/. Embryol. exp. Morph. Vol. 23, 2, pp. 359-74, J 970
Printed in Great Britain
359
Desynchronization of
cell divisions in the course of egg cleavage and an
attempt at experimental shift of its onset
By E. V. C H U L I T S K A I A 1
From the Institute of Developmental Biology
Academy of Sciences of the U.S.S.R., Moscow
There is considerable current interest in the study of regularities and modes of
regulation of cell cycles in the course of development (Dettlaff, 1964; Graham,
Arms & Gurdon, 1966; Chulitskaia, 1967Ö). The period of cleavage divisions
is a very suitable object for such investigations. It consists of two different
periods known as periods of synchronous and asynchronous cell divisions
(Syngayewskaya, 1931; Balinsky, 1931; Costello, 1955; Sirakami, 1958;
Neyfakh & Rott, 1959; Dan, 1960; Pankova, 1963; Agrell, 1964; Engelberg,
1964; Skoblina, 1965; Chulitskaia, 19676; Rott & Sheveleva, 1968). Peculiarities
of these two periods are known rather well but regularities of the transition from
synchronous to asynchronous divisions are still imperfectly understood (Riickert,
1899; Hoff-J0rgensen & Zeuthen, 1952; Agrell & Person, 1956; Neyfakh &
Rott, 1959; Neyfakh, 1961; Agrell, 1961, 1962; Pankova, 1963; Brown, 1965;
Bachvarova, Davidson, Allfrey & Mirsky, 1966; Timofeeva, Kafiani & Neyfakh,
1967; Chulitskaia, 1967a, b; Rott & Sheveleva, 1968).
The present investigation was designed to study the time and character of the
transition from synchronous to asynchronous cleavage divisions at different
temperatures. The correlation between the time of appearance of different
mitotic phases in various cells and the time of increase in interphase duration as
expressed by a decrease of the mitotic index has been studied.
Three aspects of cell division have been studied: (a) the stage when nuclei in
more than two neighbouring mitotic phases at any one time were first observed
in one embryo; (b) the stage when the mitotic index first decreased; and (c) the
duration of the period between stage (a) and stage (b).
On the other hand we studied the influence of the cytoplasm on cell cycle in
this transitional period. We concluded from data previously obtained (Dettlaff,
Nikitina & Stroeva, 1964) that the cytoplasm of immature oocytes inhibits
division of somatic nuclei transplanted into it and the cytoplasm of mature
1
Author's address: Institute of Agricultural Microbiology, Konjushkovskaya St. 31,
Moscow D242, U.S.S.R.
360
E. V. CHULITSKAIA
oocytes stimulates the division of nuclei and exerts a synchronizing effect on
nuclei of different origin. Somewhat later these results were confirmed by
Graham et al. (1966) and Gurdon (1967). They discovered a new very important
phenomenon—the induction of DNA synthesis in somatic nuclei under the
influence of the cytoplasm of mature oocytes. In all the experiments mentioned
above, the influence of the cytoplasm on nuclei was studied by means of transplantation of somatic cell nuclei into the cytoplasm of oocytes or mature eggs.
In the present investigation a converse experiment was carried out—the cytoplasm of mature eggs was injected into developing embryos. We studied the
effect of the cytoplasm on desynchronization of cell divisions and on the mitotic
index in the period of the transition from synchronous to asynchronous cell
divisions.
