THE ANATOMICAL RECORD 201~463-469(1981) Satellite Cell Distribution Within the Soleus Muscle of the Adu It Mouse MIKEL H. SNOW Department of Anatomy, University of Southern California School of Medicine, L o s Angeles, California XI033 ABSTRACT The frequency of satellite cells was quantitated by electron microscopy in five proximal to distal regions of the soleus muscle of adult mice. In all, 236 satellite cell nuclei and 4,475 myonuclei were counted on 51 transverse thin sections. The mean percentage of satellite cells, as a ratio of satellite cells to myonuclei, per region was found to be 5.4%, 5.3%, 5.0%, 5.2% and 4.9% for the most proximal to distal areas, respectively. Analysis of variance revealed no significant differences between either the regions or the animals studied. The number of satellite cell nuclei per cross-sectional area of muscle was also calculated for each of the five regions, and these values did not vary significantly from the proximal to distal ends of the muscle. Despite the fact that satellite cells were frequently noted in close association with cross-sectional profiles of myoneural junctions, this study establishes that the number of such perisynaptic satellite cells was not large enough to affect significantly the mean percentages of all satellite cells counted within the motor endplate regions (areas 3 and 4) of the soleus muscle. I t is concluded from this study that satellite cells are uniformly distributed throughout the whole muscle. Since Mauro's ('61) original description of satellite cells in uninjured skeletal muscle of the frog, numerous investigators have quantified such cells within normal muscles of a wide variety of vertebrates (see reviews by Muir, '70; Schmalbruch and Hellhammer, '76). Most of these quantitative studies were carried out utilizing the electron microscope to identify and count satellite cell nuclei and myonuclei within randomly selected thin sections. Such fine structural studies were necessarily limited to small sample sizes, and, therefore, the validity of the quantitative data was dependent on a uniform distribution of satellite cells throughout individual muscles. However, documentation that satellite cells are in fact evenly dispersed within a normal muscle has not been adequately provided. Muir et al. ('65)reported that satellite cells are uniformly distributed throughout muscles of bats and mice, although they provided no specific quantitative data to support this statement. Kelly ('78) reported that in the rat soleus muscle, satellite cells are more frequently encountered on muscle fibers displaying myoneural junctions than on fibers whose motor endplates are not in the plane of the section. Since myoneural junctions are concentrated in the midregion of the soleus muscle, the frequency of satellite cells may vary between sections cut near the center of the muscle and those near myotendinous junctions. Castillo de Maruenda and Franzini-Armstrong ('78) also questioned the assumed even distribution of satellite cells when they reported a higher incidence of such cells in the distal region of the frog sartorius muscle as compared to the central and proximal regions. Fine structural quantitative techniques have also been employed to determine the frequency of satellite cells during skeletal muscle growth (Allbrook et al., '71; Schultz, '74). ageing (Schmalbruch and Hellhammer, '76; Snow, '77a), regeneration (Snow, '77b), denervation (Aloisiet al., '73; Ontell, '74) and hypertrophy (Aloisi et al., '73). Since these studies were carried out on a few, randomly selected thin sections, the results and those of future quantitative studies of satellite cells in experimental muscle may be inaccurate if satellite cells are not uniformly distributed throughout normal muscles. Thus, the present fine structural study was undertaken to determine whether the frequency of satellite cells varies significantly along the proximo-distal axis of the normal soleus muscle of the mouse. 0003-276X/81/2013-0463$02.50 0 1981 ALAN R. LISS. INC. Received July 15. 1980; accepted May 7, 1981 464 MIKEL H. SNOW MATERIALS AND METHODS The soleus muscles were removed from two, four-month old (28 gm),male, C57 black Jackson mice under sodium pentobarbital anesthesia. The four muscles were pinned at resting length on wax plates and immersed in 2.0% glutaraldehyde plus 2.5% paraformaldehyde (modifiedfrom Karnovsky, '65) buffered to pH 7.4 with 0.1 M sodium cacodylate. After approximately 20 minutes, the proximal and distal tendons were trimmed away and the remaining portion of muscle, which was about 10 nun long, was cut transversely into five segments (regions) each about 2 mm in length (Fig. 1).Each of the five segments from the four muscles sampled was then divided lengthwise into two or three pieces (approximately 1 X 2 mm), which