A RADIOAUTOGRAPHIC STUDY OF
POSTNATAL GROWTH OF SKELETAL
MUSCLE IN MlCE

Thesis for the Degree of M. S.
Mififiiafifl STATE UNWERSITY
.DEANE CAROL iORDAN
1970

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A RADIOAUTOGRAPHIC STUDY OF POSTNATAL GROWTH

OF SKELETAL MUSCLE IN MICE

BY

Diane Carol Jordan

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Anatomy

1970

ACKNOWLEDGEMENTS

The author wishes to thank Dr. Bruce E. Walker
for his generous help, guidance and encouragement while
serving as major professor and advisor in the graduate
program.

I also wish to thank Dr. Rexford Carrow and Dr.
Robert Echt for serving on the guidance committee during
the graduate program, and for their comments on the thesis.

Appreciation is also extended to the faculty and
staff of the Anatomy Department for assistance and encourage-
ment received during the program. Special thanks goes to
Robert Paulson for his help with photography, Mary Grace for
typing the thesis, and especially Patricia Patterson and
Heather Murray for typing, proof-reading, encouragement, and
valuable friendship throughout the program.

Finally, I wish to thank my parents for their

confidence and love.

ii

TABLE OF CONTENTS

Page

INTRODUCTION 0 I O O O O O O O O O O O O O O O O O O O O O 0' ...... O O O O 0 O O O O O 0
LITERATURE REVIEW 0 O O O O O O O O 0 O O O O O O I ...... O O O O O O O O O O O O 0
Muscle Development ........ ....... ...........
Nuclear Proliferation .......................
Fiber Increase O O O O O O O O I O I O O O O O O O O O O O O O O O I O O 0
Location Of Growth 0 O O O O O O O O O O O O O 0 O O O O O O O O O O 0
Radioautography O O O O O O O I O O 0 O O I O ..... O ........
MATERIALS AND METHODS 0 O 0 O O O O O O O O O O 0 O O O O O O O O 0 O I O O O O O O 0
RESULTS O...0.0.0.00000000000000000000000000000000000.
DISCUSSION 0 O O O O O O O I O O O O O O O O O O O 0 O O O O O O O O O O O O O O O O I O O O O 0
CONCLUSION O O O O O O O O O O O O O O O O O O 0 O O O O O O O O O O O O O O O O O O O O O O O 0
LITERATURE CITED 0 O O O O O O O O O O O O O O O O I O O O O O O O O O O O O O O O O O O 0
APPENDIX A

Original data collected on muscles labeled with
tritiated thymidine OOOOOOOOOOOOOOIOOOOOCOOOO

APPENDIX B

Original data collected on muscles labeled with
tritiated proline ............................

iii

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10
16
36
44

46

49

78

10.

11.

LIST OF TABLES
Page

Original data collected on a triceps surae
muscle labeled with tritiated thymidine ..... 51

Original data collected on a tibialis anterior
muscle labeled with tritiated thymidine ..... 55

Original data collected on a triceps surae
muscle labeled with tritiated thymidine ..... 59

Original data collected on a gracilis muscle
labeled with tritiated thymidine ............ 65

Statistical results for tritiated-thymidine
labeled muSCleS 00.0.0...OOOOOOOOOOOOOOOIOOOO 25

Original data collected on a triceps surae
muscle labeled with tritiated thymidine ..... 73

Statistical results for tritiated-thymidine
labeledmuSCIeS 0.0.0....OCOOOOOOOOOOOOOOOOOO 28

Original data collected on a gracilis muscle
labeled with tritiated proline 1 hour prior
to fixation .... .............. ............... 79

Original data collected on a gracilis muscle
labeled with tritiated proline 1 day prior to
fixation 0.0.0...OOOIOOOOOIOOOOOOOOIOOOOOO... 80

Original data collected on a gracilis muscle
labeled with tritiated proline 6 days prior
to fixation ......... ....... . ...... .... ...... 82

Original data collected on a gracilis muscle

labeled with tritiated proline 14 days prior
to fixation OOOOIOOOOOOOIOOOOOOOOOO0.00...... 85

iv

Figure

LIST OF FIGURES

Page
Frequency polygons for the distribution of
nuclei along the length of a triceps surae
muscle labeled with tritiated thymidine ..... 19

Frequency polygons for the distribution of nu-
clei along the length of a tibialis anterior

muscle labeled with tritiated thymidine ..... 20

Frequency polygons for the distribution of
nuclei along the length of a triceps surae
muscle labeled with tritiated thymidine ..... 21

Frequency polygons for the distribution of
nuclei along the length of a gracilis muscle
labeled with tritiated thymidine .. ..... ..... 22

Frequency polygons for the distribution of
nuclei across the width of a triceps surae
muscle labeled with tritiated thymidine ..... 27

Frequency polygons for the distribution of

labeled protein along the length of a gracilis
muscle labeled with tritiated proline 1 hour

prior to fixation ...... ..................... 30

Frequency polygons for the distribution of

labeled protein along the length of a gracilis
muscle labeled with tritiated proline 1 day

prior to fixation ................... ........ 31

Frequency polygons for the distribution of

labeled protein along the length of a gracilis
muscle labeled with tritiated proline 6 days

prior to fixation ................ ..... ...... 32

Frequency polygons for the distribution of

labeled protein along the length of a gracilis
muscle labeled with tritiated proline 14 days
prior to fixation ........................... 33

INTRODUCTION

Basic research in the area of skeletal muscle and
the application of such information to muscle—related
diseases is a growing field of interest. A variety of
procedures have been used to study the development, structure,
and regeneration of muscle. However, relatively little is
clearly understood as to how additional material is added
on structurally during postnatal growth.

As an animal grows, its skeletal muscle must increase
in size proportionally. But how and where the increase occurs
has not been adequately investigated. Several possibilities
do exist. Nuclear proliferation may continue for some time
after birth, either in distinct zones of growth (Kitiyakara
and Angevine, 1963a), or randomly throughout the length of the
individual muscle fiber. In addition, synthesis of new cytoplasm
may occur in specific areas or along the entire length of the
fiber. Some information is available which suggests that
skeletal muscles increase in size by the addition of entirely
new fibers while other reports indicate the increase in size is
due to enlargement of existing fibers. These possibilities are
as yet inconclusive and must be resolved in order that problems
related to muscle growth and development, muscle diseases and
rehabilitation may be effectively dealt with.

l

By utilizing radioisotopically-labeled bases and
amino acids, nuclei preparing to divide and newly synthesized
protein can be evaluated as to their distributions along the
length of the muscle fiber. These distributions at different
time intervals after administration of the label, will be
compared to determine which materials are proliferating,

and where the new materials are located in the muscle fiber.

REVIEW OF LITERATURE

Muscle Development

During early deve10pment, the mesoderm differentiates
into three segments, an epimere, a mesomere, and a hypomere.
The epimere, or somite, further differentiates into three
segments. First, a cavity develops, the myocoele, which
has no function in the adult. The segment of the epimere
which is dorsal to the myocoele is called the dermatome,
and gives rise to the dermis of the adult. The segment
ventral to the myocoele is called the myotome. Then groups
of cells bud off from the ventral portion of the myotome, to
become the sclerotome, which gives rise to the vertebrae.
The myotome gives rise to the axial musculature of the adult
animal. The mesenchymal cells in this area become myoblasts,
which differentiate to form mature muscle fibers. The limb
musculature is also a derivative of mesenchymal cells, but
develops from the lateral plate mesoderm (hypomere). This is
separated into two portions by the developing coelom, which
associates with the ectoderm and endoderm, and is called
somatopleure and splanchnopleure, respectively. The mesenchyme
destined to become limb musculature buds off from the
somatopleure (Boyd, 1960).

There are three theories as to the formation of
multinucleated muscle fibers (Bloom and Fawcett, 1968). The

3

first and most widely accepted theory proposes that the
mononucleated myoblasts fuse together to form a syncytium.
The second theory postulates rapid growth of the myoblast
with proliferation of nuclei, but no division of cytoplasm,
therefore resulting in a long multinucleated muscle fiber.
Amitosis of muscle nuclei has also been proposed. Another
possibility, is that some combination of these processes is
occurring.

Hay (1963) defines three stages in the development
of skeletal muscle in the salamander tail. The myoblast, "a
cell that can be identified by morphological criteria
(spindle shape, abundant basophilic cytoplasm, and close
proximity to muscle) as a muscle cell precursor", becomes an
"early muscle fiber", which may or may not be multinucleated,
and which may contain a few myofibrils. These cells then
become "formed muscle fibers", which are defined as cells "in
which myofibrils fill most of the cytoplasm and are regularly
aligned".

Similar stages of development have been reported for
human muscle (Ishikawa, 1965), chick muscle (Dessouky and
Hibbs, 1965, and Przybylski and Blumberg, 1966), and in tissue
culture (Shimada, Fischman, and Moscona, 1967, Coleman and
Coleman, 1968, and Birschoff and Holtzer, 1969). The most
prevalent theory as to the mechanism involved in the forma-
tion of the multinucleated fibers is cell fusion. This
process has already been shown to be operating during muscle

regeneration (Bintliff and Walker, 1960).

Nuclear Proliferation

Mitoses are frequently seen in the mononucleated
myoblasts, but it is generally agreed that mitotic activity
does not occur in the multinucleated fiber containing myo-
fibrils (Stockdale and Holtzer, 1961, and Zhinkin and
Andreeva, 1963).

Therefore, after birth, we are mainly interested in
the potentials of the nuclei and the cytoplasm of muscle
fibers to provide for growth in muscle. Several authors have
reported increases in the number of skeletal muscle nuclei
after birth. Leblond, Messier and Kopriwa (1959)
found that muscle nuclei could be labeled with tritiated
thymidine in 3-day-old rats, but not in young adults. Laird
and Walker (1964), however, failed to label any muscle nuclei
when 3-day-old mice were injected with tritiated thymidine.
This seems to indicate that the cessation of mitotic activity
in muscle occurs within 3 days after birth.

Enesco and Puddy (1964) found the amount of DNA in
skeletal muscle to increase with age, and that the increase
is due to a rise in the number of muscle nuclei. MacConnachie,
Enesco, and Leblond (1964) report finding mitotic figures in
young rat skeletal muscle, indicating that the nuclei are
capable of dividing.

Shafiq, Gorycki, and Mauro (1968), however, using
electron micrographs, found mitoses only in satellite cells
and cells outside the muscle fibers. No myonuclei were seen

in mitosis. Moss and Leblond (1970), using tritiated thymidine

in young rats, found that the only cells labeled during the
first 24 hours after injection were the satellite cells.
Thereafter, an increasing number of myonuclei became labeled.
They concluded, therefore, that the satellite cell gives

rise to new muscle nuclei providing for the increase in their

number.

Fiber Increase

Some work has been done on the role of the cytOplasm
in the growth of skeletal muscle. Fennichel(l965) states that
the adult number of human muscle fibers is established by the
16th week of development. Enesco and Puddy (1964) found no
difference in the number of muscle fibers in suckling and
adult rats. Rowe and Goldspink (1969) also reported no
increase in the number of fibers in mice from newborn to 24
weeks of age, but did observe an increase in fiber diameter.
Several other authors also support this finding (see Moss,
1968). A conflicting report by Chiakulas and Pauly (1965)
found that, in rats, during the first 3 weeks after birth,
there is an increase in the number of fibers, but after that,

only an increase in fiber size.