MATERIALS AND METHODS
Experiments were carried out on the eggs of Acipenser güldenstädti and Rana
temporaria, in Spring 1964-66. Sturgeon and frog eggs were inseminated and
incubated in a Heppler's ultrathermostat at different temperatures : frog eggs—
at 7°, 13°, 18°, 23 °C and sturgeon eggs at 17°, 20° and 23 °C. Fixations of
sturgeon and frog eggs were performed at all the temperatures studied, from the
7th cell division (which is still quite synchronous at all the temperatures studied)
onwards during the subsequent eight cell cycles. The interval between fixations
was equal to one cell cycle (T 0 ); T0—means the duration of the shortest cell
cycle, i.e. the cell cycle during synchronous cleavage divisions (Dettlaff &
Dettlaff, 1961). it corresponds to the time between the appearance of two
successive cleavage furrows on the egg surface. Using the previously obtained
values of r0 at different temperatures (Dettlaff & Dettlaff, 1961; Chulitskaia,
1965) we estimated the time of fixation of embryos. The first fixation was performed after the appearance of the 7th furrow on the egg surface, i.e. after the
time necessary for the transition of the cell nucleus from interphase to metaphase
(Dettlaff, 1963).
However, as this time is not estimated exactly for various temperatures,
sometimes embryos proved to be fixed not at metaphase, but at meta- and anaor prometa- and metaphase. As successive cleavage stages during the period of
asynchronous divisions cannot be characterized by the number of nuclear
divisions, we designated them by the number of T0 from the onset of nuclear
divisions, i.e. from metaphase of the first cleavage division.
Sturgeon embryos were fixed with Sanfelice fluid, frog embryos with Bouin's
fluid. Sections of sturgeon embryos 10 /i thick were stained with haematoxylin
by the Heidenhain method; sections of frog embryos were stained according to
Mann. 5-10 embryos were studied for each temperature and each time of
fixation.
In order to study the influence of the cytoplasm on the cell cycle, sturgeon eggs
were inseminated and incubated in Heppler's ultrathermostat at 20°. The cyto-
Desynchronization of cleavage divisions
361
plasm to be injected was taken with a glass micropipette from the animal region
of a mature unfertilized egg (4-5 % of the total volume of cytoplasm) and injected into the animal portion of developing embryos of the same female, in the
region between furrows at the stage of the 4th cleavage. At this stage the furrows
do not completely divide the cytoplasm and the endoplasm of the blastomeres is
not completely disconnected.
In addition, experiments were carried out using an injection of the same
volume consisting of the supernatant fluid obtained by homogenization of
250-300 mature eggs in 4 ml Tris-buffer (pH 7-8), followed by centrifugation of
the homogenate for 20 min at 5000 g. The supernatant while on ice was evaporated under an air stream to approximately \-\ of the initial volume. As the
volume of the sturgeon egg is about 0014 ml, it follows that the concentration of
substances which passed from the cytoplasm into the supernatant increased
approximately 2-5 times. After the appearance of the fourth cleavage furrows
control embryos were injected with cytoplasm from embryos of the same age.
All the injected embryos were cultivated in Holtfreter solution with sulphasin and
penicillin. Most of them cleaved normally, those with abnormal cleavage were
excluded.
Embryos were fixed from 8 T0 onwards till 11 T0 at intervals equal to 1 r0.
Some eggs were fixed at 15T 0 and 16r 0 for estimation of the mitotic index
(MI), as in normal eggs it decreases sharply at 15 T0. Finally the time of gastrulation was determined in some eggs from the groups injected with cytoplasm, and
some eggs from the control group and untreated group. Embryos were fixed with
a mixture containing 7-5 ml 70 % alcohol, 2-5 ml 40 % formalin and 0-5 ml
glacial acetic acid. The use of this method of fixation excluded the passage of the
egg contents through the holes in the membranes, produced by the micropipette,
which took place after fixation with Sanfelice fluid, Bouin, Zenker and 4 %
formalin. The mixture used did not change the form of embryos and gave
satisfactory results with subsequent treatment. Sections 10 ju, thick were stained
with haematoxylin according to Regaud and Heidenhain. Material fixed at
15-16 T0 was counter stained with methylgreenpyronin. 15-20 embryos were
studied at each cell division in the period of the transition from synchronous to
asynchronous divisions.
