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The visualization of myosatellite cells in normal and denervated muscleA new light microscopic staining technique.

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THE ANATOMICAL RECORD 196~373-385(1980)
The Visualization of Myosatellite Cells
in Normal and Denervated Muscle: A New
Light Microscopic Staining Technique
ALAN A. LAROCQUE, ALBERT0 L. POLITOFF, AND ALAN PETERS
Departments ofdnatomy and Physiology, Boston Uniuersity School of
Medicine, Boston, Massachusetts 02118
ABSTRACT
A new light microscopic staining technique allows the visualization of satellite cells on the surface of myofibers. Either prior to or during fixation,
whole frog sartorius muscles are bathed in an acidic buffered solution containing
lead nitrate and subsequently exposed to ammonium sulfide. The staining of the
satellite cells resulting from this procedure reveals their positions, and the outlines
of their cell processes which occasionally branch. Electron microscopy shows that
the staining is due to lead deposits localized between apposing membranes of
satellite cells and associated myofibers. Prior exposure to N-ethyl-maleimide
(NEM) does not alter the formation of the lead deposits on the satellite cell, but
reduces the amount of Pb deposits on the muscle surface and connective tissue.
This technique has been applied to determine the effects of denervation on the
satellite cells of frog sartorius muscles. Four weeks after denervation, the number
of satellite cells is essentially the same in both denervated muscles and the intact
muscles of the contralateral side. However, denervation results in a subpopulation
of satellite cells with altered shapes. They have elongated cytoplasmic processes
which often branch. It is suggested that these supernumerary cytoplasmic processes represent a n intermediate phase in the transition of satellite cells to myoblasts.
Mauro ('61) first noticed the satellite cell in
a n electron microscopic study of frog and rat
striated muscle, and Ishikawa ('66) reported
the presence of the satellite cell in early postnatal and mature human muscle. Since its discovery, the satellite cell has been implicated in
numerous physiological and pathological
events, including muscle development and
growth (Allbrook et al., '66, '71; Church, '69;
Hellmuth and Allbrook, '73; Ishikawa, '66, '70;
Moss and Leblond, '70, '71; Muir et al., '65;
Schultz, '76; Shafiq et al., '681, response to injury and regeneration (Carlson, '73; Church et
al., '66; Conen and Bell, '70; Kasper et al., '69;
Mastaglia et al., '75; Price et al., '64; Fkznik,
'69a,b, '73, '76; Schmalbruch, '76, '77; Shafiq,
'70; Shafiq and Gorycki, '65; Shafiq et al., '67;
Snow, '77a,b, '78; Teravainen, '70; Walker, '63),
diseases of human skeletal muscle (Aloisi,'70;
Carlson, '73; Conen and Bell, '70; Laguens, '63;
Mastaglia and Kakulas, '70; Mastaglia et al.,
'70; Shafiq e t al., '67; Van Haelst, '70;
Wakayama, '76) and response to denervation
(Aloisi, '70; Aloisi et al., '73; DeRecondo et al.,
000-3276X/80/1964-0373$02.60
0 1980 ALAN R. LISS, INC.
'66; Hess and Rosner, '70; Lee, '65; Schultz, '74,
'76, '78). The response to denervation has been
described as changes in the cytoplasm of satellite cells in growing (Schultz, '78) and adult
(Chou and Nonaka, '77; Hess and Rosner, '70)
muscles and as a n increase in satellite cell population (Aloisi, '70; Aloisi et al., '73;Hess and
Rosner, '70; Ontell, '74).
All of the studies cited above had to rely on
electron microscopic techniques, so that without reconstructions made from serial thin sections, they could not provide a complete image
of the form of the whole cell, and especially of its
extensive cytoplasmic processes. Neither was
this possible through the use of a light microscopic staining technique described by Ontell
('74) that allows the identification and visualization of only the nuclei of these cells.