Location of Growth

Some authors propose that muscle growth, regardless
of the mechanisms involved, occurs at distinct locations in
the muscle as a whole. Kitiyakara and Angevine (1963a and
1963b), using both radioautographic techniques and gross

marking procedures, prOposed that growth of the muscle as a

whole, and probably of the individual fiber as well, takes
place in small zones near the terminals of the muscle. By
counting labeled nuclei in cross sections of muscle, they
observed two peaks of concentration of labeled myonuclei.
However, no statistical tests were reported to verify the
significance of these concentrations. Also, when employing
the gross marking techniques, they drew illogical conclusions
concerning the role of the connective tissue scar, which was
formed. Mackay and Harrop (1969) with similar gross marking
techniques, obtained contradictory results, but attributed
them to growth of the connective tissue and possibly the
sarcolemma. They also concluded that the major growth takes
place at the muscle-tendon junction. Further support for this
hypothesis can be found in Hay (1963) and Goldspink (1968).
Montgomery (1962) found no differences between the
terminals and the midpoints of human striated muscle, with
regard to morphology and nuclear counts. Marchok and Herrmann
(1967), in contradiction of Kitiyakara and Angevine (1963a),
observed no significant difference in the labeling of muscle
nuclei between the ends and the central portions of embryonic
chick muscles, but offered no statistical evidence. Farrell
and Fedde (1969), also reported uniformity in morphology and
enzyme activity throughout the length of the muscle fiber.
Uniformity in fiber number in the central and end portions of
muscles was reported by Enesco and Puddy (1964). Therefore,
in light of these contradictions, before accepting any pro-
posal for the method of growth, further studies should be

done.

Radioautography

Radioautography is a technique used to study uptake
and utilization of radioisotopically labeled compounds.
Briefly, it entails the labeling of certain cell components
(protein, DNA, RNA) with a radioactive precursor. The
labeled tissue is then subjected to routine processing for
the production of histological slides. The slides may then
be exposed to a photographic emulsion, developed, stained,
and examined. Those components which were labeled are re-
vealed by an overlay of silver grains.

The mitotic cycle is characterized by four periods:
G1, the period between the end of the preceding mitosis and
the beginning of DNA synthesis; S, the period of DNA syn-
thesis; G2, the period between the end of DNA synthesis and
the beginning of mitosis; and M, the period of mitosis (Bloom
and Fawcett, 1968). If a DNA precursor is injected just
prior to the S-period, it will be utilized by those nuclei
which are synthesizing DNA in preparation for division. Any
compound which is utilized during synthesis, and can be
labeled with an isotope, may be used as a precursor. However,
when labeling with an isotope such as C14, radiation damage
may become a factor due to the large dose required. Also,
some precursors are not specific for DNA, and therefore com-
plicated techniques for identification of the DNA are necessary
(Leblond, Messier, and Kopriwa, 1959).

The use of tritium as a label for thymidine, has

made it relatively easy to obtain good radioautographs

(Leblond, Messier, and Kopriwa, 1959). Tritium is a low
beta—emitter with a range of 2-3p, and a half-life of 12.5
years (Rogers, 1968). Therefore, it is possible to obtain a
very clear-cut picture of where the label is being utilized,
and to retain the results for a relatively long time.
Thymidine is a nucleic acid base which is specific for DNA.
This provides a sure method of labeling only those nuclei
which are synthesizing DNA in preparation for division.
Another advantage, is the availability time of thymidine.

It has been shown that all available labeled thymidine is
removed by the end of one hour after injection, in adult
animals (Hughes, Bond, Brecher, Cronkite, Painter, Quastler,
and Sherman, 1958). Therefore, all the nuclei which are
labeled were synthesizing DNA just after the time of injec-
tion. This provides an adequate means for studying proli-
feration and migration of nuclei. The same sequence of
reasoning is followed in choosing a precursor for other cell

components.

MATERIALS AND METHODS

C3H/HEJ strain mice (Jackson Memorial Laboratory,
Bar Harbor, Maine) were maintained in a temperature-controlled
room, at 22°C.:20. Lighting was automatically regulated
to give 10 hours of darkness, from 6:00 AM to 4:00 PM, and
14 hours of light, from 4:00 PM to 6:00 AM. The only source
of light permitted during the dark period was a 30 watt
Sylvania infrared light, used during cleaning and feeding.

The mice were kept in 7 1/4" x 11 1/2" x 5" plastic
cages, containing wood shavings. Two males or four females
were kept in each cage. Food1 and water were provided ad
libitum.

Mice approximately 7-10 weeks old, and weighing
18-209 were used for mating. Mating occurred during the
dark hours, between 8:00 AM and 4:00 PM, twice weekly. At
these times, two females were placed in each cage containing
two males. At the end of the mating period, females found

to have vaginal plugs were separated for experimentation.

 

lOld Guilford Laboratory Animal Diets, manufactured by the
Emory Morse Co., Guilford, Conn. Protein, minimum - 19.0%;
Fat, minimum - 7.5%; Fiber, maximum - 2.5%; N.F.E., minimum -
52.0%.

10

11

During the last 2-3 days of gestation, pregnant females were
put in separate cages in which nesting material was provided.
To determine the time of birth, cages containing pregnant
females were checked every 4 hours. This made it possible

to record the time of birth with a range of error of 0 to
-4 hours.

The first experiment involved labeling nuclear DNA
in growing skeletal muscle with radioactively-labeled thymidine.
Ten‘pc (0.01ml.) per gram body weight of tritiated thymidine
(specific activity of 2c/mM) were injected subcutaneously
with a Hamilton Microliter Syringe #725 and a 27Gl/2" dis—
posable needle, into 3 young mice when they were 24 hours old
(1.5g). Following injection, the mice were returned to the
mother, and the cage was transferred to a separate room, used
only for radioactive animals.

Fourteen days after injection of thymidine, the mice

were killed by an overdose of ether. The skin of both hind
limbs was incised, and M. gracilis, M. tibilis anterior,
M. triceps surae, and M. quadriceps femoris were removed.
Dissection was carried out by sectioning the proximal and
distal attachments of the muscles, to insure complete re-
moval.

The excised muscles were stretched on small card—
board squares, and immediately immersed in 10% formalin
buffered with CaCO3, for overnight fixation. Dehydration of
the tissues in graded alcohols, and infiltration with

toluene and paraffin, was followed by embedding in paraffin

12

(Paraplast, Scientific Products, Evanston, Illinois). Muscles
from the left limbs of the animals were oriented for longi-
tudinal sections in paraffin blocks, while the muscles from
the right limbs were oriented for cross sections. The blocks
were cut into 7p sections on a rotary microtome. The sections
were mounted on pre—cleaned, albuminized slides, and were
dried overnight at 37°C.

One-third of the slides (every third slide) were
stained with periodic acid-Schiff (PAS) reagents (Gridley,
1960), one-third with PAS reagents after diastase treatment,
and the remaining one-third were deparaffinized only. After
drying, all slides were coated with Kodak NTB2 liquid
emulsion, using the method reported by Walker in 1959, except
for the omission of the subbing fluid and celloidin treatments.
After 4 to 6 weeks of exposure at 5°C., the slides were
developed for 5 minutes in Kodak Microdol X, and fixed for
5 minutes in Kodak acid fixer. All slides were stained after
development with Harris Hematoxylin and Eosin Y. The slides
were then coverslipped with Permount, dried and cleaned.

Using an A/O Spencer binocular microscope, slides
with longitudinally—oriented muscle sections were analyzed
histometrically. Nuclei and the associated silver grains
were counted under a 97X oil immersion objective and a 10x
ocular lens. Nuclear counts were recorded by optical fields
having an area of 31,400);2 per field. Each consecutive
field along the entire length of the muscle was included.

Therefore, each count was made on a strip of fibers through

13

the bulk of the muscle, but not necessarily in the center,
the width of an Optical field and the length of the muscle.
The position, in relation to the sarcolemma, and grain count
of each nucleus were recorded. Nuclear position had one of
three designations: I, indicating the nucleus was inside the
fiber, and not touching the sarcolemma; P, indicating the
nucleus was at the edge of the fiber; and 0, indicating the
nucleus was outside the limits of the muscle fiber. The
number of silver grains over each nucleus was recorded at
the same time as its position. Similar counts were taken

by crossing the muscle section at various levels.

Grain counts were recorded for non-specific back-
ground reaction (fog). The microscope was equipped with a 10x
ocular fitted with a 1421 A Net Reticule (20mm. dia. 5mm. sq.
ruled into 1.0mm sq.'s - American Optical Company, Buffalo,
New York) measuring 55p x 55p, under the lens system described.
Fog counts were recorded for open spaces away from the tissue
sections, open spaces within the tissue sections, and over
tissue with no specific reaction, in individual areas measuring
11p x 11p. These counts were averaged to determine the
average fog count per 121p2. These data were then used to
determine which labeled nuclei could be attributed to back-
ground rather than a specific reaction.

The second experiment involved labeling of muscle
protein in growing skeletal muscle with radioactively-labeled
amino acids. Three amino acids were used: tritiated proline,

specific activity of 5.1c/mM (New England Nuclear Corp.,

14

Boston, Mass.); tritiated leucine, specific activity of
58.20/mM (New England Nuclear Corp., Boston, Mass.); and
tritiated histidine, specific activity of 3.0c/mM (Schwarz
Bioresearch, Orangeburg, New York). All three compounds were
packaged as aqueous solutions, lmc/mM. Fifty pc (0.05m1.)
per gram body weight of each amino acid were injected sub-
cutaneously with a Bristol Universal Syringe and a 27Gl/2"
disposable needle, into mice which were 24 hours old. A
total of 30 mice received treatment, each of the three amino
acids being administered to 10 mice. These mice were also
returned to their mothers, and transferred to the room for
radioactive animals.

The mice were killed by an overdose of ether at
various time intervals. Three mice in each amino acid group
were killed at 1 hour after injection, 2 mice at 1 day,

2 mice at 6 days, and 3 mice were killed at 14 days after
injection. The same muscles were removed as described above
for the previous experiment. Tissue processing, staining, and
autoradiographic procedures were the same as those described
above.

Grain counts were done on an A/O Spencer binocular
microscope under a 97x oil immersion objective and a 10x
ocular equipped with a 1421 A Net Reticule. Beginning at one
end of a longitudinally-oriented muscle section, grain counts
were recorded for areas of lzlpz, throughout the length of
the muscle, in groups of five areas at a time. Each group

of five areas was totaled to give a grain count per 605p2.

15

This type of count was performed at each edge of the section,
and also through the center. Counts were also taken in
transverse areas perpendicular to the longitudinal axis of
the same muscle section, at regular intervals. The total

area in this case varied with the width of the muscle.