In all experiments, regularities of the transition from synchronous to asynchronous divisions were studied on blastomeres of the animal region where
there were a lot of nuclei by this time. Mitotic phases were determined in all the
cell nuclei of the animal region and marginal zone in all the sections of each
embryo. In order to follow changes of the mitotic index in the period of transition from synchronous to asynchronous cell divisions, we compared the number
of different mitotic phases in nuclei of different cells at successive cleavage
stages in the period of asynchronous divisions, with their relative duration in the
period of synchronous divisions (Dettlaff, 1963). The number of nuclei at each
phase was expressed in % of the total number of nuclei counted. After 15 r0,
362
E. V. CHULITSKAIA
when there were already a lot of nuclei in embryos, the percentage of dividing
nuclei (at prometa-, meta-, ana- and telophase) was estimated in 1000 nuclei.
As fixation could be performed at the stage of the transition from one mitotic
phase to another, the occurrence of two neighbouring phases was still considered
characteristic of a synchronous division. Simultaneous occurrence in an embryo
of nuclei at three mitotic phases was considered as the onset of desynchronization
of cell divisions. A division in which the number of nuclei at the third mitotic
phase was more than 5 % of the total number of nuclei was regarded as the first
desynchronized division.
In addition, in sturgeon embryos the structure of the nucleus was studied at
the stages of transition from synchronous to asynchronous cell divisions.
100
u
9
10
11
12
13
Time from metaphase of the first division (T 0 )
14
Fig. .1. Occurrence of mitotic phases (in %) in successive cleavage divisions at
different temperatures in embryos of Rana temporaria. • , 1 = interphase + prophase;
E2, 2 = prometaphase; ^ , 3 = metaphase; H, 4 = anaphase; • , 5 = telophase.
Desynchronization
of cleavage divisions
363
RESULTS
I. The character of the transition from synchronous to asynchronous cleavage
divisions in sturgeon and frog embryos at different temperatures
Figs. 1 and 2 give the number of different mitotic phases in the animal region
and marginal zone in frog and sturgeon embryos at successive cleavage stages at
different temperatures (in % of the total number of nuclei). In both species the
transition from the synchronous to asynchronous divisions has some common
Table 1. Number of nuclei at different mitotic phases {in % of the total number of
nuclei) in embryos of Rana temporaria in the period between 11 and 14 r 0 at
different temperatures
Temperature
(°C)
Total
number
of nuclei
Interphase+
prophase
Prometaphase
Metaphase
Anaphase
23
18
13
7
1668
1547
3723
1753
43-8
51-5
47-4
45-5
2-1
91
14-8
111
27-0
28-1
19-2
20-8
161
9-5
12-1
10 9
110
1-8
6-5
11-7
9-3
23-8
12-1
7-7
47-1
Average
8691
values
0-47
The same in prortions of the total
number of nuclei
taken as 1
Relative duration
0-50
of mitotic phases
during one nuclear
division in the
period of synchronous
cleavage divisions
(in T 0) after
Dettlaff (1964)
Telophas
009
0-24
012
008
010
0-20
014
006
features. At the optimum temperatures (14-20 °C for the sturgeon and 13-21 °C
for the frog) the first single nuclei at neighbouring mitotic phases appear among
synchronously dividing nuclei and at the next division many nuclei are already at
different mitotic phases. At the higher temperatures this transition proceeds
within one division when many nuclei are simultaneously at different mitotic
phases. At the lower temperatures single nuclei at the third mitotic phase appear
during two divisions and only at the third division are nuclei at different mitotic
phases (note the distance between lines b and c in Fig. 3). After that, synchronous
divisions are still predominant. Later, after complete desynchronization of
nuclear divisions for some period the nuclei occur at all mitotic phases but in the
same ratio as that of the duration of each mitotic phase during synchronous
364
E. V. CHULITSKAIA
cleavage divisions. The end of this period is characterized by a decrease of
mitotic index and occurs at all the temperatures synchronously, at the same
division.