Clearly, a method for visualizing the complete myosatellite cell in light microscopic
preparations would greatly accelerate the
Received November 20, 1978; accepted August 29, 1979
373
374
ALAN A. LAROCQUE, ALBERT0 L. POLITOFF, AND ALAN PETERS
elucidation of a number of basic questions in
both muscle biology and pathology. A purpose
of this present article is to report such a
method. Another purpose of this paper is to
describe changes in satellite cell morphology
seen after muscle denervation.
MATERIALS AND METHODS
The experiments were performed on the frog
sartorius muscle ofRanapipiens (northern and
southern species, Carolina Biological and Connecticut Valley Supply Companies). Mature
animals were used to assure that all mononucleated cells underlying the external lamina
were myosatellite cells and not mast cells, fibroblasts, or other unclassified cells found to be
contained by the external lamina in neonatal
muscle (Ishikawa, ’66; Ontell, ’77). Animals
were used shortly after arrival in the laboratory; when frogs had to be kept in captivity for
long periods (as in the denervation experiments), they were frequently fed live tenebrio
larvae. The sartorius muscles were dissected
under a low power stereo microscope while
bathed in amphibian Ringer’s solution (NaC1,
118 mM; KCl, 2.8 mM; CaCl,, 2.8 mM; Tris
buffer, 5.0 mM, pH 7.4).Dissectedmuscles were
then pinned (minuten pins) to the bottom of
transparent Sylgard (Dow Corning, Midland,
Michigan) chambers, where they were incubated, fixed, stained, examined under the light
microscope, and photographed. Similar chambers were used to prepare the specimens for
electron microscopy. The chambers were made
by pouring 3 ml of uncured Sylgard mixture on
a 1007 Falcon plastic Petri dish.
Care was taken to avoid muscle damage during dissection. Damage of the sarcolemma results in the entry of large quantities of Pb,
which upon exposure to (NH,),S obliterates visualization of any cell surface structures.
A . Light microscopy
The following method gave the clearest and
most consistent results. The sartorius muscle
was dissected and then incubated for 30 min in
Ringer’s, buffered with 5 mM Tris, pH 6.15,
containing 1mM NEM. Incubation in Ringer’s
a t pH 6.15 reduces the formation of insoluble
lead hydroxide precipitate during Pb incubation, while the addition of NEM prevents the
formation of granular Pb precipitates associated with the muscle surface and connective
tissue. After incubation, the tissue was briefly
washed (5 sec) in a solution of 115 mM sodium
cacodylate (NaCaco) buffer, pH 6.15 (titrated
with nitric acid), to decrease the concentration
of free chloride ions. This wash was followed by
incubation in a 60 mM solution of lead nitrate
(Pb(NO,),) in 70 mM NaCaco, pH 6.15, for 30
min. (The Pb incubation medium was prepared
by the addition of NaCaco to a solution of
Pb(NO,), in bidistilled water. Attempts to raise
the pH of this solution above pH 6.15 resulted
in the formation of lead hydroxide, insoluble
white precipitate.) The Sylgard chamber holding the muscle was then inverted and immersed in the incubation medium to prevent
deposition of any insoluble precipitates formed
in the bulk solution during incubation. Furthermore, the incubation container was covered to reduce exposure t o atmospheric CO,
which might result in the formation of insoluble carbonate salts. Next, the tissue was briefly
washed (5 sec) in a 115 mM NaCaco buffer
solution, pH 6.15, and fixed in 2.5% glutaraldehyde and 115mMNaCaco, pH 7.4, for 2 hr a t
room temperature. Following fixation, the
muscle was exposed to a solution of 1.1%ammonium sulfide [(NH,),S] in 115 mM NaCaco
for 10-20 sec, depending upon the density of the
reaction product (lead sulfide) produced. The
muscle was then washed in NaCaco buffer, pH
7.4, and viewed under the light microscope.