were placed in coded vials to maintain their positional identity. All samples were fixed an additional 2-3 hours in fresh prefixative, postfixed for one hour in buffered 1% osmium tetroxide, dehydrated in ethanol, and embedded in Epon (Luft, '61). Transverse thin sections were cut from each of the five regions sampled from each of the four muscles until a total of ten, or in one case eleven, sections had been cut and pooled for each region. Whenever possible, one thin section was cut per block, but occasionally two sections were taken from one block whenever there were fewer than ten blocks pooled in a region; in such cases the second section was cut approximately 1mm distal to the first section. All sections were mounted on carbon-coated 100 mesh grids, stained with uranyl acetate and lead citrate, and coded so that the region of origin was unknown until after the sections had been examined with a JEOL lOOC electron microscope. All satellite cell nuclei and myonuclei not covered by grid bars were counted in only one of the ribbon sections per grid; the grid squares served as a counting reticle. Identification of satellite cells was verified by means of high magnification electron micrographs. The frequency of satellite cells was expressed as a percentage of the total nuclei (satellite cell nuclei plus myonuclei) observed beneath the external lamina. Low magnification electron micrographs were taken for each section, and the total cross-sectional area of muscle was determined by using a Summagraphics ID digitizer. All nonmuscle tissue was excluded from this analysis. The number of satellite cell nuclei and myonuclei per crosssectional area was calculated for each section. The quantitative d a t a were analyzed statistically using the Kruskal-Wallis nonparametric analysis of variance (Sokal and Rohlf, '69). A probability of 0.05 was used to determine significant differences between groups. RESULTS Quantification of satellite cell nuclei and myonuclei in the soleus muscles of adult mice is presented in Table 1. A total of 236 satellite cell nuclei and 4,475 myonuclei was counted on 51 sections. Nucleated satellite cells were observed in every section, and they appeared to be randomly distributed within the transverse plane of the sections. The percentage of satellite cells per section (calculated by dividing the number of satellite cell nuclei counted per section by the number of satellite cell nuclei plus myonuclei per section) varied from 1.2% to 13.3%. The cross-sectional area of muscle calculated for each section ranged from 0.021 mm2 to 0.219 mm2. The majority of sections cut near the ends of the muscle were smaller in area than those cut more centrally due to the marked tapering of the soleus muscle, particularly near the proximal tendon (Fig. 1).The observed numbers of myonuclei and satellite cell nuclei per section were also less for the smaller sections. When the number of satellite cell nuclei was calculated per cross-sectional area for each section and pooled according to the five regions, the mean values were found to be 63, 55, 54, 53 and 56 from the proximal to distal areas, respectively (Table 1).Analysis of variance of these data revealed no significant difference between the five regions studied (P > 0.05). When satellite cell percentages for each section were pooled according to the five regions mapped out, the mean percentages were found to be 5.4%, 5.3%, 5.0%, 5.2% and 4.9% from the proximal to distal areas, respectively (Fig. 1).Analysis of variance of these data indicated no significant differences between the mean percentages of satellite cells for the five regions studied (P > 0.05). The overall mean percentage of satellite cells for the entire muscle was calculated from the pooled values of both animals and found to be 5.2%. No significant difference was found between the mean satellite cell percentages of 5.4% and 4.9% calculated for both animals (P > 0.05). Profiles of motor endplates were noted in association with 30 of the 4,981 myofibers observed in this study. The thirty myofibers displaying motor endplates were noted in 12 of SATELLITE CELL DISTRIBUTION IN SOLEUS 465 8- - 1 7- 6- 5- 4- 3- 2- 1- 0 2mm 4mm 6mm Fig. 1. Comparison of the mean percentages of satellite cells, as a ratio of satellite cells per myonuclei, in five regions sampled from the proximal to distal tendons of the soleus muscle of adult mice. Each stippled rectangle represents the pooled percentages from ten or eleven thin sections, and the vertical bars represent two standard errors of the mean. Motor endplates were observed only in areas 3 and 4. the 51 sections examined and all were observed cantly different from the 5.0% calculated for in regions three and four, with the majority the remaining 39 sections without motor being in area four (Table 1).Nineteen of