These regularities are most clearly revealed in frog embryos. At 8 T 0 only
synchronously dividing nuclei are found in these embryos: in the animal
region all the nuclei still divide with the same rhythm and are at the same, or at
two neighbouring mitotic phases. Desynchronized nuclei appear at 23 °C at
9 r 0 , at 18 °C—at 10 r 0 , at 13 °C and 7 °C—at 11 r 0 from metaphase of the first
cleavage division (see Fig. 1 and Table 2). After complete desynchronization
within the interval between 11 and 14 r 0 (see Table 1) at all the temperatures
Table 2. Time of the onset of desynchronization of nuclear divisions and of the
decrease in mitotic index in different nuclei in blastomeres of the animal region and
marginal zone at different temperatures in frog embryos
Temperature
(°C)
Number of
embryos
studied
23
18
13
7
37
40
37
38
Occurrence in %
Time in T0 of the (prometa-, meta-,
first appearance of ana- and telophase)
desynchronized
in the period
nuclei in different between 11-14 T0
cells of the same from the beginning
embryo
of cell divisions
9
10
11 (10)*
11 (9)
56
48
52
54
(mean value 52)
MI at the 15 T0
stage from metaphase of the first
cleavage division
23-9
17-4
15-9
20-2
(mean value .19)
* In brackets—time (in T0) of desynchronization of cell divisions in single nuclei (meaning
that the number of nuclei in a third mitotic phase does not exceed 5 %).
about a half of the nuclei (47 %) are at interphase -I- prophase, 9 % at prometaphase, 24 % at metaphase, 12 % at anaphase and 8 % at telophase. Relative
duration of mitotic phases during one nuclear division in the period of synchronous cleavage divisions equals 0-50, 0 1 0 , 0-20, 0 1 4 and 0-06 correspondingly.
Although in the period of 11—14 r 0 , the number of interphase and prophase
nuclei amounts to about 50 % of the total number of nuclei, the mitotic index is
still close to the average mitotic index during synchronous division. The average
mitotic index can be considered equal to 50 during the period of synchronous
cleavage divisions as half of this time all the nuclei are in active mitotic phases
(MI = 100) and the other half of the time they are at interphase and prophase
(MI = 0).
A sharp decrease in MI occurring within the one cell division is observed only
in the embryos fixed at 15 T 0 from metaphase of the first cleavage division. The
percentage of nuclei in the active mitotic phases decreases sharply (Table 2:
Desynchronization of cleavage divisions
365
from 52 % in the period of l l - 1 4 r 0 to 19 % at the 15 r 0 stage). Accordingly,
the percentage of cells at interphase and prophase increases.
Similar but less precise data were obtained for sturgeon embryos (Figs. 2, 3).
In sturgeon embryos, the onset of desynchronization also depends on temperature :
at 23 °C desynchronization begins at 8 T0, at 20 °C—at 9 T 0 and at 17 °C—at
10 r 0 . At subsequent stages in the interval between 12-14 T 0 from metaphase
100
23
UT- Uh-
=£Sfc=M.
20
J
J •it
10
a
Lii
17
11
j
12
13
14
Time from metaphase of the first division ( T 0 )
Fig. 2. Occurrence of mitotic phases (in %) in successive cleavage divisions at
different temperatures in embryos of Acipenser güldenstädti. Key as in Fig. .1.
12
10
12
15
20-5
Time from metaphase of first cleavage division (TQ)
Fig. 3. Time of the onset of desynchronization of cell divisions and of decrease in
mitotic index in sturgeon embryos at different temperatures, a = Period of synchronous cleavage divisions; b = first appearance of desynchronized nuclei; c =
onset of desynchronization; d = period of desynchronization; e = asynchronous
period; ƒ = onset of gastrulation.