B. Electron microscopy
Muscles were dissected and incubated as described above, except t h a t the incubation
period in the Pb(NO,), solution was varied between 15 to 30 minutes, and the concentration
of Pb(NO,), was reduced to between 15mM and
60 mM. The purpose of these changes was to
find the conditions that resulted in the best
morphological preservation and that produced
adequate quantities of Pb deposits between the
apposing membranes. The muscles were then
fixed in 2.5% glutaraldehyde in a buffer solution of 115 mM NaCaco at pH 7.4. After 2 hr of
fixation a t room temperature, specimens (2
mm3)containing nerve terminals were cut from
the sartorius muscle, postfixed in 29’0 OsO, in
115 mM NaCaco buffer, pH 7.4, for 2 hr, dehydrated in graded alcohol solutions, and embedded in a n Epon 812-Araldite mixture. For
orientation, thick sections (0.5 pm) were
mounted on glass slides and stained in 0.5%
toluidine blue-0, or according to the method of
Ontell (‘74),and after trimming the block face,
thin sections were cut with a diamond knife.
(Ontell’s method consists of staining with basic
fuchsin in methanol, followed by azure 11,
methylene blue, Na,CO,, and methyl alcohol.)
The thin sections were mounted on bare grids
and stained with uranyl acetate and lead citrate before examination in an AEI-6Belectron
microscope.
375
MYOSATELLITE CELLS
C.l. Concentrations of PbCNOJ, in the
incubation medium for light microscopy
To determine the Pb(NO,), concentration
needed for optimal staining during incubation,
the Pb(NO,), was varied between 30 mM and 90
mM. Dissection, incubation, fixation, and exposure to (NH,),S were performed as in method A.
C.2. Concentrations of PbWO,), in the
fixative medium for light microscopy
Concentrations between 30 mM and 90 mM
Pb(NO,), in 2.5% glutaraldehyde buffered with
70 mM NaCaco at pH 6.15 were tested for optimal staining. Muscles were dissected in normal
Ringer’s, pH 7.4, rinsed briefly in a solution of
115 mM NaCaco buffer, pH 6.15, and fixed for 2
h r without prior incubation.
disturb the vasculature of the hindlimb. The
skin incision was sutured with 5.0 silk. Four
weeks post denervation, the animals were sacrificed and the sartorius muscles were dissected
and stained accordingto method A. Microscopic
examination of each denervated muscle showed
degeneration of the distal segment of the sartorius nerve and absence of reinnervation. The
intact sartorius muscles of the contralateral
limbs served as controls.
RESULTS
C.5. Incubation and fixation with
Pb(NOJ, buffered by Bis Tris
To obtain a stable Pb buffered solution a t pH
7 . 2 , i t was necessary to dissolve 15 mM
Pb(NO,), and 100 mM Bis Tris in two separate
volumes of bidistilled water with the addition
of the Bis Tris solution to the Pb(NO,), solution
immediately before use. For incubation with Pb
and Bis Tris prior to fixation, method A was
followed with a Bis Tris buffered wash, pH 7.2.
Fixation with Pb and Bis Tris plus 2.5%
glutaraldehyde was performed as in method
C.2., with a Bis Tris buffered wash, pH 7.2.
Light microscopic observations
The spindle shaped entities (Figs. 1-4) revealed by Pb salts lie on the surface of the
muscle fibers. As shown below, by means of the
electron microscope, these spindle shaped entities are Pb-stained satellite cells. These cells
usually occur individually, so that few of them
are found in close proximity to each other (Fig.
4).
The majority of satellite cells lie parallel to
the lengths of the muscle fibers (Figs. 3, 4;
Table 1).They are mostly fusiform in shape
with a n elongated, central nuclear region and
one cytoplasmic process (Figs. 1,2,4)projecting
from each pole of the cell body. These cytoplasmic processes are usually of similar length.
Figure 3 shows a cell that is unusual, being
asymmetric in shape and having one large
cytoplasmic process. In the frog sartorius muscle, the satellite cells are approximately 40 to
90 p m in length and 6 to 10 pm in their widest
transverse diameter.
The number of cells that can be seen on the
femoral surface of Pb-stained sartorius muscles
using a 40 x water immersion objective is remarkably constant, with counts of satellite
cells on four control muscles ranging between
75 and 100 cells. It should be noted that after
exposure to (NH4),S,the muscle fibers vary in
color from yellowish to light brown. These
slight variations in color are noted within individual fibers and from one fiber to another.