RESULTS

In order to evaluate radioautographs accurately, it
is necessary to eliminate the non-specific background reactions
called fog. Fog is caused by many diverse stimuli: heat,
light, chemicals, agitation, radiation in the atmosphere, even
radiation inherent in the glass slides themselves. After
all possible precautions have been exercised, a small amount
of fog is still evident. It then becomes necessary to de-
termine which reactions are due to incorporation of the tritium
label and which are background reactions.

Since fog is assumed to be random, its distribution
should fit a normal curve, which is the limit of the binomial
(p + q)N as N approaches infinity (Croxton, 1953). We cannot,
however, fit a binomial. First, in fitting a binomial, it is
necessary to know the proportion of occurrences (p) and non-
occurrences (q). By using the Poisson distribution, it is not
necessary to know the actual or theoretical proportions. The
only restriction is that the proportion of occurrences be
very small and the number of observations be quite large. A
second factor which restricts the use of the binomial is the
number of observations. This figure must be known since it
determines the number of terms in the binomial. With the

Poisson distribution, it is possible to use data with a certain

16

17

number of observed categories, but also an indefinite number
of additional categories.

Background reactions in radioautographs fall into the
class of Poisson distributions. The probability that any one
nucleus will be labeled due to fog is quite small, e.g., the
average fog count per nucleus may be .3 grains. Since the
total number of observed nuclei is large, we would expect
only a few nuclei to be labeled due to fog. Also, although
the nuclei in question are those with only 1-4 grains, the
total number of grains possible per nucleus is not defined.
Therefore, we can use the Poisson distribution to determine
the proportion of labeled nuclei in a sample that should be
disregarded as labeled due to fog. It is necessary to know
only the mean (average fog count/nucleus) which is determined
by histometrics. With this figure and the following formula
(Bailey, 1959), it is possible to calculate the relative
proportions in each category, i.e., the proportion of total
nuclei with 0, l, 2 ... grains that are so labeled because of

fog:

e — the base of the natural loga-
rithms (2.71828)

(D
9’ I
.- xl
xl
I

NI
u

the mean (average fog count/
nucleus)

a = the number of grains in each
category: 0, l, 2 ... (by
definition, 0! = 1)

18

These frequencies can then be compared to the observed fre-
quencies and the necessary deductions can be made to eliminate

those reactions estimated to be due to fog.

All animals receiving thymidine treatment at the age
of 24 hours survived to the 15th day. Histological slides
were prepared from the autopsied hind limb muscles. Longitu-
dinal muscle sections, prepared for radioautography, were
coated with emulsion, exposed and developed. The slides were
then analyzed histometrically by counting labeled and unlabeled
nuclei. General observation of the sections revealed no con-
spicuous concentrations of label in any area of any of the
muscles. However, in order to obtain a more accurate account
of the distribution of the label, actual counts were made of
the number of each kind of nucleus and the number of grains

over each nucleus.

Counts were made in a lengthwise direction by Optical
fields, 31,400u2 at 970x, from one end of the muscle to the
opposite end. By referring to the Poisson distribution, that
proportion of nuclei overlaid by 0-4 silver grains and not
actually radioactive, was subtracted from the radioactively
labeled nuclei. Frequency polygons (Figures 1-4) were then
constructed from the corrected data (Tables 1-4, Appendix A).
Four nuclear groups were used for the graphs: the total number
of radioactive nuclei observed; all the peripherally located
nuclei, no attempt being made to distinguish between myonuclei,
satellite cell nuclei, and endomysial nuclei closely adherent to
the fiber; all nuclei observed lying outside the limits of the
fiber; and all nuclei which appeared to be well within the limits

of the fiber.

19

FIGURE 1

Frequency polygons for the distribution of nuclei along
the length of a triceps surae muscle labeled with tri-
tiated thymidine.

The graphs in this figure (Figure l) and in Figures 2-4
represent the distributions of four groups of nuclei:
total nuclei, peripherally-located nuclei, nuclei located
outside the muscle fiber, and nuclei located inside the
muscle fiber (top to bottom, respectively). The solid
line in each graph represents the observed radioactive
nuclei. The broken line represents the calculated
theoretical frequency of radioactive nuclei, if the label
were uniformly distributed throughout the muscle. The
units of the abscissa in each graph are the successive
optical fields, 31,400p2 at 970x, viewed along the length
of the muscle to include the entire length.

 

100

 

 

 

 

 

 

 

 

80
60
to
.1
2 h 6 8 1O 12
60
3 Peripherally-leceted nuclei
0
a 40
g I ~
I: 20 I \" " \v x \ ° ‘
:g
u o 2 A 6 8 1o 12
H
o
"‘ 6O
Inclei located
‘0 outside fiber ”
20
° 2 4 6 a 10 12
14.0
Nuclei located.
20 inside fiber
‘s a ‘— \\‘ ’
O

 

 

2 4 6 ,8 1O 12
Location along the length of the muscle
Figure 1.

20

FIGURE 2

Frequency polygons for the distribution of nuclei along
the length of a tibialis anterior muscle labeled with
tritiated thymidine.

 

120

Ibtal nuclei

 

 

 

 

 

llhclei located
cuteide fiber

   

lulber of radioactive nuclei
c:

 

 

‘° luclei located
ineide fiber

 

 

 

2 4 6 8 10 12 14 16 18

Location along the length of the uuecle

Figure 2.

21

FIGURE 3

Frequency polygons for the distribution of nuclei along
the length of a triceps surae muscle labeled with tri-
tiated thymidine.

In this muscle, two lengthwise nuclear counts were made.
The four graphs at the left represent the distributions
obtained from the first count. The four graphs at the
right represent the distributions obtained from the
second count.

 

luaber of radioactive nuclei

 

 

60

b0

20

° 2 a 6 3
‘° Peripheral:-

20 nucil/ ~ \
O

 

 

 

 

‘0 luclei located
outeide fiber
20
/ \/ ’
O

luclei located

mi 1%
a ”I \‘

° 2 a 6 8

20

G

, Ibtal nuclei

 

 

 

f Peripherally—located nuclei

” ‘

 

 

luclei located
cuteide fiber

 

luclei located
inaide fiber

[W

2 h 6 T

Location along the length of the nuacle

Figure 5e

22

FIGURE 4

Frequency polygons for the distribution of nuclei along
the length of a gracilis muscle labeled with tritiated

thymidine.
In this muscle, two lengthwise nuclear counts were made.
The four graphs at the left represent the distributions

obtained from the first count. The four graphs at the
right represent the distributions obtained from the

second count.

 

Numb-r of radioactivc nucloi

100

40

20

20

Number of radioactive nuclei

20

Total nuclei

 

 

 

 

Peripherally-lccated nuclei

-e

 

 

Nuclei located
outaide fiber

 

‘0

20

 

luclei located
ineide fiber

 

'180re b.

100

80

60

‘0

20

‘0

20

60

20

‘0

20

Total nuclei

 

 

 

 

 

 

Inclei located outaide
fiber

 

clei located
ide fiber

 

Location along the length of the nuacle

23

Distributions varied between the different muscles
observed, between different areas in the same muscle, and
between the nuclear groups. The variations, however, were
inconsistent. For example, in a gracilis muscle, Figure 4,
right, the distributions of total nuclei and nuclei lying
outside the fiber resemble each other. In a triceps surae
muscle, Figure l, the peripherally located nuclei and the
total nuclei had similar distributions. In a tibialis
anterior muscle, Figure 2, the nuclei located outside the
fiber and those peripherally located had distributions re-
sembling the distribution of the total nuclear count. These
observations were to be expected, since a prominent peak in
any of the three separate groups would also be observed in
the distribution of the total number of nuclei.

In order to compare the distributions of nuclei in
different areas of the same muscle, two lengthwise counts
were done in a triceps surae muscle and a gracilis muscle
in the same manner as before. Both counts were in the body
of the muscle, but not necessarily in the center. When these
data (Tables 3 and 4, Appendix A) were plotted (Figures 3 and
4), the distributions within each muscle showed little
similarity. In both muscles many of the peaks found in the
first distribution corresponded to a depression in the dis-
tribution of the other area.

The number of peaks observed in each distribution
was inconsistent, and the general area long the length of

the muscle where the peaks were located indicated no consistent

24

growth pattern. To determine if the observed peaks and de-
pressions did, in fact, indicate real concentrations of label
in the muscle, the observed distributions were compared to
theoretical distributions, in which the labeled nuclei in each
group were evenly distributed along the length of the muscle.
To do this, the total number of labeled nuclei in each group
was divided by the total number of nuclei (labeled and un-
labeled) counted in that group. This calculation yielded the
proportion of nuclei labeled in the muscle. The proportion
was then multiplied by the number of nuclei in each group
which was observed in each field along the length of the
muscle. This calculation gave the number of labeled nuclei
that could be expected in each field, if they were evenly
distributed throughout the muscle. These data were plotted
with the observed data (Figures 1-4) to facilitate comparison.
In all cases, the observed distribution corresponded closely
to the theoretical distributions which were calculated. The
deviations that did occur were not consistent in that at some
levels the observed distribution fell above the theoretical
and at other levels it was below. The peaks and depressions
did coincide in the two distributions.

Although the graphs revealed some information about
the distribution of labeled nuclei, it was felt that the data
should be statistically tested. A'x? "goodness of fit" test
(Croxton, 1953) was employed to test the difference between
the observed and theoretical distributions. The x? values

obtained were converted to p-values to determine the

25

significance of the differences.

corresponding p-values are recorded in Table 5.

TABLE 5

The‘x? results and the

Statistical results for tritiated-thymidine labeled muscles.
1? tests were performed to determine the significance of
differences seen between the observed and theoretical dis-

tributions of nuclei in Figures 1-4.

x? and p for distribution of nuclei

 

 

 

Total Peripheral Outside Inside
Muscle nuclei nuclei nuclei nuclei
triceps x2=20.3 302=9.78 x2=12.33 x2=8.23
surae p(.02 p).30 p>.10 p>.50
tibialis x2=20.43 x2=8.58 x2=7.l7 «2:17.12
anterior p).20 p).90 p).95 p).30
triceps x2=12.39 x2=3.55 x2=6.56 «2:24.?
surae p).05 p>.70 p).30 p(.001
- first
lengthwise
count
- second x2=4.87 x2=7.79 x2=l.01 «2:4.63
lengthwise p ).50 p>.25 p>.98 p) .50
count
gracilis ‘x?=5.48 x?=2.33 x?=2.06 'x2=4.55
lengthwise
count
- second x2=9.07 x2=2.58 x2=9.45 «2:5.77
lengthwise p > .50 p ) .98 p ) .30 p ) .80

count

26

The difference between the observed and theoretical
disbributions was considered non-significant if the p-value
was greater than 0.05. Nearly all the p-values were greater
than 0.05. This means that at least 5% of the time, and usually
more often, the observed distribution was not significantly
different from the theoretical distribution in which the
labeled nuclei were distributed evenly throughout the length
of the muscle.