366
E. V. CHULITSKAIA
of the first cleavage division in each embryo, nuclei occur simultaneously at all
mitotic phases and the average relative number of different mitotic phases
approximately corresponds to their relative duration in the period of synchronous cleavage divisions. Only at 23 °C, i.e. at a temperature close to harmful
ones, is there a high percentage of interphases in the early stages of cleavage
divisions.
The study of the structure of interphase nuclei in sturgeon embryos at different
times during desynchronization has shown that at the beginning of this period
nuclei have karyomere structure and nucleoli are absent at 20 °C. Nucleoli appear
in 4-5 T0 after the onset of desynchronization although nuclei still have karyomere
structure; the latter disappears only after the decrease of the mitotic index
(Chulitskaia, 1967a). Thus, the appearance of typical interphase nuclei is not
connected with the onset of desynchronization.
The data obtained on frog and sturgeon embryos show that there is a certain
transition period between synchronous and asynchronous cleavage divisions
which may be called the period of desynchronization. It differs from the period of
synchronous cleavage divisions by desynchronization of mitotic phases in
nuclei of different blastomeres, and from the period of asynchronous cleavage
divisions—by the absence of a decrease of the mitotic index. Desynchronization
begins either at earlier or later cleavage stages at different temperatures. The end
of the period of desynchronization is characterized by a sharp decrease of the
mitotic index and does not depend on temperature. Hence, there is no strict
correlation between the onset of desynchronization and the decrease of the
mitotic index at different temperatures.
Unlike the period of desynchronization, the asynchronous period always
starts at all the temperatures at the same time (expressed in T0). The end of this
period being at the same time the onset of gastrulation is attributed to a definite
r 0 only at the optimum temperatures, but varies markedly at extreme temperatures (Fig. 3).
II. Injection of the cytoplasm into developing sturgeon embryos
In all the experiments, the injection into developing embryos of the cytoplasm
or the supernatant of the mature egg homogenate resulted in prolongation of the
period of synchronous cleavage divisions. The data obtained are presented in
Figs. 4 and 5.
In normally developing embryos nuclei in all the cells of the animal region
still divide synchronously at the stages 7 T0 and 8 T0 at 20 °C. Desynchronized
nuclei first appear at the 9 T0 stage. At this stage nuclei occur in four mitotic
phases. In embryos injected with the cytoplasm of mature eggs, nuclei
divide synchronously not only at the 8 T0 stage, but also at 9 T0. At the 8 r0
stage in all the embryos nuclei are in the same or in neighbouring mitotic
phases; at the 9 T0 stage in more than half the embryos nuclei occur in a third
mitotic phase, but their number does not exceed 3 %. At the 10 r0 stage in all the
Desynchronization
of cleavage divisions
367
embryos nuclei occur in three or four mitotic phases simultaneously. At the
11 T0 stage in all the embryos studied nuclei occur in five mitotic phases.
In embryos injected with the supernatant from homogenized mature eggs,
nuclei divide synchronously not only at the 8 r0 and 9 r0 stages, as was the case
with the cytoplasm injection, but also at the 10 T0 stage. At the 8 r 0 and 9 r0
stages nuclei in all the embryos are in the same or in two neighbouring mitotic
a
b
e
1 II
e
d
N
I I
\
1
9
11
15
18
Time from metaphase of first cleavage division (To)
Fig. 4. Time of the onset of desynchronization of cell divisions and of decrease in
mitotic index in sturgeon embryos injected with the cytoplasm of mature eggs.
TV = normal untreated embryos; K = embryos injected with cytoplasm of the same
age; C = embryos injected with cytoplasm of mature eggs; H - embryos injected
with the supernatant of mature egg homogenate. a, b, c, d, e-as in Fig. 3.
phases. At the 10 r0 stage in the majority of embryos nuclei are still in one or two
neighbouring mitotic phases and only in some embryos are there single nuclei in
a third or fourth mitotic phase: the number of such nuclei does not exceed
4 % of the total number of nuclei counted. At the 11 r0 stage nuclei occur in
four mitotic phases.