these endplates. myofibers were in close association with either The cytological features of the satellite cells nucleated or non-nucleated profiles of satellite were found to be similar to those reported in cells (i.e., perisynaptic satellite cells). Thus, previous studies of adult mammalian muscle 63% of muscle fibers having myoneural junc- (Schultz, ’76; Snow, ’77a; Kelly, ’78) and need tions in the plane of the section also had satel- not be repeated here. lite cells within the limits of their external lamina. In contrast, only 4.4% of myofibers DISCUSSION without motor endplates in the section were associated with satellite cells. Since only nucleThis study provides quantitative data docuated satellite cells were included in the quanti- menting that satellite cells are uniformly distative data presented here, it is important to tributed throughout the soleus muscle of adult note that of the 236 nucleated satellite cells mice. The mean percentages of satellite cells counted in all sections, only 18 (7.6%) were (as a ratio of satellite cell nuclei to myonuclei), “perisynaptic” satellite cells. When the 1 2 as well as the number of satellite cell nuclei per motor endplate sections were pooled their cross-sectional area of muscle, did not vary mean satellite cell percentage was calculated significantly from the proximal to distal ends to be 5.7% which was not found to be signifi- of the muscle. (1) 3 2 (proximal) 1 Region Percent SCN per section 4.8 7.4 10.3 7.0 4.5 2 2 3 4 3 40 25 26 53 63 A A B B 2 6 9 8 5 99 129 83 114 120 76 115 139 185 75 1 8 7 2 7 6 58 116 55 46 82 2.6 5.0 6.1' 4.1 6.2 7.24 8.40 13.31 21.89 6.58 13.76 12.92 8.12 9.07 14.40 7.5' 5.1' 2.4 5.8l 4.8 1050 1368 1044 845 1140 719 999 1022 1257 833 1231 487 980 968 9.42 11.29 4.70 8.47 28 71 68 37 76 58 54 25 71 42 21 53 106 12 - 90 30 97 62 28 1107 1352 1068 1228 856 61 93 97 88 51 (8) SCNimmz the mouse. 31 64 31 26 88 of 1301 1051 907 1046 1252 1212 1160 841 1164 1077 - 6.68 6.73 8.24 9.69 10.87 6.38 3.14 6.51 3.82 4.55 3.30 2.15 3.10 4.55 5.85 (7) MNimml - A (6) Area of muscle per section (mmz X 10-2) 4.9 1.7 9.8 9.8 1.2 7.5 2.2 8.3 4.8 3.1 6 2 8 6 3 74 91 88 119 93 B 3 2 6 5 2.4 5.7 3.3 2.4 6.6 2 2 2 1 4 83 33 59 40 57 (4) SCNisection (3) MNlsection 12) Animal (5) TABLE 1. Distribution of satellite cell nuclei (SCN) and myonuclei (MN) in the normal soleus muscle 54&6.2 55&11.8 63*8.7 (9) Mean SCN per m m 2 per region + standard error B A 'Indicates sections displaying motor endplates. Totals (distal) 5 4 103 51 17 102 99 4475 168 107 98 55 93 71 107 94 127 85 98 94 98 93 108 111 236 10 5 3 2 5 5.0 5.6 4.5 3.0 3.5 5.1 18.04 5.19 15.53 7.86 10.16 10.41 5.86 9.04 10.59 5.08 8.0 5.6 6.1 2.8 4.8 9 3 5 3 5 3.0' 13.3' 9.92 11.13 9.98 13.09 5.22 11.76 5.02 15.42 13.80 15.25 9.82 4.0' 3.6l 5.0' 3.0 6.0 10.11 4.1 2.7' 2.6' 3 4 5 4 13 4 3 3 11 3 6 TABLE 1 (cont.) 915 700 931 2063 631 989 870 851 963 1949 1413 693 681 833 865 988 844 982 710 2067 944 55 96 19 25 49 86 51 55 28 98 60 26 36 26 132 30 54 110 31 57 26 56h9.1 53 f 10.9 468 MIKEL H. SNOW profiles associated with 100%of the myofibers Muir et al. (’65)examined the frequency of having myoneural junctions in the plane of the satellite cells in web muscles of the bat, but section, whereas in the present study, only they did not provide specific quantitative data to support their conclusion that such cells are 63% of the observed synaptic myofibers had evenly distributed along an entire muscle. satellite cells; approximately 4.4% of Castillo de Maruenda and Franzini-Armstrong myofibers not displaying a synapse in the (‘78)quantified, at both the light and electron section had satellite cells. Based on these microscopic levels of resolution, satellite cells results, it might also seem logical to expect a higher incidence of satellite cells in samples in the frog sartorius muscle, and they reported taken from the central region of the soleus a significantly higher incidence of such cells in the distal as compared to the central and proxi- muscle since motor endplates for this muscle mal regions of that muscle. Their light micro- are distributed in a “U-shapedband across the midbelly region (Albuquerque and McIsaac, scopic data, however, are difficult to interpret ’70). Yet, in spite of the fact that myoneural since the percentages of satellite cells junctions were only found in central areas 3 presented in column seven of their first table were not accurately calculated. When this and 4 (Table l), the mean percentages of author calculated the correct values based on satellite cells, as well as the number of satellite the number of myonuclei and satellite cell cell nuclei per area of muscle, for all five nuclei presented in the same table, the mean regions were not found to be significantly percentages for the distal, central and prox- different. Even