In addition to the nuclear counts made in a lengthwise
direction, parallel to the longitudinal axis of the muscle,
counts were made proceeding from one edge of the muscle section
to the other in a direction perpendicular to the longitudinal
axis of the muscle. Counts were done at three different
levels in one of the muscles to determine if there were any
concentration of label across the width of the muscle. Ob-
served and theoretical distributions were plotted (Figure 5)
in the same manner as the lengthwise counts from original data
after fog was subtracted (Table 6, Appendix A). Although the
observed and theoretical distributions corresponded closely,
the three levels were not comparable in number or location of
peaks. As before,'x? tests were used to determine the sig-
nificance of the differences between the observed and theoreti-
cal distributions. The results and p-values revealed non-
significance, indicating that the observed and theoretical

distributions are essentially the same.

27

FIGURE 5

Frequency polygons for the distribution of nuclei across
the width of a triceps surae muscle labeled with tritiated
thymidine.

The twelve graphs represent the distributions of the four
groups of nuclei at each of the three levels observed, and
grouped as one. The solid line in each graph represents the
observed radioactive nuclei. The broken line represents

the calculated theoretical frequency of radioactive nuclei,
if the label were uniformly distributed throughout the
muscle. The units of the abscissa are the successive fields,
31,400’12 at 970x, viewed across the width of the muscle.

 

 

number of radioactive nuclei

Figure 5.

LOV01 10

L070]. 2e

Level 5.

Grouped

Location of nuclei

 

 

 

 

Tbtel Peripheral Outside Inside
60
J
I
Inc ’ to 1+0
20 20' N20 20
/ l
o (J
0' 2 11° 2 I: 2 b 2
80
6° r
I
I
to ho no
20 20 A20 A20
I
,_____ o o
o 2 h 2

 

 

 

 

Location across the width of the muacle

28

TABLE 7

Statistical results for tritiated-thymidine labeled muscles

:x2 tests were performed to determine the significance of
differences seen between the observed and theoretical dis-

tributions of nuclei in Figure 5.

‘x2 and p for distribution of nuclei across muscle

 

 

Triceps Total Peripheral Outside Inside
surae nuclei nuclei nuclei nuclei
Level 1 x2=8.2 x2=l.53 «2:7.1944 x2=0.8590
p<.02 p).30 p(.05 p).50
Level 2 x2=l.00 x2=2.00 x2=2.8184 x2=2.0797
p).50 p).50 p).20 p).30
Level 3 x2=0.04 x2=0.38 x2=0.327l x2=1.0574
p).98 p).80 p).80 p>.50
3 levels x2=1.3 x2=1.89 x2=2.0002 oez=o.l327
grouped as l p >.50 p ).30 p').30 p>).90

29

As with the thymidine treated animals, all animals
receiving tritiated amino acids survived to the end of the
experimental procedures. Muscle autopsies were performed
and slides were prepared for radioautography. After coating,
exposure, and development, the slides were scanned for con-
centrations of silver grains over the cytoplasm of the muscle.
None of the sections observed showed pronounced accumula-
tions of silver grains in any particular area. The reaction
did seem to increase from 1 hour after injection to 1 day
after injection, but then decreased on the 6th day, and de-
creased again slightly on the 14th day after injection.

The intensity of reaction was also different depending upon
the amino acid which was injected. Histidine showed the least
uptake and leucine the greatest, with proline being inter-
mediate, according to visual estimate. In no case was there

a concentration of grains at a specific region of the muscle,
as far as could be determined by visual scanning of the
sections.

To search for evidence of subtle concentrations that
might be missed without quantitation, grain counts were per-
formed on muscles labeled with tritiated proline. The counts
were made in three areas parallel to the longitudinal axis
of the muscle, one at each periphery of the section and one
through the center of the section. The muscle used for these
observations was in all cases, the gracilis muscle.

. Frequency polygons (Figures 6-9) were constructed

from original data (Tables 8-11, Appendix B) as follows.

30

FIGURE 6

Frequency polygons for the districution of labeled pro—
tein along the length of a gracilis muscle labeled with
tritiated proline 1 hour prior to fixation.

The four graphs in this figure and in Figures 7-9, re-
present the distribution of labeled protein in muscle
labeled with tritiated proline. The first and third
graphs in each figure represent the distribution of
label at the edges of the muscle, while the second
graph represents the distribution in the center of the
muscle. The fourth graph represents the distribution
of label in the muscle in general. The counts taken
across the width of the muscle were averaged at each
level and plotted against the length of the muscle.

The solid line in each graph represents the actual count
at each point. The broken line represents the averaged
grain count if the label were evenly distributed. The
units of the abscissa are the successive areas, 55p x
11p at 970x, Viewed along the length of the muscle to
include the entire length.

 

140

 

 

 

 

 

 

 

 

 

 

 

 

Peripheral
120
100 W --
80
01
2 4 6 8 lo 12 lb 16 18 20 22 26
160
120 Central
100 _____ . 4‘£:7 \\\_!:.‘=—__-
80
N
9
I11
0
O
H
a 2 I. e a lO 12 ll. 16 la 20
3
vi
I
H
on
‘3 140
H
O
'i 120 Peripheral
z
100
P— A
80
60
0!
2 4 6 8 10 12 14 16 18 20 22 26
40
20 "" "" ""
0

 

Location along the length of the muscle

1'igure 6.

31

FIGURE 7

Frequency polygons for the distribution of labeled pro—
tein along the length of a gracilis muscle labeled with
tritiated proline 1 day prior to fixation.

 

180

Peripheral

 

 

 

180

Central
160

1.3.1 ....

120

 

 

100

 

2
c:

200

Peripheral

Nueber of graina per 605p

160

140

120

100

 

 

b0

20

 

 

Location along the length of the muscle

Piflure 7.

32

FIGURE 8

Frequency polygons for the distribution of labeled protein

along the length of a gracilis muscle labeled with tritiated
proline 6 days prior to fixation.

Nueber of graine per 605;:2

70

 

 

 

 

 

 

 

60 Peripheral
50
b — ' _ A ‘ ‘ -
.0 Y
30
J
O"
6 8 12 16 20 26 28 32 36 60 66 68 52 56 60
60
Central
5° .___ ____ .a vi . ____ 4‘
v v _ v
.. 1
30
A
0
70 6 8 12 16 20 26 28 32 36 60 66 68 52 56 60
60 Peripheral
5° 1 A
A
v v — _ —
60
30
20
0 ‘1
6 9 12 16 on 24 78 32 36 Iao 44 as 52 56 60
20
0
6 8 12 16 20 26 28 32 36 60 66 68 52 56 60

Location along the length of the muscle

figure 8.

33

FIGURE 9

Frequency polygons for the distribution of labeled protein
along the length of a gracilis muscle labeled with tritiated

proline 14 days prior to fixation.

 

 

 

A AA /\ "r1phor-l

H¢VAIOAL\/(\//la/\14 AA

ll)

.m enough

eaves! an» no conned ecu anode ecuueooq

on en an on an on «N «N c~ an on «a ad 0A a o

 

 

non can no on no a. nu as no 00 nn on nc 0e nn on n~ on nu on n

 

 

o
«ea-nan...— ow
on

C)

1232,, .

«ewe-e0 o~

on nu on nu oh no 00 nn on no 00 nn on nu ON nu OH n

ngog zed enters JO Jaqunn

Z

34

The number of silver grains observed over an area of 605p2
(55» x 11p), was plotted against the level of the muscle,
proceeding from one end of the muscle to the Opposite end.

A theoretical number of grains per 605p2, based on an assump-
tion of uniform distribution, was calculated and added to

the graph. These data were not corrected for fog, since it
would have been subtracted evenly at each point in the dis-
tribution. The distributions varied between the different
areas in the same muscle and between the different time in-
tervals observed. Numerous peaks were observed at all times,
but the number increased from 1 hour after injection of the
isotOpe to 14 days after injection. At the earliest time in-
terval, there was a great variability in grain count about the
mean. The mean grain counts/605}:2 at the four time intervals
studied were approximately 95 at 1 hour, 135 at 1 day, 45 at
6 days, and 10 at 14 days after injection of the labeled
amino acid.

NO consistent peaks and depressions were Observed
in the three lengthwise counts at any one time interval or
in the four successive time intervals. However, as mentioned
before, at 6 days and 14 days after injection, the intensity
of reaction had decreased and was less variable about the
mean.

1? "goodness of fit" tests were done to determine
the significance of the difference observed between the actual
counts per area at each level, and the mean count per area.
The p-values indicated significant differences in some cases,

but non—significance in others.

35

As an additional observation, counts were done at
regular intervals along the length of the muscle, perpendi-
cular to the longitudinal axis of the muscle. These counts
were averaged to yield an average grain count per 121p2 at
intervals of 55p, except in the muscle observed 14 days after
injection where the interval was 165p. These data (Tables
8-11, Appendix B) were also tested by these2 test in a
lengthwise direction from one end of the muscle to the other.
In this case, the difference was non-significant at all time

intervals.

DISCUSSION

Growth centers (Kitiyakara and Angevine, 1963a)
have been proposed as a factor influencing the early post-
natal growth of skeletal muscle in the mouse. Although
muscles grow in diameter as well as in length, the growth
center was prOposed only in relation to longitudinal growth.
Kitiyakara and Angevine (1963a) studied the gracilis muscle
in the mouse and proposed that growth in skeletal muscles
takes place only in certain zones located near the muscle-
tendon junction at each end of the muscle. They applied
sutures and India ink marks to the muscles of mice, 1-10
days of age and observed the movement of the markers during
the animal's growth. They observed that the markers moved
progressively away from the end of the muscle but not any
nearer the midpoint. In a second experiment, they injected
tritiated thymidine into one-day-old mice and studied the
areas of nuclear labeling in relation to the length of the
muscle at different time intervals. They reported a concen-
tration of label at each end of the muscle at early time
intervals. The peaks were later reported at locations farther
from the ends but maintaining the original interval between
them. From these deductions, they proposed that growth, as

interpreted from the movement of the markers and uptake of

36

37

the labeled thymidine by the nuclei, takes place only in
sub-terminal zones near the muscle-tendon junction.

Marchok and Herrmann (1967) studied muscle develop-
ment in chick embryos and examined the uptake of tritiated
thymidine by muscle nuclei. "No significant differences in
LI [labeling index] were found between terminal and central
areas of the whole muscle."

In the work presented here, no growth center was
observed. Frequency polygons (Figures 1-4) for the distri-
bution of four groups of thymidine labeled nuclei, revealed
no consistent concentrations in the muscles examined. In fact,
the distributions of labeled nuclei closely resembled the
distributions one would expect if the thymidine had been
utilized by nuclei evenly throughout the length of the muscle.
When the data were tested statistically, the same conclusion
was reached. No significant difference was found between the
observed and theoretical distribution of the labeled nuclei.

