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Satellite cell distribution within the soleus muscle of the adult mouse.

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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
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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.
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469
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99435-444.
Ontell, M. (1974) Muscle satellite cells: A validated technique for light microscopic identification and a quantitative study of changes in their population following
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Schmalbruch, H., and U. Hellhammer (1976)The number of
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Rw., 180~589-596.
Schultz, E. (1976)Fine structure of satellite cells in growing
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Snow, M.H. (1977a)The effects of ageing on satellite cells in
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Snow, M.H.. (1977b) Myogenic cell formation in regenerating rat skeletal muscle injured by mincing. I. A fine
structural study. Anat. Rec., 188:181-200.
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