/ . Embryol. exp. Morph., Vol. 11, Part 2, pp. 353-367, June 1963
Printed in Great Britain
DNA Synthesis and Nuclear Reproduction during
Embryonic Development and Regeneration of
Muscle Tissue
by L. N . ZHINKIN and L. F. ANDREEVA 1
From the Laboratory of Cell Morphology, Institute of Cytology, Academy of Sciences of
the USSR, Leningrad
WITH ONE PLATE
INTRODUCTION
D E S P I T E a large number of investigations devoted to the development and
regeneration of skeletal musculature, the problem of the mode of reproduction
of the muscle nuclei remains unsolved (Boyd, 1960; Murray, 1960; Holtzer,
1961; and many others). The majority of investigators believe that the symplast
nuclei reproduce by amitosis (Bucher, 1959). Only a few investigations have
shown the presence of mitoses in developing muscle fibres. Purely morphological investigations of the development and regeneration of muscle tissue seem
to be unable to solve this problem.
The nuclei of muscle fibres developing in vitro have recently been shown to
synthesize DNA, moreover, the experiments showed the synthesis of DNA by
the nuclei to be antagonistic to that of specialized muscle proteins (Stockdale &
Holtzer, 1961). Bintliff & Walker (1960) showed that a considerable percentage
of de-differentiated nuclei synthesized DNA upon regeneration of the skeletal
musculature. These observations make it possible to assume that nuclei in
developing and regenerating muscle fibres reproduce in the same manner as
those in any other tissue.
The present investigation, carried out in two experimental series, concerns the
relationships between reproduction by mitosis or amitosis and DNA synthesis
in muscle nuclei, and their relation to muscle differentiation.
The first series was devoted to a study of nuclear reproduction during
embryonic development, while the second dealt with nuclear reproduction
during the regeneration of striated muscles. To detect DNA synthesis, tritiated
thymidine was applied as a specific indicator.
1
Authors' address: Institute of Cytology, Academy of Sciences of the USSR, Maklin
Avenue, 32, Leningrad, F-121, USSR.
354
L. N. ZHINKIN AND L. F. ANDREEVA
MATERIAL AND METHODS
3
H-thymidine (from the Radiochemical Centre, Amersham, Buckinghamshire,
England) with a specific activity of 3-8 C./mM, diluted with distilled water to
200 mc./ml., was used in the experimental studies on muscle development and
regeneration. 3H-thymidine (from France) having specific activity of 300
mc./mM., diluted with distilled water to 200 mc./ml. was used in the series of
experiments on the mitotic cycle. In all the experiments 3H-thymidine was
injected subcutaneously in doses of 0-5 /xc./g.
The first series of experiments was devoted to the investigation of developing
muscles in 14-21-day-old albino rat embryos. Albino rats with a precisely
dated pregnancy were injected with 3H-thymidine and killed 4 or 24 hr. after
the injection.
Several embryos from each rat were fixed in Bourn's solution and used for
the study of the structure of developing muscles and for counting mitoses.
Other embryos were fixed by the Carnoy method and used for autoradiography.
The mitotic index was determined on the tongue muscles of 14-, 15-, 17- and
19-day-old embryos, while the index of labelled nuclei was determined on
autoradiographs. In addition, the intensity of the label per nucleus and the
mitotic cycle of nuclei were determined in 17-day-old embryos.
The second series of experiments, on muscle regeneration, was carried out
with four rats and eight albino mice. The sartorius muscle was cut in the rats
on the right and the left legs. In mice the rectus femoris muscle was transected. The rats were killed 2, 4 and 6 days after operation; 4 hr. before death
thymidine was injected into each of them. One rat was treated with thymidine 4 days after the cut, and killed 2 days later. Tritiated thymidine was
injected into albino mice 2, 4 and 6 days after operation, 4, 24 and 48 hr.
prior to fixation. Material was fixed in Carnoy's solution and embedded in
paraffin.
The investigation was carried out by means of autoradiography on a liquid
emulsion of ' R ' and ' M ' types supplied by the NIKFI, Moscow. The emulsion
was applied to sections without a sublayer, following a method described earlier
(Zhinkin, Zavarzin, Lebedeva & Andreeva, 1961). The sections with the
emulsion applied were, as a rule, exposed for 20 days. After the development
of preparations they were stained with hematoxyrin-eosin, azur-II-eosin or
methyl green-pyronin. Staining by Feulgen was carried out prior to the application of the emulsion. The data obtained were treated statistically (Bailey, 1959).