When Kitiyakara and Angevine proposed that muscle
grows only in sub-terminal zones, several factors were not
considered. First, they failed to consider the different
types of nuclei which were counted in their study. When
counting muscle nuclei with a light microsc0pe, it is not
possible to distinguish between the actual myonuclei, the
satellite cell nuclei, and the endomysial connective tissue
nuclei. The myonuclei appear at the edge of the muscle
fiber, closely applied to the plasma membrane. The satellite

cell, first observed by Mauro (1961) in the frog tibialis

38

anticus muscle, is a cell which is found between the plasma
membrane and the basement membrane of the muscle fiber. The
satellite cell has very little cytoplasm and therefore appears
in the light microscope as a peripherally located nucleus,
indistinguishable from the myonuclei. A third nuclear type
which can be confused with the myonuclei, is the endomysial
connective tissue nucleus. Each muscle fiber is closely
invested with a very thin sheet of connective tissue, the
endomysium. The nuclei of fibroblasts, which are present

in this sheath are also indistinguishable from the myonuclei.
Although Kitiyakara and Angevine disregarded any nuclei whose
location was questionable, they failed to mention the fact
that their counts did include nuclei other than actual myo-
nuclei.

In addition, no statistical tests were employed to
determine the significance of the concentrations of label
which were found. In the present study, peaks of concentra-
tion were found, but when statistically tested, were shown to
be not significantly different from the theoretical distribu-
tion. Therefore, had Kitiyakara and Angevine tested the
distribution of the labeled nuclei, they may or may not have
found a significant difference. In either case, the results
would have been more complete and valid.

A third factor which was not considered in Kitiyakara
and Angevine's study, was the type of section used for ob-
servation. Since the counts were done on cross sections of

muscles at intervals of 30-80p, it is possible that additional

39

counts within the intervals would have evened out the con-
centrations of label, and consequently would have revealed no
growth center.

In this work, these factors were taken into con-
sideration. Rather than disregarding nuclei with questionable
location, all nuclei were counted, but were tabulated according
to location with respect to the sarcolemma of the muscle
fiber. In this way, it was possible to study the behavior
of all the nuclear types involved. Although it was still not
possible to separate myonuclei from satellite cell nuclei or
endomyseal nuclei, we are aware of their existence in the
counts.

The need for statistical evidence has already been
discussed. Only by a statistical test can one accurately
determine the significance of the data. The'xz "goodness of
fit" test used in this paper revealed that even though peaks
in the number of labeled nuclei were found, these peaks were
to be expected even if the labeled nuclei had been evenly dis-
tributed. Therefore, in order to document data accurately,
statistical evaluation must be included.

In the present study, longitudinal sections rather
than cross sections were used for the histometric analysis.
This made it possible to Observe a strip of fibers and their
nuclei from one end of the muscle to the opposite end with
no intervals along the length of the muscle. This may be one

of the reasons no growth center was found in this study.

40

In the other paper (Marchok and Herrmann, 1967)
reporting on the distribution of labeled nuclei in muscle, the
observation was a side issue and offered no quantitative data,
nor provided support for the presence of a growth center.

Recently, studies have shown that myonuclei do not
proliferate after birth. If this is true, and evidence
indicates this, Kitiyakara and Angevine and the authors of
this paper were not labeling myonuclei directly. It has been
shown in adult animals that of the nuclei located within the
basement membrane of the muscle fiber, only the satellite
cells undergo mitosis (Shafiq, Gorycki, and Mauro, 1968).
More recently, Moss and Leblond (1970) observed that during
the first 10 hours after labeling, only satellite cells had
utilized the tritiated thymidine. However, at later periods
of time, labeled myonuclei were also observed. If this is,
in fact, the method used by myonuclei for proliferation, the
Kitiyakara and Angevine study and the present study are not
documenting the proliferation of myonuclei at all, but the
distribution of myonuclei derived from the satellite cells which
were preparing for mitosis at the time of injection of the
label. Since satellite cells are distributed evenly along
the length of the fiber (Muir, Kanji and Allbrook, 1965), the
formation of new myonuclei from satellite cells should also
be evenly distributed along the length of the fiber. The
results presented here support this statement since the dis-
tribution of labeled nuclei observed was not significantly
different from the distribution expected if the nuclei were

evenly distributed.

41

Since no significant concentrations of labeled nuclei
were observed in this study, and because of deficiencies noted
in the growth center study by Kitiyakara and Angevine (1963a),
it is reasonable to propose that additional muscle nuclei are
incorporated into the muscle fiber evenly throughout its
length during postnatal growth. Other reports on mitosis and
satellite cells also lend evidence in support of this proposal.

Studies of growth in tissues should not be limited
to one cellular structure, e.g., the nucleus. Therefore, in
an attempt to gain information as to the role of muscle
cytOplasm in the growth of the muscle as a whole, the uptake
and dispersion of radioactive amino acids were studied.

Three amino acids, proline, leucine, and histidine, were
chosen on the basis of their presence in muscle protein.
Although at present quantitative data have been taken only
from the proline treated muscle, it is felt this provides a
preliminary look at the areas of protein synthesis and the
subsequent dispersion of the newly-formed protein. If growth
centers do exist in muscle, it would be reasonable to assume
that not only nuclei but other cell components, such as pro-
tein, are involved. If the apprOpriate radioactive label
were applied, one would expect to see major concentrations
of labeled cytoplasm in those areas synthesizing protein at
the time of labeling. At later time intervals, depending on
the dispersion of the newly-formed protein, it would be
possible to document the growth of the muscle due to the in-

crease in muscle protein.

42

In the present study, no consistent concentrations
were found at any of the intervals examined. The frequency
polygons (Figures 6-9) reveal several areas of high and low
concentrations of label, but since these concentrations are
inconsistent from one area of the muscle to another and from
one time interval to the next, it does not seem reasonable
to call them growth centers or centers of protein synthesis.
Since at later time intervals the peaks have increased in
number and have appeared to decrease in intentisy, it seems
likely that newly-formed protein is being dispersed rather
evenly throughout the length of the muscle fiber. This is
also indicated by the transverse counts, since when these
counts were averaged to give one value for each level, the
variability about the mean decreased considerably. Therefore,
it is believed that although areas of high labeling can be
found, these represent very localized areas of protein
synthesis. When taken as a whole, the muscle reveals no
pronounced center of protein synthesis or accumulation.
Farrell and Fedde (1969) report uniformity in fiber diameter
and some enzyme activities throughout the length of the muscle
fiber, which might be expected if protein synthesis and
accumulation were so distributed.

The distributions of labeled protein were tested
statistically by means of the‘x? test. The differences were
significant in some cases, and non-significant in others.
The «9 tests done on the transverse counts revealed no

significance. These results were interpreted to indicate

43

localized sites of growth in the individual areas counted,
but no such area of growth in the muscle as a whole.

In light of these results, no center of growth is
proposed for muscle protein. Although areas of high degrees
of labeling were found, due to their inconsistency throughout
the muscle, they can be interpreted only as localized areas of
growth and not as part of a major growth center for the muscle
as a whole. The observed distribution of labeled protein
indicates an evenly distributed uptake and dispersion of the
label.

No growth center was indicated in the distribution
of labeled nuclei or labeled protein during the first 15 days
of postnatal growth in the skeletal muscle of mice. All
apparent concentrations in the number of labeled nuclei along
the length of the muscle were found to be only random
deviations of a proposed theoretical distribution in which all
the labeled nuclei were evenly distributed throughout the
muscle. Therefore, no growth center could be proposed due
to proliferation of nuclei. Likewise, no growth center was
proposed for protein growth, since only inconsistent localized
grOWth sites were observed.

The proposal of a specialized area in the muscle as
a whole for the proliferation of nuclei or synthesis of
protein does not appear reasonable at this time. The results
presented here indicate that both nuclear proliferation and
protein synthesis and dispersion take place evenly along the

length of the muscle.

CONCLUS ION

By utilizing radioisotopically labeled compounds,
postnatal growth in skeletal muscles of mice was investigated.
Nuclear proliferation was observed in animals treated with
tritium-labeled thymidine. Tritiated amino acids, proline,
leucine and histidine, were used to document areas of protein
synthesis and the subsequent dispersion of the newly-formed
protein.

Animals were injected with the labeled compounds
when they reached the age of 24 hours. They were killed at
various time intervals up to 14 days after injection of the
label. Muscles from the hind limbs were removed and prepared
for radioautography. After exposure and development, the
radioautographs were examined for concentrations of silver
grains. Counts were made and the distributions of label
were plotted. Although peaks were observed in the distribu-
tions of labeled nuclei, they were found to be expected if
the label had been utilized uniformly throughout the muscle.
Statistical analysis revealed no significant difference
between the observed and theoretical distributions. Several
peaks were also seen in the muscles labeled with tritiated
amino acids. However, these peaks increased in number and
decreased in intensity with time, indicating an even dispersal

44

45

of the label. Since the distributions showed variability within
the same muscle, it appears that the peaks in labeling re-
present localized areas of growth, and not a major growth

zone for the muscle as a whole. Therefore, there appears to

be no specialized areas in muscles where growth takes place,

as shown by the distributions of labeled nuclei and cytoplasm

in the skeletal muscles of mice.

LITERATURE CITED

Bailey, N.T.J. 1959. Statistical Methods in Biology. John
Wiley and Sons, Inc. New York. Pp. 6-20.

Bintliff, S. and B.E. Walker. 1960. Radioautographic Study
of Skeletal Muscle Regeneration. Am. J. Anat. 106:
233—245.

Bischoff, R. and H. Holtzer. 1969. Mitosis and the Processes
of Differentiation of Myogenic Cells in Vitro. J.
Cell Biol. 41:188-200.

Bloom, W. and D.W. Fawcett. 1968. A Textbook of Histology.
9th Edition. W.B. Saunders Co., Philadelphia, London,
Toronto. Pp. 61, 270-285 and 298.

Boyd, J.D. 1960. Development of Striated Muscle. In:
Structure and Function of Muscle. Vol. 1. G.H.
Bourne (Ed.) Academic Press. New York and London.
Pp. 63-85.

Chiakulas, J.J. and J.E. Pauly. 1965. A Study of Postnatal
Growth of Skeletal Muscle in the Rat. Anat. Record.
152:55-62.

Coleman, J.R. and A.W. Coleman. 1968. Muscle Differentiation
and Macromolecular Synthesis. J. Cell Physiol. 72:
(Suppl 1.) 19-34.

Croxton, F.E. 1953. Elementary statistics with Applications
in Medicine and the Biological Sciences. Dover
publications, Inc., New York. Pp. 180-206 and 267-
283.

Dessouky, D.A. and R.G. Hibbs. 1965. An Electron Microscope
Study of the Development of the Somatic Muscle of the
Chick Embryo. Am. J. Anat. 116:523-566.

.Enesco, ML and D. Puddy. 1964. Increase in the Number of
Nuclei and Weight in Skeletal Muscle of Rats of
Various Ages. Am. J. Anat. 114:235-244.

lFarrell, P.R. and M.R. Tedde. 1969. Uniformity of Structural
Characteristics Throughout the Length of Skeletal
Muscle Fibers. Anat. Record. 164:219-230.

46

47

Fennichel, G.M. 1965. The Development of Human Skeletal
Muscle. Develop. Med. Child Nuerol. 7:69-72.

Goldspink, G. 1968. Sarcomere Length During Postnatal
Growth of Mammalian Muscle Fibres. J. Cell Sci.
3:539-548.

