The source of the new nuclei appearing during the growth of muscle fibers was examined in the tibialis anterior muscle of young Sherman rats (14-17 days of age) using radioautography at various intervals after a single injection of a small, non-toxic dose of 3H-thymidine ( 2 pCi/g body weight). Two techniques were employed: (1) labeled nuclei were detected in 1 $L thick radioautographs examined in the light microscope, and identified by simultaneous electron microscope examination of an adjacent section. The nuclei were then classified either as "true" muscle nuclei (within the plasmalemma of the fibers) or as belonging to "satellite cells" (which are mononucleated cells with scanty cytoplasm wedged between plasmalemma and basement membrane). (2) Muscle fibers freed by collagenase digestion were radioautographed one hour after 3H-thymidine injection in order to determine the total number of labeled nuclei (true muscle nuclei plus those of satellite cells) per unit length of fiber.Certain nuclei within the basement membrane of muscle fibers are labeled one hour after 3H-thymidine and, therefore, synthesize DNA. The electron microscope demonstrates that these nuclei invariably belong to satellite cells, never to true muscle nuclei. Furthermore, the total number of labeled nuclei per unit length of fiber doubles between 1 and 24 hours; and, therefore, the labeled satellite cell nuclei undergo mitosis.Following mitosis, half of the daughters of satellite cells are incorporated into the fibers to become true muscle nuclei. The remaining half divides again later; and half of their daughter cells are incorporated. Thus, satellite cells in young rats divide repeatedly and function as a source of true muscle nuclei.The early stages of muscle development are now well understood. At the start there is proli€eration of mononucleated cells, the myoblasts; groups of these fuse to form a multinucleated myotube, which then acquires myofibrils to become a muscle fiber. It is also known that the number of fibers in a muscle is fixed at birth or soon after (MacCallum, 1898;Morpurgo, 1898; Enesco and Puddy, '64), and that the extensive muscle growth which occurs during development in young animals is due to hypertrophy of the fibers (Enesco and Puddy, '64; MOSS, '68).However, it is not clear how this hypertrophy takes place. In vitro studies have shown that, once myoblasts have been incorporated into myotubes to become muscle fibers, the nuclei lose the ability to divide (Bintliff and Walker, '60; Stockdale and Holtzer, '61). Consequently, it is generally assumed that the nuclei of muscle fibers, unlike those of other tissues, do not divide and that their number does not increase during growth. On the other hand, in vivo studies of muscles of growing animals do not support this belief: DNA determinations have demonstrated that there is a considerable increase in the number of nuclei during muscle growth in rats (Enesco and Puddy, '64) and chickens (Moss, '68). Furthermore, radioautography with 3H-thymidine has shown that...

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Between 0 and 266 days of age the weight of the pectoral and gastrocnemius muscles of chickens increased 300-600-and 40-90-fold respectively depending on the breed and sex. In both muscles the mean cross-sectional area of the fibres and the total number of nuclei (estimated from DNA determination) maintained a constant ratio during growth. This suggests that for individual fibres the cross-sectional area increased in proportion to the number of nuclei. This phenomenon is discussed in relation to current knowledge concerning the mode of growth and multinucleation of skeletal muscle fibres.In the pectoral muscle, between 0 and 266 days, the cross-sectional area of the fibres increased in proportion to the two-thirds power of the muscle weight, which suggests that the length and diameter of the fibres maintained a constant ratio. The same relationship existed for the gastrocnemius for two months, after which the fibre cross-sectional area increased in proportion to the muscle weight, which suggests that the fibre length was then constant.

SUMMARYEighty-two Merino sheep from birth to 517 days old were dissected into individual muscles. A system of overlapping growth coefficients was used to classify the growth patterns of 96 muscles and nine muscle groups. The relative size of muscles and groups at birth and one year are expressed as percentages of total half-carcass muscle weight.Some differences were noted from the previously recorded growth patterns of bovine muscles. The groups of muscles in the proximal part of the pelvic limb and those surrounding the spinal column were relatively faster-growing in sheep than in cattle.The patterns of the muscle groups are discussed relative to their function and it is shown that the patterns of growth of muscles within some groups are markedly different from the pattern of the whole group. This is most apparent in those groups with complex functions, and it is clear that anatomical grouping of muscles and functional grouping of muscles will be similar only in those regions of the body with relatively simple actions.

During unrestricted growth of the pectoral muscle of chickens, the mean cross-sectional area of the fibres increased in proporiton to the total number of nuclei and in proportion to the two thirds power of the weight of the muscle. Continuous restricted feeding from 0 day, which limited the weight of the muscle at 16 days to 44% or 69% of that of chickens fed ad libitum, did not affect these relationships.A sudden restriction of feed from eight days of age retarded growth of the muscle to approximately 70% of the weight of the controls at 16 days. It did not affect the relationship between the number of nuclei and the fibre cross-sectional area, both of which were also limited to about 70% of those of the controls; but it did disrupt the relationship of these two variates to the weight of the muscle. Subsequent ad libitum feeding caused compensatory growth and restored the relationship.Starvation or severe undernutrition for a few days, which reduced weight of the muscle and the fibre cross-sectional area by approximately 2 5 % , caused no loss of nuclei, thereby disrupting both relationships. Subsequent ad l i b i t u m feeding initially caused an increase of muscle weight and fibre cross-sectional area but no increase in the number of nuclei. This restored the relationships, which were then maintained during subsequent compensatory growth.

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The relationship between the weight of the breast muscle and the number of nuclei

A technique is described for the radioautography of whole mounts of muscle fibers, which are approximately 15µ in diameter and too small to be handled individually. The fixed muscle is digested with collagenase to separate the fibers; these are then stained with hematoxylin, thoroughly washed and suspended in radioautographic emulsion on microscope slides. Thus the nuclei, which are all at the surface of the fibers, are close to the surrounding emulsion. This ensures that all of the nuclei containing the radioisotope will be labeled in the radioautograph, irrespective of their position, above, below or at the side of the fibers.