Since thymidine is eliminated from the organism approximately 1 hr. after
its injection (Hughes, 1958), 4 hr. after the injection of tritiated thymidine, the
nuclei that have just synthesized DNA could be seen at various sites in muscle
fibres. Later on it was possible to observe the movements of nuclei which had
earlier synthesized DNA, a change in their number and a change in the intensity
of the label over the nuclei.
DNA SYNTHESIS IN MUSCLE TISSUE
355
RESULTS
Development of muscle tissue
The counting of mitoses on longitudinal sections of developing muscles of
the tongue and back showed a decrease in the number of mitoses as the
development of the embryos proceeded. Thus, in the 14-day-old rat embryo
whose future tongue muscles consist mainly of myoblasts, 3 • 1 per cent, of
mitoses were found. On the 15th day of development when myosymplasts have
already formed, and when mainly smooth myofibrils are seen in the peripheral
portions of the bulk of them, there are 3 • 6 per cent, of mitoses. The difference
in the number of mitoses between this and the preceding stage is not statistically
significant. In tongue myotubes formed in 17-day-old embryos from 1-5 to
0-9 per cent, of mitoses were found in different series, while on the 19th day
0 • 3 per cent, of mitoses were found. The calculation of mitosis percentage at
the three first stages was carried out on the basis of an examination of 3000
nuclei and at the last stage on the examination of 10,000.
The index (percentage) of labelled nuclei was determined on the basis of the
examination of 1000 nuclei in the embryos 4 hr. after 3H-thymidine injection.
The label obtained in all three cases was sufficiently intense. As the development
of the embryos proceeded the percentage of labelled muscle nuclei, as well as
that of mitoses, decreased. Thus, on the 14th day of development 33 • 5 per cent,
of labelled nuclei were observed in the tongue muscles, while on the 21st day
they were only 1 • 5 per cent.
As can be seen in Text-fig. 1, the fall in the percentage of labelled nuclei runs
parallel to a decrease in mitotic activity. The similarity of these two curves
shows that they reflect one and the same process. In the tongue muscles of
adult rats labelled nuclei are rarely seen: only a few labelled nuclei were found
in many sections examined.
By counting the grains produced by the label over the nuclei, the intensity of
the label was calculated in 17-day-old embryos 4 and 24 hr. after 3H-thymidine
injection. It turned out that after 4 hr. labelled nuclei were 14 • 3 per cent, of the
total, the average number of grains per nucleus being 15-5. After 24 hr.
labelled nuclei were 32-3 per cent., the number of the grains per nucleus being
7-35. Thus the number of labelled nuclei doubled in 24 hr., and the intensity of
the label decreased by one-half. Therefore, during 24 hr. most labelled nuclei
of the developing muscle fibres in a 17-day embryo have divided at least once,
some of them possibly twice.
With a view to a more detailed analysis of nuclear reproduction, the mitotic
cycle was studied. Howard & Pelc (1953) were the first to study the mitotic
cycle with the use of 32P. With the use of tritiated thymidine a detailed determination of the mitotic cycle, as well as the subdivision of the interphase into
individual periods, was developed by Quastler & Sherman (1959); Quastler
23
L. N. ZHINKIN AND L. F. ANDREEVA
356
(I960); Painter & Drew (1959); Stanners & Till (1960); Kisieleski, Baserga &
Lisco (1961).
In order to determine mitotic cycle of the nuclei in developing muscles,
3
H-thymidine was injected into female rats on the 17th day of pregnancy; the
embryos were fixed after 30 min., 1, 2, 4, 6, 8, 9, 18, 20 and 24 hr. A hundred
mitoses were counted in tongue muscles for each time, and the number of
- 3
1
60
60
B
B
§
§
- 1
21
1. A graph illustrating a change in mitotic activity
and in the number of tritium labelled nuclei at different developmental stages of tongue muscle in rat embryos. Abscissa—
days (of development). Ordinate—percentage of dividing or
labelled nuclei. Continuous line—change in mitotic activity.
Dotted line—change in the percentage of labelled nuclei.