Gridley, M.F. 1960. Manual of Histologic and Special Staining
Technics. Second Edition. Histopathology Laboratory
of the Armed Forces Institute of Pathology (ed.)

The Blakiston Division, McGraw-Hill Book Company, Inc.
New York, Toronto, London. Pp. 132-133 and 142.

Hay, E.D. 1963. The Fine Structure of Differentiating
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Acta. Anat. 72:38-49.

48

Marchok, A.C. and H. Herrmann. 1967. Studies of Muscle
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J. Biophys. Biochem. Cytol. 9:493-495.

 

Moss, F.P. 1968. The Relationship between the Dimensions
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Exp. Morphol. 11:353-367.

APPENDIX A

 

APPENDIX A. Original data collected on muscles labeled with
tritiated thymidine

Tables 1-4 contain original data obtained from the
thymidine-labeled muscles. The data were collected by counting
the nuclei in consecutive fields, 31,400}12 at 970x, along the
length of the muscle. Nuclei were separated on the basis of
their location in relation to the muscle fiber. Fog was
subtracted, and the corrected data were used in preparation
of Figures 1-4.

The first column in each table contains the total
number of nuclei, labeled and unlabeled, counted in each field
along the length of the muscle. The next group of columns
contain the number of nuclei overlaid by l or 2 silver grains
before and after subtraction of fog. The proportion of nuclei
'with l or 2 silver grains that could be considered labeled
due to fog was computed by the Poisson distribution. With a
Inean fog count of 0.3 grains per nucleus, 22% of the total
‘would have 1 grain per nucleus which, in most cases, included
all nuclei in that category. 3% of the total would have 2
grains per nucleus due to fog. These proportions were then
subtracted from the observed number of nuclei to yield the
Innmber of labeled nuclei in each category. Nuclei overlaid

Iby 3 or 4 grains were not corrected for fog, since the proportion

49

 

 

50

expected in each class was very small, 0.3% and 0.02%
respectively. The next two columns contain the total number
of observed labeled nuclei in each field before and after
subtraction of fog. The last column contains the number of
labeled nuclei which could be expected in each field if the
label were uniformly distributed throughout the length of the

muscle.

 

51

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.OHO>HHoommmH tmucsoo Ozoomm Ono HmHHH may OH HOHOH SOHHOH

£0H£3 mumu Ho mHom 03H map .OHOHOHmnp One .OOME OHO3 munsoo OmH3SHOGOH 03H .OHOOSE mHsu OH

OGHOHEOSH OOHOHHHMH SHHB OOHOQMH OHOmDE mHHHowHO m :0 OOHOOHHOO OHOU HmcHOHHo

O WHmdB

66

 

 

 

O0.0H O0.0H OH O0.0 5 O NN
N5.00 50.HO NO 50.0 OH N OO
OO.NO OO.5O OO OO.N O N OO
OO.NO N0.00 NO OO.5 O OO.5 OH HO
N5.00 O0.00 OO O0.0H HH O O5
N5.00 OO.NO NO OO.5 O ON.N HH O5
N0.00 O0.00 OO NO.HH NH O0.0 OH OO
O0.00 O0.00 OO OO.HH NH OH.N O 5O
O0.00 OH.OO OO O0.0 5 O0.0 OH HO
O0.0N O0.0N OO NO.H N OO.H 5 NO
OOH OOH OOH OOH OOHHOOHHOO OOHHOOHHOO
HOHHO OHOHom HOHH4 OHOHom OOH HOHHH OOH ononm
OHOHH
mom HOHO
Ids OOHOQOH UHOHH Mom HOHOSC OHOHH mom

HMOHHOMOOSB OOHOQOH UO>HOOQO msHmHO N £HH3 HOHODZ GHOHO H anz HOHODZ HOHOSG HMHOB

 

HOHOSG OOHOOOHIOHHOHOQQHHOH

A.O.Lcoov O OHOOH

67

 

 

 

NO.5H O5.0N HN O5.0 O N ON
O0.0H NO.NH OH NO.N O H ON
O0.0H H0.0H 5H H0.0 H H HN
O0.0N OO.5N NO OO.N O O0.0 O OO
O0.00 OH.OO OO N0.0H HH OO.H O OO
50.0N 50.0N OO O0.0 O NO.N 5 OO
O0.00 N0.0N ON N0.0 O O NO
OO.HN OH.NN ON O5.H N O0.0 O OO
H5.0H O5.HN NN O5.0 O O 5N
O0.00 50.00 5O 50.0 O O OO
OOH OOH OOH OOH GOHHOOHHOO COHHOOHHOO
HOHH< OHOHom HOHHO ouoHom OOH HOHHO OOH OHOHom
OHOHH
mom HOHO
In: OOHOQOH OHOHH Mom HOHOOn OHOHH Hon

HOOHHOHOOQB OOHOQOH Om>nomno mchHO N SHHz HmHosz QHOHO H £HH3 HOHOSZ HOHOOG HMHOB

 

HOQHH OHOODE may OOHmuso OOHOOOH HOHOSZ

A.O.usoov O mamas

68

 

 

 

O5.0 OO.H N OO.H N O O
O5.0 OO.N N O O O
O0.0 OH.O OH 50.N O NO.N O OH
OO.5 H0.0 HH O0.0 H OO.N O 5H
50.0 ON.OH OH H0.0 O O0.0 O HN
O0.0H OO.5 O O0.0 H H NN
5H.O O0.0 O O OO.N O HH
O0.0 O0.0 O O O0.0 N NH
O0.0H OO.NN ON O0.0 O ON.O O OO
O5.HH 55.0H 5H 55.0 O OO.5 OH ON
OOH OOH OOH OOH soHHOOHHOO GOHHOOHHOU
HOHHO OHOHom MOHH4 OHOHmm OOH HOHHH OOH OHOHmm
OHOHH
mom HOHO
Isa UOHOQOH UHOHH mom HOHOSC UHOHH Hmm

HOOHHOHOmne OOHOQOH Oo>nmmno mnHmuO N QHH3 HOHODZ GHOHO H £HH3 HOHODZ HOHOSG HMHOB

 

HOQHH OHOmDE on» OOHmcH UOHMOOH HOHODZ

A.O.Hcoov O mqmde

69

 

 

 

O0.00 O0.00 OO O0.0 OH O OO
ON.OO N0.00 OO ON.5 O OH.OH ON NO
OO.NO O0.00 OO HN.HH NH O0.0 OH OO
O0.00 OO.NO NO O0.0 5 NH.O NH O5
OO.5O O0.05 O5 O0.0H OH OH OHH
O0.05 O0.00 NO OO.NN ON HH OOH
ON.O5 ON.NO OO OO.NN ON OO.N 5H NNH
ON.HO OO.HO HHH O0.0H OH O5.0 ON NOH
O0.00 50.00 HOH O5.HN ON NO.N OH OOH
O0.00 OO.5O OHH OO.HN ON ON.H ON OOH
O0.00 50.00 OO O5.0H HN N0.0 ON OOH
O0.00 ON.OO 5O O0.0H HH O0.0 OH OO
OOH OOH OOH OOH OOHHOOHHOO COHHOOHHOO
HOHHO OHOHOm HOHHH OHOHom OOH HOHHH OOH OHOHOm
OHOHH
mom HOHO
Inc OOHOQOH UHOHH mom HOHOSO OHOHH mom

HOOHHOHomsa OOHOQmH Oo>nomno mchnO N anB HOHODZ GHOHO H £HH3 HOHOOZ HOHODO HOHOB

 

HOHOOG Hmuoa

A.U.HCOUV O MHmNB

70

 

 

 

OO.HN O0.0N HN O0.0 O O OO
OO.5N 50.5N OO O0.0 O O0.0 OH OO
OO.HO OH.HO OO NO.5 O O0.0 5 OO
OO.HN O0.0H HN O0.0 O O0.0 O OO
ON.5O O0.00 OO OO.5 O 5 NO
O0.00 ON.OO HO O0.0H HH O0.0 O OO
O0.00 OO.5O OO OO.NH OH O OO
OO.NO OO.NO NO O0.0 OH O0.0 OH H5
OO.HO ON.OO OO ON.OH OH OH OO
O0.00 H0.00 OO HO.5 O O OO
O0.00 O0.00 OO O0.0H OH ON.O OH O5
ON.OH N0.0H NN O5.N O O5.N O 5N
OOH OOH OOH OOH OOHHOOHHOO OOHHOOHHOO
HOHHm onoHom HOHHN OHOHom OOH HOHHO OOH OHOHOm
OHOHH
mom HOHO
1:: OOHOQOH OHOHH mom HOHODG OHOHH mom

HOOHHOHOOBB OOHOQOH Om>uomno mchHO N £HH3 HOHOOZ :HonO H OHH3 HOHosz HOHOOG HOHOB

 

HOHOOQ OOHMOOHIOHHOHOSQHHOO

A.O.usoov O mqmme

71

 

 

 

NO.NN N5.0N 5N N5.0 O N HO
ON.5H 50.5 O O5.0 O NH.O O ON
O0.0H OO.HH NH OO.N O N OH
ON.NH OO.HH NH O0.0 H H 5H
O0.0N OO.NO OO O0.0 O N OO
O0.00 H0.00 OO HO.5 O H OO
N0.0N OO.NN 5N O0.0 O O0.0 O OO
OO.5O O0.00 HO O0.0 O O NO
O5.0N O5.0N ON O5.0 5 OO.N O OO
O0.00 O0.00 OO OO.HH NH O OO
ON.OO O0.0N NO N0.0 O O0.0 O NO
OO.5N OH.OO OO O0.0 O OO.H O OO
OOH OOH OOH OOH GOHHOOHHOO GOHHOOHHOO
HOHHO OHOHOm HOHHO OHOHom OOH HOHHH OOH OHOHOm
OHOHH
mom HOHO
Is: OOHOQOH OHOHH mom HOHOOG OHOHH mom

HOOHHOHOOSB OOHOQMH OO>MOOQO msHmHO N £HH3 HmHosz QHMHO H nqu HOHOSZ HOHODG Hmuoa

 

HOQHH mHomsE onu OOHOHOO OOHOOOH HOHOOZ

A.O.ucoov O memes

72

 

 

 

OO.H OO.H H O O O
O0.0 O0.0 O O O0.0 N OH
O0.0 O0.0 O OO.H H O0.0 O OH
OO.5 N0.0 O NO.H N O0.0 O ON
O0.0 O0.0 5 OO.H N H OH
OH.O 55.HH NH 55.0 O N ON
OH.O 55.0 O 55.0 O O ON
O0.0H OH.OH OH O5.0 H OO.H O OO
OO.5 ON.O O H0.0 H O0.0 O HN
O0.0 OO.HH OH O5.H N ON.O O ON
O5.0 O0.0 O O OO.H O ON
O5.H OO.H N O OO.H N O
OOH OOH OOH OOH GOHHOOHHOO COHHOOHHOO
HOHHO mHoHom HOHH< OHOHmm OOH HOHHO OOH OHOHmm
OHOHH
mom HOHO
In: OOHOQMH OHOHH Hmm HOHODO OHOHH mom

