DEGENERATHH€CHiLARGEIMYOTUBESPTHLOWHNG REMOVAL OE TRANSFERRIN FROM CULTURE MEDIUM

Chick myoblasts multiplied and formed large myotubes showing cross-striations and spontaneous twitching when incubated with a medium composed of Eagle’s minimum essential medium, horse serum, and a small amount of chick transferrin (Tf). Large myotubes degenerated after Tf was removed from the medium. Fat droplets appeared in the cells within a. few days and various changes took place. Most degenerated cells became detached from the culture dish within 1 or 2 weeks. Myoblasts were relatively resistant to Tf deficiency. Large myotubes were formed again when the myoblasts were again incubated with Tf. These newly formed large myotubes quickly degenerated following the removal of Tf. Chick myogenic cells do not grow in a medium composed of Eagle’s minimum essential medium (MEM) and horse serum, but grow well when a small amount of chick embryo extract is added to the medium. Following this addition, myoblasts multiply and form large myotubes with cross-striations and spontaneous twitching (5, 13). Embryo extract can be replaced either by chick serum or by muscle trophic factor (MTF), which is a component of chick serum and embryo extract (5, 14). Very recently, MTF was identified in our laboratory as transferrin (Tf), an iron—binding protein in serum (8). We also found that Tf had class specificity in its muscle trophic effects (12), i.e., chick and rat myogenio cells in primary culture do not grow in the absence of avian and mammalian Tfs, respectively, but do grow well in their presence. Therefore, horse Tf contained in the culture medium does not affect chick myogenic cell growth. Since Tf is also an indispensable component of serumfree culture medium for the culture of various kinds of mammalian cell lines (1, 2), the name ‘muscle trophic factor’ should be withdrawn and the substance should be referred to as transferrin, or Tf. We have shown that Tf promotes myoblast multiplication in proportion to its concentration, resulting in an increase in the number of nuclei contained in myogenic cells (14). We have found that large myotubes degenerated when cultured without chick Tf. Myoblasts and small slender myotubes, however, persisted for a fairly long period and grew into large myotubes following the readdition of chick Tf. In this paper, we report on this process as observed under a light. microscope. Parts of this work have been published in a preliminary report (10). MATERIALS AND METHODS Methods for chick Tf isolation, myoblast preparation from the 11th day chick embryo breast muscles, cell harvesting and creatine kinase (CK) assay have been described elsewhere (5, 11, 14). The schedule of the experiments was as follows: 8><104 dissociated chick embryo breast muscle cells, purified by the differential adhesion technique, were inoculated onto 35 mm Falcon plastic Petri dishes precoated with gelatin. One hundred and four dishes were prepared. The cells were incubated with a medium composed of 85 ‘X, MEM and 15 % horse serum containing MYOTUBE DEGENERATION IN VITRO Table 1 Coiicerztmrion of Tf(,ug/ml) in the llledium Days Days Days I Days 4-8 8-10 10-15 15-39 Group I --Group Ia — — Group II O O Group IIa 3 7.5 Group IIb 0 25 @@@L»JL»J C>u->CJC>u. > 10 /. tg/ml chick Tf; they were photographed and harvested for CK assay on selected days. (Each point shown in Fig. 1 denotes the mean of CK activity of 4 dishes.) On day 4, the dishes were divided into two groups (Table 1). The cells of group I and II were incubated further with and without 3 ,ug/ml Tf, respectively. On day 8, group Ia was formed from group I by the removal of Tf and group Ha was formed from group II by the addition of 3 pg/ml Tf. On day 15, group IIb was formed from group II by the addition of 25 /. tg/ml Tf. On the same day, the Tf concentration in group Ila was raised to 7.5 ,ug/ml. The medium of all the groups was changed every 3 or 4 days. RESULTS On day 4, CK activity in the myogenic cells was detectable (Fig. 1). This activity increased in group I until day 8, and then decreased slightly, whereas the activity in group Ia decreased substantially with time. The CK activity in group II increased slightly from day 4 to day 6, but then decreased to an almost. undetectable level by day 15. This temporal increase in CK activity was