TEXT-FIG.
labelled ones determined. No labelled mitoses were found before the 2nd hour.
After 4 hr. 96 per cent, of mitoses were labelled, after 6 hr. 97 per cent.; later on
the percentage of labelled mitoses began to decrease, reaching 40 per cent,
after 24 hr. The change in the percentage of labelled mitoses is shown in
Text-fig. 2.
If the duration of a mitotic cycle (T) is calculated from one maximum of
labelled mitoses to the other, it takes about 18-20 hr. The pre-mitotic period
(G2), from the cessation of the synthesis of DNA to the onset of mitosis, takes
DNA SYNTHESIS IN MUSCLE TISSUE
357
more than 2 and less than 4 hr., so that it can be assumed to take nearly 3 hr.
The period of synthesis of DNA, or that of chromosome duplication, (5),
takes 6-7 hr., or, including the time of 3H-thymidine circulation in the organism,
about 7-8 hr. The pre-synthetic period (G^, from the end of mitosis to the
start of DNA synthesis, is determined from the difference Gx = T-(G2 + S+
mitosis). Therefore, when using the designations accepted, it can be thought
100
80
I
60
a
40
20
10
12 14
Hours
16
18
20
22
24
2. The number of labelled mitoses in the tongue muscle of rat embryos fixed
at different time after 3H-thymidine injection to their mothers on the seventeenth day
of pregnancy. Abscissa—the time between 3H-thymidine injection to a pregnant and the
fixation of its embryos. Ordinate—percentage of labelled mitoses.
TEXT-FIG.
that <?!+mitosis = 8-10 hr.; S = 6-7 hr.; G2 = 3 hr. the whole mitotic cycle
T ^ minimum 18 hr., maximum ~ 21 hr. The above calculated values of the
duration of the mitotic cycle and of its phases should be regarded as average
values estimated from the data obtained.
With the aim of determining the duration of mitosis itself, colchicine was
subcutaneously injected into female rats on the 17th day of pregnancy, at a dose
of 1 mg./kg. weight. The embryos were fixed 10, 20 min. and 4 hr. after colchicine injection; control embryos were fixed concurrently. The counting of
individual mitotic phases per 10,000 nuclei is shown in Table 1.
358
L. N. ZHINKIN AND L. F. ANDREEVA
TABLE 1
Ratio of mitotic phases at different times after colchicine injection
After colchicine injection
Prophase
Metaphase
Anaphse
Telophase
Total mitotic
index
Control
0-84
0-43
008
015
10 min.
0-83
0-43
0-0
005
20 min.
0-80
0-47
00
00
4 h.
0-74
1-20
00
00
1-5
1-37
1-27
1-94
It follows from the counts that anaphases disappear after 10 min. and telophases after 20 min. Therefore, anaphase takes 10 min. or somewhat less,
while telophase takes about 20 min. On the basis of the data on the duration of
anaphase and telophase, and on the frequency of occurrence of different mitotic
stages, it can be calculated that the entire mitosis requires at the most 3 hr.
But since each anaphase takes less than 10 min., while telophases disappear
after 20 min., 8 min. for the anaphase and 15 min. for the telophase may be
assumed as more probable values; then the durations of the prophase, metaphase
and of the entire mitosis would be 84 min., 43 min. and 2-5 hr. respectively.
The latter of these values coincides with that calculated by Manina & Bystrov
from the cinematographic records in myosymplasts developing in vitro. However, this value might be somewhat of an overestimate, but if it is accepted, then
the nuclei might be assumed to require about 6 days to double in number. But,
judging by 3H-thymidine incorporation, myotube nuclei of a 17-day-old embryo
divide at least one in 24 hr. The discrepancies between the data obtained by
colchicine and autoradiographic procedure are so great that they cannot be
ascribed to the insufficient precision of the procedures.
According to the data of Stockdale & Holtzer (1961), it can be assumed that
the 'differentiation' of nuclei during the development of muscles in an organism
proceeds gradually and consists in some nuclei synthesizing specialized contractile proteins, while others undergo reproduction. Since mitotic activity, as
well as the number of 3H-thymidine labelled nuclei, decreases with embryonic
development, it can be thought that the number of differentiated nuclei participating in the synthesis of specialized proteins increases concurrently. If the
whole cycle from one division to another takes about 20 hr., while the synthesis
of DNA takes 6-7 hr., i.e. one-third of the cycle, then the total number of
reproductive nuclei is three times that found labelled 4 hr. after 3H-thymidine
injection. Labelled nuclei in a 17-day-old embryo are 14 • 3 per cent, of the total
number, therefore the number of reproducing ones were 42-9 per cent, of the
total, while the remaining ones turn out to be 'differentiated', i.e. they do not
undergo reproduction and are engaged in the synthesis of specialized proteins.