Muscles exposed to (NH,),S without prior exposure to Pb result in the fiber acquiring a yellowish color.
D . Deneruation
The effect of muscle denervation on the morphology of satellite cells was studied in frog
sartorius muscles. Frogs were anesthesized by
immersion in a 0.1% solution of tricaine methanesulfonate (Finquel, Ayerst Laboratories).
Denervation was accomplished under the dissection microscope by removal of a 10-20 mm
section of the sartorius nerve, taking care not to
Electron microscopic identification of lead
stained cells
Satellite cells of mature muscle are mononucleated and contained beneath the external
lamina of the myofiber (Figs. 5-7). The external lamina does not intrude between the apposing cell membranes (sarcolemma and satellite
cell membrane). Associated with both membranes are numerous caveolae with lucent contents opening to the intercellular space (Figs.5,
C.3. Duration of Incubation
To determine the optimum duration of incubation, muscles were incubated in 60 mM
Pb(NO,,),for 5-45 min. Dissection, incubation,
fixation, and exposure to (NH,),S were performed as in method A.
C.4. Buffers
Various buffers were tested with the purpose
of creating a more physiologically compatible
incubation medium (increasing the pH and removing the arsenic based NaCaco). PIPES,
ADA, ACES, MES, BES, MOPS, and Bis Tris
were tested for their ability to buffer a 15 mM
concentration of Pb(NO,), a t pH 7.2
376
ALAN A. LAROCQUE, ALBERT0 L. POLITOFF, AND ALAN PETERS
MYOSATELLITE CELLS
TABLE I . Percentages of 357 cells showing varying
degrees of deviation from the longitudinal axes of
myofibers measured in the region of the cell body
(Measurements done on photographs using
a protractor)
Control
Denervated
0"-3"
4"-lo"
11"-30"
91.5
91.4
4.5
4.0
2.7
5.9
7, 8). The plasmalemma of the satellite cell is
separated from the sarcolemma by intercellular space, 15 to 20 nm wide.
Lead deposits are located between the apposing membranes of the satellite cell and
myofiber, and within numerous caveolae extending from these membranes (Figs. 7,8).The
cleft deposits do not form a continuous layer
between the apposing membranes but appear
as sporadic dense patches.
All satellite cells, including cytoplasmic
processes examined with the electron microscope after Pb(NO,), exposure, exhibited these
electron-dense deposits. Other mononucleated
cells present in muscle, such as fibroblasts,
pericytes, and endothelial cells described by
Ontell ('74) do not exhibit Pb deposits around
them or in association with their cell surfaces.
It should be noted that after exposure to NEM,
regions of the muscle fiber continue to exhibit
Pb deposits associated with the external
lamina and associated collagen matrix; however, their quantity is reduced.
Occasional membranous whorls are present
between apposing membranes of satellite cells
and myofibers (Fig. 6, arrow). These whorls
decrease in size and number with decreasing
periods of Pb(NO,), incubation.
Effects of p H , buffer, and P b concentration
To avoid the precipitation of Pb in the incubating solution or in the fixing solution, it is
necessary to use buffers that do not signifi-
377
cantly react with Pb t o form insoluble complexes. The best light microscope visualizations
are obtained by incubating the muscles for 15to
30 minutes before fixation (method A) in 60
mM Pb(NO,,),in a buffer of 70 mM NaCaco a t
pH 6.15 (Figs. 1-3). Higher pH values result in
precipitation of Pb, while greater concentrations of lead salts (90 mM) result in intense
Pb infiltration of fibers, so that it is difficult to
identify satellite cells after the addition of ammonium sulfide. Concentrations lower than 60
mM Pb produce only partial staining of the
cells.
Adequate light microscopic visualization is
also obtained with 60 mM Pb(NO,J, in a fixative solution buffered with 70 mM NaCaco, pH
6.15, with no prior incubation (method C.2.;
Fig. 4). However, incubation in Pb(NO,), prior
to fixation has been found t o be the method of
choice because of the homogeneous lead staining of muscle fibers and the clarity of the satellite cell demarcation.
