin as a fetal growth factor: acquisition of responsiveness related to embryonic induction.

“…Transferrin is thus a molecularly defined effector in this model system. It was recently shown that the "muscle trophic factor" required for myogenesis (38) is identical to transferrin (39,40). Evidence from these two developmental systems therefore suggests that transferrin is an important serum factor for organogenesis.…”

The adult fast isozyme of myosin is present in a nerve-muscle tissue culture system

“…An alternative explanation is that some neurotrophic effect of the spinal cord is important in the organotypic cultures. Adult myosins were not seen in cultures grown in medium containing chicken embryo extract [I, 2, 281 which contains some nerve-derived components [16]. Such medium additives may not substitute for the actual presence of the cord, however, because the necessary factors are labile, become too dilute, or may need to be applied topically by the axon terminals.…”