Since the curves reflecting changes in mitotic activity and the index of labelled
DNA SYNTHESIS IN MUSCLE TISSUE
359
nuclei (Text-fig. 1) run almost in parallel, the mitotic cycle can be assumed to
remain constant at all the investigated stages of muscle development; therefore,
proceeding from the data obtained for the 17th day of development, the ratio
of 'differentiated' and reproducing nuclei can be calculated for other developmental stages as well (Table 2).
TABLE 2
Changes in the number of reproducing nuclei and in that of' differentiated' ones
participating in the synthesis of specialized proteins, in the embryonic development
of rat tongue muscle
Ratio of reproducing to
differentiated nuclei
Development
(days)
14
17
19
21
Labelled
nuclei
(%)
Reproducing
nuclei
(%)
33-5
14-3
100-0
42-9
23-2
8-4
1-5
4-5
Differentiated
nuclei
(%)
0
57-1
74-8
95-5
The stage of 14 days of development when presumptive muscles are represented by myoblasts whose nuclei, as all authors agree, divide mitotically,
turned out to be an initial stage when all the nuclei undergo reproduction.
Thereafter, the nuclei, in developing muscle fibres which are acellular structures,
represent a mixed population providing simultaneously differentiation of myofibrils, growth of fibres and an increase in the number of nuclei.
The data obtained show the development of muscles to be provided by
mitotic division of nuclei, while the bulk of described 'amitoses' seems to be
explained by a movement of nuclei within the developing muscle fibre and their
temporary pairing. Had the amitosis actually taken place and had the nuclei
not shifted within the muscle fibre, the number of labelled nuclei arranged in
pairs should have increased sharply after an interval following 3H-thymidine
injection, long in comparison to the brief period during which the indicator stays
within the organism.
Counts in 17-day embryos showed, however, the number of labelled nuclei
to reach 14-7 per cent, after 2 hr., and that of paired labelled ones to make
3 per cent, of all the paired nuclei, the probability of proximity of two labelled
nuclei, their number being 14-7 per cent., would be 2-2 per cent. After 19 hr.
the share of labelled nuclei was 25-0 per cent, that of paired labelled ones
8 per cent., while the theoretically expected value was 6-3 per cent. As can be
seen, the expected number of paired labelled nuclei is very close to that actually
observed. Therefore, the pairs of nuclei are formed not as a result of the
division of one nucleus (mitotically or amitotically) but due to a random collision
of nuclei during movement within the fibre.
360
L. N. ZHINKIN AND L. F. ANDREEVA
Regeneration of muscle tissue
In order to compare changes taking place in the sarcoplasm and in the nuclei
in which the synthesis of DNA starts, it is necessary to describe briefly the
morphological changes in injured muscle fibres which were observed at the three
stages studied. Two days after operation on mice an intensive disintegration of
muscle fibres, which proceeds by the Zenker type of necrosis, ceases. A large
number of connective tissue cells, mainly of hematogenic origin, accumulates in
the region of the wound, at the place of muscles that had earlier undergone
necrosis. Myotubes filled with phagocytes or individual smaller or larger parts
of disintegrating fibres are to be found in the vicinity of the site of inflammation.
The majority of muscle fibres are rather clearly separated from the zone of
inflammation and necrosis. As a rule, no myonbrils are seen at the ends of
disintegrating fibrils, the cytoplasm is feebly basophilic, locally vacuolated, the
ends of the fibres somewhat swollen. The nuclei are large with one or two
nucleoli, and the sarcoplasm around the nuclei is more basophilic. The changes
in the nuclei are revealed at a considerable distance from the cut ends of the fibres.