It should be noted that for electron microscopy, incubation in media containing 15 to 60
mM Pb(NOJ, either before or during fixation
resulted in visualization of Pb deposits (Figs.
6-8).
In an effort to create a more physiologically
compatible incubation medium, numerous buffers were tested (method C.4.). Empirically, it
was found that with Bis Tris, i t was possible to
obtain a solution of 15 mM Pb(NO,), and 100
mM Bis Tris at pH 7.2. However, no Pb deposits
could be visualized with light or electron microscopy after incubation in this medium, followed by fixation and (NH,),S treatment
(method C.5.). However, 30 minute incubation
in Bis Tris and PB(NO,J, produces an increase
in membrane clarity and flocculent appearance
of the dense chromatin seen in the electron
microscope. In contrast, after fixation (without
prior incubation) in the presence of Pb and Bis
Tris at pH 7.2 followed by ammonium sulfide
(method C.5.1, numerous satellite cells and
Fig. 1. Satellite cell exhibiting two cytoplasmic processes. Incubated in 1mM N-ethylmaleimide and 60 mM
lead prior to fixation, followed by exposure to ammonium sulfide. Water immersion objective. x 1,000, Frog
sartorius.
Fig. 2. Satellite cell exhibiting two cytoplasmic processes. Incubated in 1mM N-ethylmaleimide and 60 mM
lead prior to fixation, followed by a 24 hour wash in sodium cacodylate buffer before exposure to ammonium
sulfide. Water immersion objective. x 1,000,Frog sartorius.
Fig. 3. Satellite cell exhibiting two cytoplasmic processes. Note the variation in size and shape of the
cytoplasmic processes. Incubated in 1 mM N-ethylmaleimide and 60 mM lead prior to fixation, followed by
exposure to ammonium sulfide. Water immersion objective. x 1,000. Frog sartorius.
Fig. 4. Two satellite cells located on one muscle fiber. Incubated in 1mM N-ethylmaleimide prior to fixation
with 60 mM lead, followed by exposure to ammonium sulfide. Water immersion objective. x 1,000.Frog sartorius.
378
ALAN A. LAROCQm, AL,BERTO L. POLITOFF, AND ALAN PETERS
Fig. 5. Electronmicrograph of a satellite cell fmm normal muscle. Dllated rough endoplasmic reticulum can be
seen in the satellite cell cytoplasm (arrow). The cell lies in a groove beneath the muscle basal lamina. x 26,000. Frog
sartorius.
Fig. 6. A satellite cell from normal muscle incubated i n 1 mM NEM and 15mM lead nitrate prior to fixation. Note
the presence of lead deposits within caveolae of the satellite cell and myofiber (arrowheads), and the presence of a
membrane whorl between apposing membranes of the satellite cell and myofiber (arrow). X 36,000. Frog sartorius.
Fig. 7. A .iatellite cell from normal muscle. The fixative contained 15 mM lead nitrate and wad buffered at pH 7.1
with lOOmM RisTris. x 36.000. Frogsartorius. Nowthe pre;ienceofPt,depositsassociatedwith thecollagenmatrix.
Fig. 8 A high mapification view of the opposing membranes illustrated in Fig. 7. Note the It~alizationof lead
between the apposing mernhranes 0 1 the satellite cell and parent myufiher (arrows,. A 69,UOU. Frog iurtorius
MYOSATELLITE CELLS
379
380
ALAN A. LAROCQUE, ALBERT0 L. POLITOFF, AND ALAN PETERS
myonuclei became visible under the light microscope. Figures 7 and 8 are without ammonium sulfide.
Effect of denervation on the cytoplasmic
processes of the satellite cell
Four weeks after denervation, the number of
satellite cells counted on the femoral surface of
the muscle is remarkably similar to the
number of cells counted on the femoral surface
of the intact contralateral muscle. Thus in two
frogs, the denervated versus control counts are
105, 82, and 99,79. No mitatic activity or cellto-cell contacts are observed following denervation.