HOOHHmHomne OOHOQOH Oo>nwmno mchuO N HHHB HOHOSZ GHOHO H SHH3 HOHODZ HOHUSG HOHOB

 

HmnHH OHomsfi OSH OOHmsH OOHOOOH HOHOOZ

A.O.H:oov O manna

73

 

 

 

 

O0.0N ON.OO HO O0.0H OH ON.O HN 5O
N5.5N ON.ON OO HH.HH OH OH.H OH OO
N5.5N HH.5N ON HH.O HH OH OO
OO.5H OO.5H OH O0.0 O 5 OO
HoHosn HouoanH m.: H Ho>oH
O0.00 OH.55 OHH N0.0N OO OH.5 OO OOH
HO.5O O5.00 O5 55.HN ON OO.N OO HOH
O0.00 O0.00 OO O0.0H ON ON OOH
55.00 O0.00 OO OO.HH OH OH 5O
OOH OOH OOH OOH noHHooHHOO nOHHooHHOo
HoHHn oHOHom HoHHn oHoHom OOH HoHHN OOH oHOHom
UHoHH
mom HoHo
Ian OoHoan OHoHH mom HoHosn OHoHH Hon
HOOHHoHOonB OoHonoH nobuomno mnHouO N nHHB HOHUDZ nHouO H nHH3 HoHosz HoHosn HOHOB

 

HoHosn HMHOH I H Ho>oH

.nonHoOOH mHo>oH O onH OGHQDOHO HO HHOmoH onH mH MHon HO How HmMH one

.OoHnnoo oHoB HonH
oHOOSE onH Ho mHo>oH O onH OH HoHoH BOHHOH HonH OHoO Ho mHom O HoHHH one

.O oHsOHm HO

noHHoummoum onH nH now: onoz OHOO OoHooHMOO onH Ono .noHooansm mmB OOH .oHoHon mo mmsouO
.mHo>oH oounH Ho oHOmsE onH HO nHOHB onH mmouom .xO5O Ho
NROOO.HO .mOHoHH o>HHsoomnoo nH HoHODn onH OnHHnsoo On OoHooHHoo ono3 OHOO OanoHHOH one

OHnH noHoHomom ouoz HoHosn one

onHOHEOnH OoHOHHHHH nHHB OoHoHOH oHomsfi ownsm mmoOHHH o no OoHooHHOo OHOO HonHOHHO

O mqmflfi

74

 

 

 

 

OO.HH O5.NH ON 55.0 N O0.0 OH HO
O5.0H ON.NH ON 50.0 O O5.H OH HO
H0.0 OH.OH NH OH.5 O O ON
OO.5 ON.5 OH O ON.H 5 ON
HoHosn oOHmnH u H Ho>oH
HN.OO ON.OO HO ON.OH OH O 5O
O5.0H O0.0 OH NN.O O O0.0 O ON
O5.NN H5.0N ON H5.N O O OO
O0.0H 50.0H OH 50.0 O O HO
OOH OOH OOH OOH nOHHooHHOO noHHooHHOO
HoHHm oHOHom uoHHn onomom OOH HoHHn OOH oHOHom
OHoHH
mom HoHo
ID: OoHonOH OHoHH Mom HoHosn OHoHH Mom
HMOHHoHoonB OoHonoH Oo>nomno onHoHO N nHH3 HoHosz GHOHO H nHH3 HoHosz HoHosn HOHOB

 

HoHosn oOHmHOO I H Ho>oH

A.O.H:oov O memes

75

 

 

 

 

 

 

 

O0.0H O0.0H OH O0.0 O N NO
O5.0H O0.0H HN OO.HH OH N OO
OH.O O0.0 5 O0.0 O N OH
O0.0 ON.O NH O0.0 O NO.H O OH
HoHosn oOHmnH I N Ho>oH
O0.0 O0.0 5 O0.0 O O OH
5N.HO HO.5O OO H0.0H NH O OO
O0.0 N0.0 5 N0.0 O N OH
N0.0H OH.OH OH OH.O 5 O ON
HoHosn oOHmHso I N Ho>oH
O0.0N O0.0N OO O0.0H NH OH OO
O0.0N O5.0N OO 5H.NH OH OO.H OH HO
OH.OH H0.0H OH HO.5 O O OO
ON.ON O0.0N OO O0.0 O N0.0 OH OO
HoHosn HouoanHomII N Ho>oH
N5.00 O0.00 OO O0.0N ON OH OHH
OO.N5 OO.55 NO O0.00 OO ON OOH
O0.00 O0.00 OO O0.0H 5H O N5
O0.00 ON.OO 5O 5N.OH OH O0.0 HN HO
OOH OOH OOH OOH nOHHoonnoo noHHoounoo
uoHHm oHoHom HoHHn oHoHom OOH uoHHd OOH oHoHom
OHoHH
mom HoHo

Inn OoHonOH OHoHH mom HoHosn
HOOHHonoonB OoHoan Oo>nomno mnHonO N nHH3 HoHosz

OHmHH ewe
nHmnO H nHHz HoHosz HoHosn HMHOB

 

HOHUDG HMD.O# I N HO>OOH

A.O.Hsoov O manna

76

 

 

 

 

 

 

NH.O N0.0 O NO.N O N OH

O0.0 OH.O O OH.O O O 5N

NH.O O0.0 OH NO.N O OO.H O OH

NH.O N0.0 5 NO.H N O OH
HoHosn oOHmnH I O Ho>oH

O5.NH O0.0H ON NN.O O O0.0 O ON

OO.NO N0.00 OO N0.0H OH OH OO

OO.NO N0.00 OO N0.0 HH OH OO

OO.5H N0.0H 5H N0.0 O 5 OO
HoHosn oOHmHDO I O Ho>oH

ON.HN OO.NN NO OO.HH OH ON.H OH OO

H0.0N O0.0N ON OO.NH OH OH 5O

50.0N O0.0N ON OO.5 O 5 OO

50.0N ON.NN NO O0.0 O NO.H OH OO
HoHosn HononMHHom I O Ho>oH

O0.00 OO.5O OO O0.0H HN O0.0 OH NO
HN.5O H5.00 H5 H5.0N OO ON OOH
N0.00 N0.00 OO N0.0H ON 5N OOH

55.NO 5N.OO OO 5N.OH OH ON HO

OOH IOOH OOH OOH nOHHooHHOO nOHHooHHOO
HoHHd oHOHom HoHHn oHOHom OOH HoHHn OOH oHoHom
OHoHH
Mom HoHo

Inn OoHoan OHoHH mom HoHosn OHoHH mom

HOOHHoHoonB OoHonOH no>uomno mnHouO N nHHB HOHODZ nHouO H nHH3 HoHosz HoHonn HOHOB

 

HoHosn HMHOH I O Ho>OH

A.O.Hnoov O mqmne

77

OH.HO
O0.00
OO.NN
OO.HN

OO.HO
O0.05
ON.OO
O0.00

O0.00
50.00
OHJOO
HO.5O

OO.HOH
O0.00H
O0.00H
O0.0HH

 

H0.00
O0.00
OO.NN
O0.0N

O0.00
O0.05
ON.OO
OH.OO

O0.00
OO.H5
O0.00
Oo.OO

O0.00H
O0.00H
H5.NOH
H0.0NH

OO
OHH
OO
HO

 

HoHosn oOHmnH I mHo>OH Oomdouw

OO
OOH
ONH
OO

 

HoHosn onHdeo I mHo>oH nomaonw

N5H
H5H
OOH
ONH

 

HoHODn HmuoanHomII mHo>oH OoQSOHO

OOO
OOO
OOO
O5N

 

 

OHoHH mom HOHODn
OoHonoH HOOHHoHoonB

moH umuHO I OHmHH ewe
HoHosn UoHoHMH no>Homno

OHOHH mom HOHODG HOHOB

 

A.O.Hcoov O mqmne

HoHosn HOHOH I mHo>oH OomsOHu

APPENDIX B

APPENDIX B. Original data collected on muscles labeled with
tritiated proline

Tables 8-11 contain original data collected on gracilis
muscles labeled with tritiated proline. The data were collected
by counting the silver grains in consecutive areas, 55p x 11p
at 970x, along the length of the muscle. Three counts were
made, one at each edge of the muscle section, and one through
the center. A fourth count was made across the width of the
muscle at intervals of 55p. The data were then used for the

preparation of Figures 6-9 respectively.

78

 

79

O5.ON oOmHo>n ON.OO oOono>< 55.00 oOono>< OO.OOH oOmno>n

 

 

 

 

 

 

 

 

OO.5N ON

O0.0H ON O5 ON

OO.5H ON 5O ON OOH ON
OO.HN ON OO ON OHH ON
O0.0H NN OO NN 5OH NN
50.0H HN OO HN OHH HN
O0.0H ON OO ON OO ON ONH ON
OO.HN OH NO OH OO OH OO OH
O0.0H OH OO OH OO OH O5 OH
H5.0N 5H O5 5H OOH 5H HOH 5H
50.0N OH OOH OH OH OOH OH
OO.NN OH HO OH OH 5NH OH
HH.NN OH O5 OH OOH OH OHH OH
O0.0N OH NO OH OO OH OOH OH
O0.0N NH OHH NH OO NH HNH NH
HN.ON HH OO HH OO HH OO HH
HH.OH OH OO OH 5O OH OO OH
O0.0N O HO O OO O OOH O
NN.ON O ONH O 5O O 5OH O
H5.HN 5 OO 5 ONH 5 ONH 5
OO.HN O NO O OO O ONH O
OO.NN O 5O O O5 O O5 O
O0.0N O OO O OOH O OOH O
OO.5H O O5 O OOH O OO O
O0.0H N 5O N HOH N OOH N
OH.OH H OO H NO H OOH H

NDHNH Mom NSOOO Hon NDOOO Mom NDOOO Mom
Hnsoo nHonw Hnsoo nHmHo Hnsoo nHonw Hnsoo nHouu

Houonoo noun Houoannom noun HmanoO noun HouoanHom noun

GOHHoxHH OH HOHHQ Hson H onHHOHm OoHOHHHHH
nHH3 OoHoan oHomsa mHHHoonO o no OoHooHHOO OHMO HmnHOHHo

m WHm<B

80

 

 

 

 

 

 

 

 

0.0N ON OHH ON ONH ON OOH ON
H.ON ON OHH ON ONH ON O5H ON
H.ON ON OOH ON OOH ON OOH ON
0.0N NN ONH NN OOH NN 5OH NN
0.0N HN OHH HN OOH HN OOH HN
0.0N ON OOH ON ONH ON OOH ON
O.5N OH NNH OH OOH OH ONH OH
H.ON OH OHH OH ONH OH OOH OH
0.0N 5H HOH 5H 5H OOH 5H
H.ON OH NHH OH OH OOH OH
5.0N OH OO OH OOH OH OOH OH
0.0N OH HNH OH OOH OH OOH OH
O.HN OH OOH OH OOH OH OOH OH
0.0N NH OO NH OOH NH ONH NH
O.5N HH 5NH HH OOH HH NOH HH
N.ON OH OOH OH OOH OH OOH OH
N.ON O HOH O OHH O ONH O
0.0N O 5HH O HOH O NOH O
N.HO 5 NOH 5 OOH 5 5OH 5
O.5N O 5OH O OOH O OOH O
0.00 O OOH O O 5NH O
0.0N O OOH O OOH O ONH O
0.0N O OOH O 5OH O ONH O
0.0N N OOH N 5OH N OOH N
5.HO H O5H H HOH H HOH H
NnHNH Hon NnOOO Hon NnOOO Hon NnOOO Hon
Hnnoo nHmno Hnnoo nHmno Hnnoo nHonw Hnnoo nHoHO
Houonow noun Houoannom monn HmanoO noun Hmnoannom noun

nOHmeHH OH HOHHQ Hon H onHHOHm OonHHHHH
nHHB noHonoH oHomnE mHHHooHO o no OoHooHHoo MHOO HonHOHHO

m mHmdB

81

 