In the areas where myofibrils which often preserve their cross-striated character
can be seen, the nuclei are conspicuously enlarged, and the sarcoplasm around
them is basophilic. Finally, in the areas of fibres which are the furthest from the
cut, myofibrils and striation are of the normal appearance but these occur
somewhat enlarged nuclei, surrounded with a small lining of basophilic sarcoplasm which is due to the presence of RNA (Roskin & Kharlova, 1944; Roskin,
1951; Dmitrieva, 1954). Therefore, an increase in nuclear size and the rise in
RNA content of the surrounding sarcoplasm seem to be the first signs of the
activation of nuclei. Four days after operation muscle buds (myotubes) are
forming at the ends of the fibres, which are basophilic, with large nuclei often
arranged in a file. The same files or chains can be found not only in the buds
but also in the fibres not directly traumatized during operation, but either located
near the focus of inflammation or adjoining traumatized fibres.
After 6 days the buds lengthen somewhat and broaden locally when coming
into contact with the granulation tissue. Similar pictures were observed in rats,
but the process here developed more slowly, and well-developed buds could be
found on the 6th day after operation. Since the cut of the muscle was not
standard, individual variations could be seen both from animal to animal and
from one regenerating muscle fibre to another.
Two days after operation and 4 hr. after 3H-thymidine injection a large
number of cells with labelled nuclei was found in autoradiographs of mouse
muscles. Many nuclei containing the label belong to connective tissue cells in
the focus of inflammation, but among them elongated cells with a large light
nucleus, i.e. myoblasts, can be seen. Many of them turn out to be labelled
(Plate, fig. A). Labelled nuclei were also found in traumatized muscle fibres.
Sometimes labelled muscle nuclei were found in the detached and disintegrating
J. Embryol. exp. Morph.
Vol. 11, Part 2
I
•*»• * *
EXPLANATION OF PLATE
FIG. A. A myoblast with 3H-thymidine labelled nucleus among connective tissue cells.
Autograph obtained two days after operation and 4 hr. after 3H-thymidine injection.
Exposure for 20 days. (All the following radio autographs obtained at the same exposure.)
F I G . B . Musclefibreswith labelled nuclei. A large light muscle nucleus. Asmall darker connective tissue nucleus. Two days after operation, 4 hr. after 3H-thymidine injection. The
fibre at the edge of the wound.
FIG. C. A part of the muscle fibre, adjoining the wound (the first part). Myofibrils are seen
at the edge of the fibre. A few labelled muscle nuclei (larger). Small connective tissue nuclei
adjoining the sarcolemma. Two days after operation, 4 hr. after 3H-thymidine injection.
FIG . D. A large labelled nucleus at the edge of the fibre. Two days after operation, 4 hr. after
thymidine injection.
FIG. E. Intensively labelled large elongated muscle nucleus in a part of the fibres with
preserved myofibrils. Two days after operation, 4 hr. after 3H-thymidine injection.
FIG. F. Chains of labelled muscle nuclei in the fibres located near preserved muscle fibres
(the bases of the bud) 4 days after operation, 2 days after 3H-thymidine injection.
L. N. ZHINKJN and L. F. ANDREEVA
(Facing page 360)
DNA SYNTHESIS IN MUSCLE TISSUE
361
portion of the muscle fibre. Elongated small nuclei adjoining the sarcolemma
of muscle fibres from outside also incorporate 3H-thymidine (Plate, figs. B & C).
The largest number of labelled, i.e. of DNA synthesizing, nuclei are found at
the ends of cut muscle fibres where they attain a particularly large size (Plate,
figs. D & E); this number gradually decreases with the distance from the wound.
In order to determine the index of labelled nuclei found in equivalent zones,
muscle fibres were divided into three parts. Part I is that adjoining the wound;
here no myofibrils are seen, the sarcoplasm is basophilic, the nuclei are the
largest. Part II, that where myofibrils are clearly seen, but cross striation is
weakly expressed or indistinguishable. Part III of the fibre is that which has
already attained the normal structure, although a basophilic lining can be seen
around many of the nuclei. The change in the nuclear size was judged by the
area calculated by measuring the greatest and the smallest diameters and was
expressed in conventional units of the ocular micrometer.