For a subpopulation of satellite cells, denervation has a clear effect on the size and number
of their cytoplasmicprocesses. This is shown by
the observation that in denervated muscles, 22
of 187 satellite cells have more than two
branches (Figs. 9, 10, 12) with a maximum of
seven cytoplasmic processes observed, while
the intact contralateral muscles had 3 of 178
cells with more than two branches, with a
maximum of three. Statistical analysis using
Fisher's exact test gives a Fisher exact
probability of 0.00008. Therefore, the increase
in branch formation seen following denervation is unlikely to be a chance occurrence, but
rather a result of denervation. The majority of
the elongated cytoplasmic processes extend
parallel to the longitudinal axis of the denervated muscle fibers (Figs. 9-11) with occasional spiraling around the myofiber. For the
population of satellite cells which proliferate
their cytoplasmic processes as a result of denervation, a high cytoplasmichuclear ratio develops (Figs. 9-12). Supernumerary cytoplasmic processes are either attached to the cell
body (Fig. 9) or to another process (Figs. 10,111.
Occasional processes extend 200 pm from the
cell body.
DISCUSSION
The present results show that incubation or
fixation of striated muscle in the presence of
Pb(NO&, under well defined ionic conditions,
leads to the light microscopic visualization of
satellite cells located on the surfaces of the
muscle fibers. Electron microscopic examination of muscle treated with Pb(NOJ, under the
same ionic conditions reveals that the
myosatellite cells are delineated by Pb deposits
contained in the cleft between the apposing cell
membranes.
Light microscopy
This new method of demonstrating satellite
cells reveals their shape, the outline of their
cell processes, and their occasional branching
pattern. Such features have escaped detection
because previous studies on satellite cells have
depended upon the use of tissue sections,rather
than upon complete muscle preparations.
Though our evidence is restricted to the frog
sartorius muscle, it is reasonable to expect that
this technique can be applied successfully to
muscles of other species.
The consistency in satellite cell counts per
muscle suggests a precise quantitative relationship between the number of satellite cells
and muscle mass. Their apparent homogeneous
distribution over the muscle surface suggests
that their function is not restricted to one specific portion of the muscle. Kelly ('78) has reported an increased incidence of satellite cells
a t the neurojunctional region in comparison
with the nonjunctional area in the rat soleus,
and has suggested that the cells near the motor
end plate may contribute to the repair of the
synapse after injury.
The difference in satellite cell dimensions reported by Muir ('70) and Muir et al. ('65) (25 pm
x 4 pm) for mammalian satellite cells and the
dimensions reported in this study (40 to 90 pm
x 6 to 10 pm) for frog satellite cells may be due
to differences in muscles, species, age, and
stretch a t time of fixation.
Previously, the cytoplasmic volume of the
satellite cell has been reported as scanty, because the observations were made on sections
through the nuclear region. These observations
have resulted in the cytoplasmic nuclear volume ratio being described as low (Carlson, '73;
Ishikawa, '66; Reznik, '76); however, the existence of elongated processes indicates that this
ratio is higher.
Numerous investigators have described the
satellite cell as being situated approximately
parallel t o the longitudinal axis of the muscle
fiber (Carlson, '73; Reznik, '761, but the Pb
staining reveals (Table 1)that a subpopulation
of cells clearly deviates from this orientation.
Possibly, cells that belong t o this minority do
not lie in longitudinal grooves on the myofiber
surface (Carlson, '73), but extend diagonally
over the surface without concomitant indentation of myofibrils.
The majority of satellite cells in normal frog
muscle possess two elongated cytoplasmic
processes. These processes project from the opposite poles of the central region and their main
MYOSATELLITE CELLS
Fig. 9. A satellite cell after four weeks denervation. Note the enlarged cytoplasmic processes (arrows).Water
immersion objective. x 1,000.Frog sartorius.
Fig. 10. A satellite cell after four weeks denervation. Note the enlarged bifurcated, cytoplasmic processes
(arrows). Water immersion objective. x 1,000. Frog sartorius.