In control embryos injected with cytoplasm of the same age, nuclei still divide
synchronously at the 8 r0 stage; only in some embryos single nuclei occur in a
third mitotic phase, but their number does not exceed 3 %. At the 9 r 0 stage in
almost all the embryos nuclei occur in three mitotic phases and the number of
such nuclei exceeds 5 %. At the 10 T0 and 11 r0 stages four or five mitotic phases
are observed simultaneously.
The desynchronization of cell divisions in control embryos, injected with
cytoplasm of the same age, does not differ from that in normal intact embryos.
As in normal embryos, in control ones stage 8 r 0 (the 9th cell division) at 20 °C is
the last synchronous division and from stage 9 T0 (the 10th cell division) desynchronization of mitotic phases in nuclei of different blastomeres is observed.
After the injection of cytoplasm from mature unfertilized eggs into the cytoplasm of cleaving embryos desynchronization starts one division later; after
injection of the supernatant of homogenized eggs—two divisions later than in
intact embryos.
368
E. V. CHULITSKAIA
In all the experimental groups, the mitotic index was estimated for 8-10
embryos. At the 15T 0 stage in normal embryos and in those injected with cytoplasm of unfertilized eggs and cytoplasm of the same age the mitotic index
decreaseds harply and equalled 11-8,108, 16-9 and 13-4 respectively. At the 16r 0
Desynchron ization
•
8<
9<
m—*
10<
*P*1— ,
(//////
11<
1 2
3
m
4
5
Number of mitotic
phases occurring simultaneously
Fig. 5. Desynchronization of cleavage divisions in sturgeon embryos under the
influence of cytoplasm. N, K, C, //-as in Fig. 4. Numbers of nuclei < 5 % at a given
mitotic phase are designated by the thick line.
stage the values of the mitotic index in the groups of embryos studied were similar
and equal to 8-1, 6-5, 6-4 and 7-9 respectively. These data indicate that the injection of 3-5 % (by volume) of the mature egg cytoplasm into cleaving embryos
shifts the onset of desynchronization to a later division, but does not influence the
timing of a sharp decrease of the mitotic index. It has no effect on the onset of
gastrulation either: in all developing embryos studied the duration of the period
Desynchronization
of cleavage divisions
369
between insemination and the onset of gastrulation was practically constant and
equal to 20 T0.
DISCUSSION
In the present paper the period of transition from synchronous to asynchronous cell divisions was studied in blastomeres of the animal region in sturgeon
and frog embryos. The period under study differs from the synchronous period
by desynchronization of cell divisions and from the asynchronous period—by
the absence of a decrease in the mitotic index.
The onset of desynchronization is characterized by the appearance, first, of
single desynchronized nuclei and at the next division many such nuclei. The onset
of desynchronization depends on the temperature: at high temperatures the
disturbance of synchrony occurs at earlier stages and occurs within one mitotic
cycle in many nuclei simultaneously. At low temperatures it occurs at a later
cleavage stage and in a smaller number of nuclei. The onset of desynchronization, when synchronously dividing nuclei are still predominant, is followed by a
complete desynchronization of cell divisions when the relative number of
different mitotic phases in the cells corresponds on the average with the relative
duration of these phases during the synchronous period. The mitotic index by
this time is approximately equal to 53 %, if telophase is considered as an active
mitotic phase, and equals about 46 %, if telophase is counted with interphase.
The end of the period of desynchronization and the onset of the asynchronous
period (the middle blastula stage) is characterized by a sharp decrease of the
mitotic index: within one cell division it decreases from 53 % to 19 %. This
decrease coincides in time, according to Neyfakh (1961), with the onset of
morphogenetic nuclear function. Relative durations of the synchronous period
and of the period of desynchronization vary at different temperatures owing to
the shift of the onset of desynchronization to earlier or later cleavage stages.