Part I
59-3
Area of nuclei
Part II
36-7
Part III
23-6
These figures, however, do not completely reflect the changes taking place,
since the volumes of nuclei increase much more than their area. Furthermore
smaller nuclei, that seem to be undergoing degeneration, occur among the large
nuclei in the first part, so that the average area of the nuclei is somewhat
underestimated. The same holds true for the third part where much larger,
clearly changed nuclei occur among small elongated ones. The area of the
largest nuclei in the first part achieves 77 units of the small unchanged ones in
the third part only 20 units. Counts of the number of labelled nuclei and of
TABLE 3
Index of labelled nuclei and number of mitoses in different parts of muscle fibres
2 days after operation and 4 hr. after zH-thymidine injection
Part of
the fibre
I
II
III
Total number
of nuclei
counted
989
980
2057
Number of
labelled
nuclei
259
142
12
Percentage
of labelled
nuclei
26-22
14-49
0-58
Number of
mitoses
27
14
0
Percentage
of mitoses
2-72
1-42
0
mitoses was carried out in these three parts of the fibre (Table 3). As can be
seen from the figures presented, 2 days after operation a relatively large number
of mitoses and of nuclei synthesizing DNA is found; a portion of mitoses turns
out to be already labelled.
Bintliff & Walker (1960) showed that the number of labelled nuclei did not
increase upon 3H-thymidine injection prior to operation or immediately after it.
A conspicuous increase in the number of labelled nuclei was found only some
time after operation.
362
L. N. ZHINKIN AND L. F. ANDREEVA
Four days after operation, when muscle buds had already formed, and 4 hr.
after 3H-thymidine injection, the number of labelled nuclei sharply diminished.
When carrying out counts in muscle buds, of 346 nuclei, 14 or 4-04 per cent.,
turned out to be labelled. Still fewer labelled nuclei are found 6 days after
operation. But when 3H-thymidine was injected 2 days after operation and the
material was fixed 2 days later, a large number of labelled nuclei was found both
in muscle buds (Plate, fig. F) and in the fibre parts behind them.
When counting labelled nuclei 2 days after operation and 2 days after
3
H-thymidine injection, the following ratios were found (Table 4). The counts
TABLE 4
Index of labelled nuclei in different parts of regenerating muscle fibres 4 days after
operation and 48 hr. after 3H-thymidine injection
Parts of
Total number
the fibre of nuclei
I
297
II
145
III
415
Number of
labelled
nuclei
116
30
14
Percentage
of labelled
nuclei
39-1
20-7
3-4
were also carried out in three parts: I, muscle buds; II, a part of the fibre
adjoining the former; III, muscle fibre of a normal structure located far from
the region of regeneration. Thus individual muscle buds can contain all or
almost all labelled nuclei, while others contain but relatively few of them.
Mitoses (mainly prophases) are seldom met with in the nuclei of muscle buds.
No counting of labelled nuclei was carried out in experimental rats, but an
examination of the preparations shows in principle almost the same regularities
as those obtained on mice; the rate of regeneration, however, seems to be
somewhat different. Thus, labelled nuclei were found in the fibres 2 and 4 days
after operation, while an aggregation of a large number of labelled nuclei in
muscle buds was observed 6 days after operation in rats which had been given
3
H-thymidine 48 hr. prior to decapitation.
The aggregation of labelled nuclei in muscle fibres of animals given ^-injection 2 days earlier shows that labelled nuclei migrate to muscle buds from
disintegrated parts of muscle fibres. A relatively small number of labelled nuclei
in muscle buds speaks in favour of the migration and aggregation of labelled
nuclei 4 hr. after thymidine injection. Therefore, the synthesis of DNA and
chromosome replication in the bulk of the nuclei have already ceased prior to
the formation of large, growing muscle buds, which is in good agreement with
the data obtained by Bintliff & Walker (1960).
DISCUSSION
In order to show to what extent the number of labelled and reproducing
nuclei during development and regeneration is able to provide an increase in
DNA SYNTHESIS IN MUSCLE TISSUE
363
the number of the nuclei, the results obtained in both experimental series should
be compared.