38 I
382
ALAN A. LAROCQUE, ALBERT0 L. POLITOFF, AND ALAN PETERS
Fig. 11. A satellite cell cytoplasmic process extends downward from the top of the field (arrows) and bifurcates
at its end (arrowheads) after four weeks denervation. Water immersion objective. x 1,000. Frog sartorius.
Fig. 12. A satellite cell after four weeks denervation. Note the enlarged cytoplasmic processes (arrows)marked
by obvious lead precipitate. Water immersion objective. x 1,000. Frog sartorius.
383
MYOSATELLITE CELLS
axis is essentially the same as the axis of the
muscle fiber (Table 1).If existing satellite cells
have the potential to become myoblasts (Bischoir, '74, '75; Church et al., '66;Jirmanova and
Thesleff, '72; Kahn and Simpson, '74; Moss and
Leblond, '70; Schmalbruch, '77; Shafiq and
Gorycki, '65; Snow, '77a,b, '78), this longitudinal orientation of the cytoplasmic processes
would be consistent with the arrangement of
the myofilaments.
Satellite cells usually occurred individually
and they were never seen making contact. This
might be correlated with the fact that we have
not observed mitotic figures, in agreement with
Aloisi et al. ('73), Ontell ('741, Reznik ('76) and
Schultz et al. ('78). In contrast, Allbrook et al.
('71) and Shdiq et al. ('68) have observed mitotic figures in normal, early postnatal muscle.
If mitotic figures are present, the Pb staining
used in our current studies is unable to reveal
them.
Electron microscopy
The most remarkable feature of the Pb deposits is the consistency of their presence in the
intercellular cleft between muscle and satellite
cell membranes, suggesting a fairly specific
phenomenon. A necessary step for the production of these deposits is the entry of Pb into the
intercellular space, probably by means of simple diffusion. The entry of Pb as well as lanthanum (Chou and Nonaka, '77) into both the
muscle and satellite cell caveolae suggest that
they are usually open to the intercellular space,
at least for some time.
It is not clear why the Pb deposits form
patches, instead of a continuous layer. Perhaps
the extracellular space between the apposed
membranes is anisotropic, due to the presence
of an extracellular or membrane-bound component that has low electron-density and high
affinity for Pb ions; or perhaps Pb ions are preferentially precipitated in specific areas because
of a localized increased biochemical activity.
Lead deposit formation
Prior exposure to NEM does not detectably
affect the formation of Pb deposits associated
with the satellite cell. This suggests that the
visualization of the satellite cell is not due to
binding of Pb to SH groups. Although NEM
decreases the quantity ofPb deposits associated
with the collagen-matrix, some Pb deposits associated with the external lamina and the
collagen-matrix persist. We assume that these
deposits are responsible for the variation in
color seen with the light microscope after exposure to (NH,),S.
Denervation
Previous studies done in mammals show an
increase in the number of satellite cells after
denervation. Aloisi et al. ('73) studied the effects of denervation on the number of satellite
cells of the soleus and the extensor digitorum
longus muscles of the adult rat. They found an
increase of 85% and 250% respectively in satellite cells six days after denervation. Hess and
Rosner ('70) examined limb and extraocular
muscles of adult guinea pigs and found an increase after seven days of denervation. Ontell
('74) reported a n increase of 500 to 610% in
adult rats 21 days after denervation of the extensor digitorum longus.
Presently, two general theories exist to explain the formation of myoblasts in regenerating muscle. One, that myoblasts form from
preexisting myosatellite cells (Snow, '77a,b,
'78) and the other, that myoblasts form from
myonuclei (Mastaglia et al., '75). Our results
show that the number of satellite cells in frog
sartorius muscle 28 days postdenervation is the
same as in the nondenervated contralateral
muscle. Provided a subpopulation of satellite
cells is not produced from myonuclei in the frog
sartorius muscle followingdenervation, the observed lack of change in the number of satellite
cells following denervation is consistent with
the absence of mitotic figures and the rare occurrence of satellite cells in close proximity to
each other (Ontell, '74). To explain the lack of
effect of denervation on satellite cell number, it
might be hypothesized that a newly formed or
latent subpopulation of satellite cells is not
made visible by Pb staining: Schultz ('74, '78)
has reported that in denervated growing muscle, a percent of satellite cells may become detached from the myofiber and if this detachment occurs in adult frog sartorius, the Pb deposits might not be formed. Additionally, this
study examines only satellite cells on the
myofiber surface. It is also possible that the
time course of the denervation changes of satellite cells in frog sartorius is slower than that of
mammalian muscle. Verma ('79) in a short
communication reports multiplication of satellite cells 38 days post denervation in the m.