when all twelve motor endplate imal regions became 7.2%, 7.2% and 6.8%,re- sections were pooled and their range (2.6% to spectively. Thus, their light microscopic 13.3%)and mean percentage (5.7%)compared results are in agreement with the findings with the range (1.2% to 10.3%) and mean recorded here. Similarly, when this author percentage (5.0%) of nonmotor endplate tested by analysis of variance the electron sections, the results showed no significant microscopic data in Table 2 of Castillo de difference. This apparent paradox can be Maruenda and Franzini-Armstrong (’78),the explained by the fact that the total number of results showed no significant difference be- nucleated “perisynaptic” satellite cells was tween the mean percentages of satellite cells only 7.6% of the total number of nucleated for the three regions quantified. Because of the satellite cells counted in all sections. Since the high variance in satellite cell percentages (i.e., number of myofibers with motor endplates per individual values ranging from 0.0% to 29.0%) section, and hence, t h e number of reported for the 15 sections they analyzed, a “perisynaptic” satellite cells represent only a much larger sample size (i.e., a greater number small fraction of the total muscle fiber or of thin sections counted) would be required satellite cell population per section, their effect before any significant difference might be de- on the overall mean percentage of satellite cells monstrated. is of little consequence. These data indicate that quantitative The present study also noted a wide range in the percentage of satellite cells (1.2%-13.2%) studies designed to determine the frequency of calculated for each of the 51 thin sections exa- satellite cells in normal and experimental musmined. Such variance may be due to the small cle need not be concerned with where tissue samples are taken with respect to the proximosurface area of the thin sections relative to the large area of the entire muscle; thus, the tech- distal axis of the muscle. However, it is clear that care should be taken to provide an adeniques employed may not be adequate to quate number of samples to assure statistical detect subtle differences or patterns in the significance in view of the wide variation in three dimensional distribution of satellite cells satellite cell percentages often seen per within a whole muscle. section. It might also be speculated that the wide range in the percentage of satellite cells per section may be correlated with the presence or ACKNOWLEDGMENTS absence of myoneural junctions per section. I t The author expresses his appreciation to Dr. has recently been shown in the soleus muscle Gene H. Albrecht for his generous help with of the rat (Kelly, ’78) that satellite cells are the experimental design, statistical analysis, more frequently found in association with and preparation of the manuscript. Special myofibers displaying motor endplates than gratitude is extended to Ms. Marian Shiba for with myofibers whose myoneural junctions are her excellent technical assistance. The study not in the plane of the section. The present was supported by grant PCM 78-15745 from results support this finding, but it is important the National Science Foundation. to note that Kelly (’78)reported satellite cell SATELLITE CELL DISTRIBUTION IN SOLEUS LITERATURE CITED Albuquerque, E.X., and R.J. McIsaac (1970)Fast and slow mammalian muscles after denervation. Exp. Neural., 26:183-202. Allbrook, D.B., M.F. Han and A.E. Hellmuth (1971)Population of muscle satellite cells in relation to age and mitotic activity. Pathology, 3233-243. Aloisi, M., I. Mussini and S. Schiaffino (1973)Activation of muscle nuclei in denervation and hypertrophy. In: Basic Research In Myology. B.A. Kakulos, ed. Excerpta Medica, Amsterdam, pp. 338-342. Castillo de Maruenda, E., and C. F r a n z i n i - h s t r o n g (1978) Satellite and invasive cells in frog sartorius muscle. Tissue Cell 10749-772. Karnovsky. M.J. (1965)A formaldehydeglutaraldehydefixative of high osmolality for use in electron microscopy. J. Cell Biol., 22137. Kelly. A.M. (1978)Perisynaptic satellite cells in the developing and mature r a t soleus muscle. Anat. Rec.. 190:891-904. Luft. J.H. (1961) Improvements in epoxy resin embedding methods. J. Biophys. Cytol., 9409-414. Mauro, A. (1961)Satellite cells of skeletal muscle fibers. J. Biophys. Biochem. Cytol., 9493-495. Muir, A.R. (1970)The structure and distribution of satellite 469 cells. In: Regeneration of Striated Muscle, and Myogenesis, A. Mauro, S.A. Shafiq, and A.T. Milhorat. eds. Excerpta Medica, Amsterdam, pp. 91-100. Muir, A.R., A.H.M. 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Rohlf (1969) Biometry, The Principles and Practice of Statistics in Biological Research. W.H. Freeman Co., San Francisco. Chap. X, 253-298.