33 • 5 per cent, labelled nuclei and 3 • 1 per cent, mitoses were found in 14-dayold embryos. During regeneration, 2 days after operation 26-2 per cent,
labelled nuclei and 2-7 per cent, mitoses are found in the first part. When
comparing these data, it can be seen that in both cases approximately one-third
of the nuclei synthesized DNA. The ratio of the number of mitoses to that of
labelled nuclei is in both cases 1:10. This comparison shows that the nuclei,
both during development and during regeneration, pass through a period of
DNA synthesis and undergo reproduction in the same manner as in any other
cellular tissue. If the period of DNA synthesis during embryonic development
takes about 7 hr., and one-third of the nuclei are in the S-period, it can be
thought that during regeneration the period of synthesis in the first zone 2 days
after operation is approximately the same, and the whole mitotic cycle of the
nuclei is the same as during development. Therefore, the onset of DNA synthesis
and the subsequent development in the bulk of the nuclei take place prior to
the formation of or at the very earliest period of the formation of muscle buds.
Lash, Holtzer & Swift (1957) showed by means of cytospectrophotometry
that upon regeneration the nuclei in the myotubes were diploid. The bulk of
the nuclei seem to have ceased to synthesize DNA at that time and to pass to
the synthesis of specialized contractile proteins; in our experiments, however,
4 per cent, of labelled muscle nuclei were found which suggest that the whole
population was heterogeneous.
In order to pass to the synthesis of DNA during regeneration, the nuclei
undergo several characteristic changes. Morphologically, these changes consist
of an increase in size and formation of a zone of RNA-containing basophilic
sarcoplasm around the nuclei, i.e. the whole complex of changes corresponds to
the differentiation of muscle fibres. The more profound the changes which the
nuclei undergo, the greater is the number of nuclei in the phase of DNA
synthesis. Synthesis commences after very slight changes in the nuclei which
are correlated with a change in both the sarcoplasm and myofibrils.
All the facts mentioned suggest that the conditions enabling nuclei to pass to
the synthesis of DNA consist in a change in myofibrils, i.e. contractile muscle
proteins.
Proceeding from the data quoted by Stockdale & Holtzer (1961) and by
Holtzer (1961), which suggest that synthesis of DNA and synthesis of contractile
proteins are mutually exclusive events, it may be thought that nuclear differentiation is related to the production of myosin and actin, while dedifferentiation is
related to a destruction of the fibre which releases nuclei from a factor blocking
the synthesis of DNA and thus allows them to undergo reproduction.
Therefore, the presence of myofibrils in developing and formed muscle fibres
is the very factor which maintains the nuclei at the differentiated state and blocks
the possibility of DNA synthesis and the possibility of their reproduction.
364
L. N. ZHINKIN AND L. F. ANDREEVA
the synthesis of specialized contractile proteins and that of DNA are antagonistic
events (Stockdale & Holtzer, 1961), then it should be assumed that, while form,
ing myofibrils, synthesized contractile proteins, in their turn, affect the nucleus
depriving it of the facility to synthesize of DNA. This connexion could be best
seen as a negative feedback. The nuclei that synthesize specialized contractile
proteins are affected by them, and it is this action which blocks the synthesis of
DNA and probably controls the rate of protein synthesis as well.
During the development of muscle fibres the nuclei seem to be more actively
involved in the synthesis of proteins, and a large nucleus with a large nucleolus
corresponds to this state. The growth of the fibres having terminated, nuclei
become smaller and change to a state in which no synthesis of DNA is possible.
In this form they maintain a very slow renewal of the myosin and actin (Velick,
1956). A negligible percentage of nuclei goes on reproducing it in the adult
organism, maintaining to some degree the renewal of nuclei. In response to
small disturbances of muscle fibres, the nuclei change to the active state, round,
enlarge and begin to synthesize DNA.
Observing the differentiation of muscle fibres in vitro, Chlopin (1940) noticed
the presence of nuclei differing in structure. In the light of more recent data,
the morphological differences he described can be regarded as correlated with
the different functions of the nuclei. These differences can be determined by
the fact that the nuclei undergo different stages of mitotic cycle, or that their
activity with respect to the synthesis of specialized proteins is different. Sometimes degenerative forms of nuclei were found upon regeneration. No degeneration was revealed in the nuclei of differentiating fibres which bears witness to
their homogeneity and to the possibility that any nucleus may change to the
synthesis of DNA and undergo division under proper conditions. Physiological
regeneration can be regarded from two aspects: 1, renewal or permanence of
muscle fibres themselves, and 2, renewal within the muscle fibre under the
sarcolemma tube. We shall not touch the first possibility, but turn to the
second one. The percentage of labelled nuclei found by Messier & Leblond
(1960) in the muscle of adult rats is sufficiently large to provide the renewal of
all the nuclei at a rate approximating that found in the liver. A few 3H-thymidine-labelled nuclei were found by Bintliff & Walker (1960) in skeletal musculature.
