NATURE OF DIVIDING NUCLEI IN SKELETAL MUSCLE OF GROWING RATS

Much is known about the early stages of muscle development-the proliferation of myoblasts, their fusion into myotubes, and the transformation of myotubes into young muscle fibers . However, the mode of growth of muscle in the young animal remains obscure . It is known that muscle growth is extensive (e .g . in the chicken the pectoralis muscle enlarges from 0.7 g at hatching to 300 g in the adult) and is mainly the result of hypertrophy of the fibers (1), with little, if any, increase in their number (2) . Moreover, it is generally assumed that, unlike what happens in most other tissues, there is no division of the nuclei and, consequently, no increase in their number (3) . This view is supported by experiments showing that, while myoblasts can divide at early stages of development, they lose this ability once they have been incorporated into myotubes (4) . Furthermore, during muscle regeneration following injury, myoblasts appear which take up thymidine-'H, indicating their ability to divide, whereas the nuclei of muscle fibers do not (5) . Hence the prevalent opinion has been that new nuclei are not produced in skeletal muscle fibers during growth . This view was questioned when DNA determinations revealed a considerable increase in the number of fiber nuclei during growth in rats (6) and chickens (1), and mitotic figures were observed within the muscle fibers of growing rats (7) . Furthermore, after an injection of thymidine3H, radioautography revealed labeled nuclei within the confines of the basement membrane of muscle fibers (7, 8). In the hope of reconciling these apparently conflicting observations, it was suggested that the muscle nuclei which take up thymidine 3H and undergo mitosis are not true muscle nuclei but belong to "muscle satellite cells" (7). Such cells, which had been observed only in the electron microscope (9), consist of a single nucleus surrounded by scanty cytoplasm and are located between the basement membrane and the plasmalemma of the fibers, whereas true muscle nuclei are within the plasmalemma . Since satellite cell nuclei are eaily distinguished from true muscle nuclei in the electron microscope, it was decided to examine radioautographs in this instrument following thymidine 3H injection . Six male rats aged 17 days and weighing 25-30 g were given a single injection of 25-60 )uCi of thymidine 3H; g body weight and sacrificed at intervals varying from I to 72 hr later . The tibialis anterior muscle was fixed by immersion in glutaraldehyde paraformaldehyde (10), postfixed in osmium tetroxide, and embedded in Epon . Thin (silver to gold) sections were prepared for radioautography in the electron microscope with Ilford L4 emulsion (11-13) and poststained with lead citrate (14) . Electron microscopy revealed the existence of two types of nuclei within the basement membrane of the fiber . The nuclei of the first type (85-90% of the total) are in direct contact with the myofibril-containing cytoplasm ; they are the true muscle nuclei. The nuclei of the second type are surrounded by a small amount of cytoplasm, which is separated from the myofibril-containing cytoplasm by an intercellular space; these are the satellite cell nuclei ; they are usually smaller and darker than true muscle nuclei . In radioautographs obtained I hr after injection of thymidine 3H, approximately 3% of the nuclei within the basement membrane were labeled . These invariably belonged to satellite cells (Fig. 1) . Not one of the true muscle nuclei was labeled at that time. The results were similar at 6 and 10 hr after injection (Table I) . It was, therefore, concluded that satellite cell nuclei, but not true muscle nuclei, were able to synthesize DNA . Moreover, since it is known that DNA synthesis precedes mitosis (8), it may be concluded that the mitotic figures previously observed in the muscle fibers of growing rats (7) must belong to satellite cells . This conclusion is in accord with a recent report that, after colchicine treatment of 30-g rats, arrested mitoses of the nuclei of satellite cells, but not of true muscle nuclei, may be observed (15) . By 24 hr, a few true muscle nuclei were also labeled (Table I). At 48 hr, the labeling frequency of true muscle nuclei was increased, and by 72