 

 

 

 

 

 

 

H.5N oOmHo>< O.ONH oOouo>< O.OOH oOouo>n 5.OOH oOouo>n
O.HN OO OO OO OHH OO
0.0N NO 5HH NO NOH NO OOH NO
0.0N HO OO HO OOH HO OOH HO
5.0N OO OO OO ONH OO HNH OO
0.0N ON ONH ON 5OH ON OOH ON
0.0N ON HOH ON OOH ON HOH ON
N.ON 5N OOH 5N OOH 5N ONH 5N
0.0N ON NHH ON OOH ON NOH ON
NnHNH Hon NnOOO non NnOOO Mom NnOOO mom
Hnnoo nHonw Hnnoo nHOHw Hnnoo nHonO Hnnoo nHoHO
Hmuonow noun Hmuonmwwom noun HmanoU noun HonoanHom noun

A.O.Hnoov O "Home

82

 

 

 

 

 

 

 

 

0.0 ON OO ON HO ON 5O ON
H.5 ON OO ON 5O ON OO ON
0.0 ON OO ON OO ON OO ON
0.0 NN OO NN NO NN OO NN
N.O HN OO HN OO HN OO HN
0.0 ON OO ON OO ON OO ON
0.0 OH 5O OH HO OH OO OH
0.0 OH OO OH OO OH OO OH
0.0 5H HO 5H 5O 5H OO 5H
5.0 OH OO OH 5O OH OO OH
5.0 OH OO OH OO OH OO OH
H.O OH OO OH OO OH HO OH
0.0 OH OO OH NO OH OO OH
0.0H NH HO NH OO NH HO NH
0.0H HH OO HH OO HH NO HH
N.OH OH OO OH OO OH OO OH
0.0 O OO O OO O OO O
0.0 O OO O OO O HO O
0.0 5 OO 5 HO 5 NO 5
N.O O NO O OO O OO O
O.5 O 5O O OO O OO O
5.0 O OO O OO O OO O
0.0 O OO O HO O OO O
0.0 N OO N OO N OO N
O.5 H OO H OO H NO H
NnHNH Hon NnOOO mom NnOOO Mom NnOOO non
Hnnoo nHmnO Hnnoo nHonw Hnnoo nHmHU Hnnoo nHonw
Hononow noun HonoanHom noun Hoanoo noun HonoanHom noun

nOHmeHH OH HOHHQ mmon O onHHonm UoHOHHHnH
nHHB OoHonoH oHomnE mHHHomuO o no OoHooHHoo OHOO HonHOHHo

OH mHmHB

83

 

 

 

 

 

 

 

 

H.O OO 5O OO OO OO OO mm
0.0 NO OO NO NO NO OO NO
O.5 HO OO HO OO HO OO HO
0.0 OO OO OO OO OO OO OO
0.0 OO OO OO OO OO ON OO
O.5 OO OO OO OO OO 5O OO
0.0 5O OO 5O HO 5O OO 5O
O.5 OO OO OO OO OO OO OO
0.0 OO OO OO 5O OO OO OO
0.0 OO OO OO OO OO HO OO
H.O OO OO OO OO OO OO OO
0.0 NO OO NO NO NO OO NO
H.OH HO OO HO 5O HO 5O HO
0.0 OO ON OO OO OO OO OO
0.0 OO OO OO OO OO 5O OO
0.0 OO OO OO OO OO OO OO
0.0 5O OO 5O OO 5O OO 5O
0.0 OO OO OO OO OO OO OO
N.O OO 5O OO OO OO OO OO
H.O OO OO OO OO OO OO OO
H.O OO 5O OO HO OO OO OO
0.0 NO OO NO 5O NO HO NO
0.0H HO 5O HO HO HO OO HO
0.0H OO OO OO NO OO OO OO
0.0 ON OO ON HO ON OO ON
0.0 ON 5O ON OO ON OO ON
0.0 5N OO 5N OO 5N 5O 5N
N.O ON OO ON OO ON NO ON
NnHNH Hon NnOOO Hon nOOO Hon NnOOO mom
Hnnoo nHmnO Hnnoo nHonw Hn oo nHono Hnnoo nHouw
Hmuonow noun Honoannom noun Hoanoo noun HonoanHom noun

H.O.Hnoov OH muons

84

0.0 oOono>n

 

 

0.0 OO

0.0 OO

O.5 OO

0.0 5O

O.HH OO

0.0H OO

O.HH OO
NnHNH mom
Hnnoo nHono

Hononow noun

 

 

O.OO oOmno>n
OO OO

NO OO

5O OO

5O

NO OO

OO OO

OO OO

NnOOO Mom

“C500 CHMHU

5.OO oOmuo>n
OO OO

OO OO

NO OO

5O

OO

OO OO

HO OO

NnOOO mom

Hnnoo nHono

 

 

HonoanHom noun

Hoanoo moHn

H.O.Hnoov OH manna

 

5.OO oOono>n

OO

OO

OO OO

OO 5O

OO OO

5O OO

5O OO
nOOO Mom

Hn Oo nHono

 

Honoannom ,oonn

85

 

 

 

 

 

 

 

 

O.H ON NH ON OH ON HH ON
O.H ON 5 ON OH ON O ON
O.N ON 5 ON OH ON NH ON
O.N NN HH NN 5H NN OH NN
O.N HN NH HN O HN 5 HN
O.N ON NH ON OH ON HH ON
H.N OH O OH OH OH OH OH
H.N OH OH OH O OH 5H OH
O.H 5H O 5H OH 5H OH 5H
5.N OH 5 OH O OH O OH
O.H OH 5 OH O OH 5 OH
O.H OH O OH O OH OH OH
N.O OH O OH NH OH OH OH
O.N NH 5H NH HH NH OH NH
O.N HH OH HH O HH O HH
O.N OH OH OH HH OH O OH
5.H O NH O NH O HH O
O.N O O O 5 O HH O
O.N 5 OH 5 OH 5 OH 5
5.H O O O NH O O O
O.N O NN O O O O O
0.0 O NH O 5 O O O
O.H O O O O O OH O
O.H N O N O N O N

H H OH H 5 H OH H

NnHNH Hon NnOOO Hon NnOOO Hon NnOOO mom
Hnnoo nHMHO Hnnoo nHmHO Hnnoo nHouo Hnnoo nHmHO

Hononow noun HouoanHom noun HoanoO noun HonoanHom noun

nOHHoxHH OH HOHHO mwon OH onHHOHm OoHOHHHnH
nHHB UoHonoH oHomnE OHHHOOHO n no OoHooHHoo OHOU HonHOHHo

HH MHHGB

86

5.H OO
O.N OO
5.H Om
O.N Om
O.N NO
H.O HO
O.N OO
O.N ON
O.N ON
H.N 5N
O.N ON

 

NnHNH Hon
Hnnoo nHono

 

HMHOGOU MOM—”4

 

 

 

 

5 OO 5 OO
OH NO 5 NO
OH HO OH HO
OH OO NH OO
OH OO OH OO
O OO HH OO
OH 5O OH 5O
NH OO OH OO
HH OO OH OO
OH OO OH OO
OH OO 5H OO
OH NO OH NO
HH HO O HO
HH OO ON OO
ON OO OH OO
OH OO 5 OO
O 5O HH 5O
HH OO OH OO
OH OO NH OO
O OO O OO
NH OO O OO
OH NO O NO
O HO 5 HO
O OO O OO
NH ON NH ON
O ON O ON
O 5N NH 5N
NN ON OH ON
NnOOO Hon NnOOO Hon
Hnnoo nHme Hnnoo nHonw
HonoanHom noun Hmanoo noun

H.O.Hnoov HH mHmHB

 

O OO
O NO
O HO
OH OO
HH OO
O OO
OH 5O
OH OO
OH OO
NH OO
OH OO
OH NO
NH HO
OH OO
O OO
OH OO
5H 5O
HH OO
5 OO
5 OO
5 OO
5 NO
OH HO
OH OO
OH ON
O ON
O 5N
OH ON
NnOOO Mom

Hnnoo nHono

 

HonoanHom noun

87

O HO 5 HO OH HO

 

 

 

 

 

 

 

 

O OO 5H OO NH OO
NH O5 NH O5 OH O5
O O5 O O5 OH O5
5 55 O 55 OH 55
HH O5 OH O5 O O5
5 O5 OH O5 OH O5
HH O5 OH O5 O O5
OH O5 O O5 HH O5
OH N5 O N5 NH N5
O H5 O H5 HH H5
OH O5 OH O5 OH O5
OH OO O OO OH OO
OH OO O OO HH OO
O 5O OH 5O OH 5O
5H OO NH OO OH OO
OH OO 5 OO OH OO
OH OO O OO 5 OO
NH OO O OO 5 OO
O NO NH NO 5 NO
OH HO OH HO O HO
O OO O OO O OO
O OO HH OO O OO
O OO OO HH OO
5 5O OH 5O NH 5O
O OO OH OO OH OO
NH OO HH OO O OO
O OO O OO 5 OO
NnHNH Hon NnOOO Hon NnOOO Hon NnOOO Hon
Hnnoo nHoHo Hnnoo nHonw Hnnoo nHmnw Hnnoo nHon
Hononoo noun HmuoanHom noun HOHHnoo noun HononMHHom moan

H.O.Hnoov HH mnmne

88

N.N mOmnm>¢ O.OH mmmnw>m O.OH mmmum>< 5.OH mOmum>m

OOH

5OH

OOH

OOH

OOH

OOH

NOH

HOH

OOH

OO

OO

5O

OO

OO

OO

OO

NO

HO

OO

OO

OO NH OO
5O OH 5O OH 5O
OH OO NH OO OH OO
OH OO OH OO O OO
O OO NH OO O OO
NH OO O OO NH OO
HH NO OH NO 5 NO

r-l

NI-lmmhmhkaI-lmmhfilmmbm
I—lt-I H H

unr4O
rqqu

ON

 

 

 

 

NDHNH Hmm NsOOO Hmm NsOOO Hmm NSOOO Hmm
assoc chuw unsoo :Hmuw ucsoo QHMHO unsoo chuw

 

 

 

 

Hmumcmw mmufi HmumanHmm mmnfi Hmngcmo mmud HmumsmHumm mmnd

A.O.ucoov HH mque

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