In our observations there are very few nuclei synthesizing DNA in the muscles
of adult rats, therefore the reliability of the average value would be small. At
present the fact of a very slow renewal of nuclei in muscle fibres of adult animals
seems to be accepted, as well as a slow renewal of contractile substance. Thus,
the half-life of H-meromyosin in the rabbit is 80 days, that of L-meromyosin
20 days, and that of actin 67 days (Velick, 1956). The renewal is so slow that
it seems to be impossible to demonstrate it in histological preparations. But any
increase in physiological regeneration in response to small disturbances seems
to show, in an accelerated form, the processes going on under normal conditions.
DNA SYNTHESIS IN MUSCLE TISSUE
365
The problem of the reproduction of muscle nuclei cannot be regarded as
solved only in respect to the form of the division itself. The synthesis of DNA
and chromosome reduplication seem to proceed in a similar way during the
development, regeneration and in the adult organism. The problem of whether
nuclear reproduction involves mitosis only or whether endomitosis with a
subsequent division of the nucleus also occurs remains unsolved.
A comparison of all the facts available at present suggests that the aggregation
of nuclei in myotubes is due to their migration from undestroyed parts of the
muscle fibre where the nuclei reproduce mitotically. The same migration
determines the formation of nuclear chains in traumatized muscle fibres which
have preserved their sarcolemma. Migration of nuclei is proved not only by
the data obtained by means of autoradiography but mainly by the observations
carried out by Manina & Bystrov (1955), Capers (1960) and Cooper & Konigsberg (1961) by means of cinematographing muscle tissue cultures. When following the behaviour of pairs of nuclei for a considerable period of time, rotational
movements and the movement of one nucleus around the other could be
observed. These motions give all the appearances regarded as amitoses in a
fixed preparation. The absence of a colchicine action upon the regeneration or
development of muscles in tissue culture is often regarded as an argument in
favour of amitoses (Godman & Murray, 1953; Godman, 1955; 1958; Murray
1960). Neither does nitrogen mustard which blocks the synthesis of DNA
(Konigsberg, McElvain, Tootle & Herrmann, 1960) affect the development of
multinuclear muscle elements in tissue culture. However, Pietsch (1961) showed
that colchicine affects mitoses in muscles if it is introduced directly into a
traumatized muscle.
All the facts mentioned show that nuclear reproduction during regeneration
proceeds in the old parts of the muscle fibre while the aggregation of already
divided diploid nuclei in muscle buds is a result of migration which gives pictures which have been considered to be amitoses. Therefore, the main mode of
the reproduction of muscle nuclei during regeneration, as well as during development, is mitosis. Whether amitosis also exists and what role it plays remains
unclear. Mitoses are sufficient to provide for the increase in the number of
nuclei required for the regeneration of muscle fibres.
SUMMARY
1. Nuclear reproduction in myosymplasts and myotubes proceeds mitotically.
Nuclei synthesizing DNA are found during the development of muscles. The
percentage of 3H-thymidine labelled nuclei diminishes with the development of
the embryo as mitotic activity decreases.
2. A mitotic cycle in the nuclei of muscle fibres of a 17-day-old rat embryo
takes about 20 hr. and the duration of mitosis itself is about 2 • 5 hr. A comparison of the number of nuclei participating in a mitotic cycle with the mitotic index
366
L. N . Z H I N K I N AND L. F . ANDREEVA
allows us to suggest that as differentiation proceeds some nuclei provide the
synthesis of specialized proteins while others undergo reproduction.
3. During regeneration of muscle fibres, 2 days after operation, a considerable percentage of 3H-thymidine labelled nuclei is found at the ends of cut
muscle fibres and, correspondingly, the mitotic index is high. The number of
labelled nuclei, as well as that of mitoses, decreases with distance from the
wound.
4. Subsequently labelled nuclei move to muscle buds where they only very
rarely undergo division.
5. All the facts obtained show that the reproduction of muscle nuclei during
embryonic development and during regeneration of muscle fibres in adult
animals proceeds in the same manner as in any other cellular tissues where cell
reproduction by mitosis is not in doubt.
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{Manuscript received 5th November, 1962)