rectus internus ofRana exculenta.
lite cells 38 days post denervation in the m.
rectus internus of R a m exculenta.
As shown by Pb staining following denervation, a subpopulation of satellite cells change
shape. This is in agreement with the observations of Hess and Rosner ('70), who noted that,
following denervation, long stretches of satellite cell cytoplasm extend away from the nu-
384
ALAN A. LAROCQUE, ALBERT0 L. POLITOFF, AND ALAN PETERS
clear region. In electron microscopic preparations, Schultz ('74, '78) also demonstrated a n
increased number of cytoplasmic processes
among a specific subpopulation of satellite cells
in denervated growing muscle. The processes
described by Schultz ('78) appear to be limited
to the nuclear region of the satellite cell and
there is no report of cytoplasmic processes extending beyond the immediate vicinity of the
cell body as described above.
There is a great variability in the number,
size, and branching patterns of the cytoplasmic
processes that are induced by denervation. The
majority of the processes extend parallel to the
longitudinal axis of the muscle fiber with occasional spiraling around the myofiber. The functional significance of the increased size and
number of cytoplasmic processes is not readily
apparent. The enlarged supernumerary cytoplasmic processes, and the resulting increase in
cytoplasmic volume and organelles may represent a n intermediate phase in the cytoplasmic
transition from satellite cell to myoblast, and
the subsequent synthesis of muscle filaments
(Chou and Nonaka, '77; Hess and Rosner, '70).
If this subpopulation of satellite cells with increased size and number of cytoplasmic processes induced by denervation represents an
intermediate phase in the transition from
satellite cell to myoblast and if the origin of this
subpopulation is from preexisting myosatellite
cells, then this report is in agreement with Bischoff, '74, '75; Church et al., '66 Jirmanova and
Thesleff, '72; Kahn and Simpson, '74; Moss and
Leblond, '70; Schmalbruch, '77; Shafiq and
Gorycki, '65; and Snow, '77a,b, '78 who indicate
that regenerating mononucleated myoblasts
form from preexisting myosatellite cells rather
than from myonuclei (Elyakova, '72; Hay, '59,
'74; Hess and Rosner, '70; Lee, '65; Mastaglia et
al., '75; Ontell, '74; Reznik, '69; Teravainen, '70;
Yanko et al., '74). If frog myoblasts are derived
from myonuclei, it is possible that the satellite
cell is a n intermediate stage. Reznik ('76)
suggests that, depending on the stimulus,
myoblasts are formed either from preexisting
satellite cells or myonuclei, and it is conceivable that both pathways might occur simultaneously. Finally, the pathway taken during
regeneration may be species-dependent.
We have demonstrated in this report the application of a Pb staining technique that results
in the light microscopic visualization of satellite cells in normal and denervated muscles. It
is reasonable to expect that this technique will
be applied successfully to other questions involving satellite cells in muscle biology, development, and regeneration.
ACKNOWLEDGMENTS
This study was supported by NIH grants NS
11588 and CA 16778 to A.L. Politoff and Training Grant GM-01979 to the Department of
Anatomy, Boston University School of Medicine. Material included in this article has been
submitted by Alan Larocque in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Anatomy a t Boston University Graduate School.
We thank the reviewers for their helpful
comments and Ms. J.M. Henry and Ms. K.A.
Larocque for their enthusiastic clerical help.
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visualization, denervated, muscle, microscopy, light, staining, norman, myosatellite, new, techniques, cells
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