Thus, according to the data presented in Fig. 3 the period of desynchronization
at 23 °C equals 35 % of the whole cleavage period (from the first cleavage
metaphase to the onset of gastrulation), at 20 °C—20 %, at 17 °C—24 %, at 12 °C—
13-15 %. The duration of the asynchronous period is rather constant at optimum
temperatures; at low temperatures it increases by 2 T0 owing to the shift of the
onset of gastrulation to later stages (Krönig, 1960).
Low temperatures have a synchronizing effect on the cells of sturgeon and
frog embryos, i.e. have the same effect as on HeLa cells (Rao & Engelberg,
1966). However, in blastomeres of frog and sturgeon embryos the frequency of
metaphase does not increase at low temperatures. Unlike low temperatures, high
temperatures accelerate the onset of desynchronization.
Many years ago Rückert (1899) found in the course of the division of different
blastomeres of Elasmobranchia embryos a period during which multiple numbers
of blastomeres were still produced, even though nuclei were already dividing
asynchronously. A similar phenomenon was found in loach embryos (Rott &
24
E M B 23
370
E. V. CHULITSKAIA
Sheveleva, 1968). They showed that a complete desynchronization of cell
divisions in loach embryos occurred one cell cycle earlier than a decrease of the
mitotic index. This suggests the occurrence of a period of desynchronization in
loach embryos also. In loach embryos it occurs within one cell cycle, whereas in
frog and sturgeon embryos it occurs over a considerable period of time
(up to 6 T0).
In the course of maturation the cytoplasm of eggs acquires the ability to
synchronize cell divisions (Dettlaff e? al. 1964) and to stimulate DNA synthesis
(Graham et al. 1966). Injection of such cytoplasm into cleaving embryos
shifts the onset of desynchronization of cell divisions but has no effect (in the
amounts used in these experiments) on the time of the decrease in mitotic index
and the onset of gastrulation. Accordingly, the duration of the asynchronous
period in all experimental groups was constant and equalled 30 % of the duration
of the whole period of cleavage (see Fig. 4). Unlike the asynchronous period,
the duration of the synchronous period and that of desynchronization vary
owing to the shift of the onset of desynchronization. Thus, the duration of
desynchronization at 20 °C in normal untreated embryos equals 30 % of the
whole period in cleavage; in embryos injected with the mature cytoplasm it
equals 25 % and in those injected with the supernatant of the homogenate of
mature eggs it equals 20 %.
The data obtained suggest the relative independence of the processes studied:
desynchronization of cell divisions on the one hand, and prolongation of
interphase and decrease of mitotic index, on the other hand. In such a way we
interpret the fact that different temperature conditions and injection of the mature
egg cytoplasm shift the onset of desynchronization but have no effect on the
time of the decrease in mitotic index.
It is rather difficult for the time being to answer the question how desynchronization occurs in cells. One can believe that desynchronization arises from a
proportional increase of the duration of all mitotic phases which does not occur
simultaneously in all cells. If it were proved experimentally, it would explain the
appearance of desynchronization independently of a decrease in the mitotic
index.
The preservation of the relative durations of different mitotic phases, which
has recently been found during the transition from the 1st maturation division
in sturgeon oocytes to the 2nd maturation division and cleavage divisions, was
accompanied by sharp changes (shortening) both in the duration of the whole
cycle and that of separate mitotic phases (Vassetsky, 1969).
The experiments with the injection of the mature egg cytoplasm lead to the
conclusion that synchronous divisions are conditioned by the presence in the
cytoplasm of mature eggs of some substances disappearing at subsequent
cleavage stages. In this connexion of great interest are the data (Crippa, Davidson & Mirsky, 1967) indicating that in Xenopus a sudden disintegration of the
long-lived RNA, synthesized during oogenesis, takes place at the blastula
Desynchromzation
of cleavage divisions
371
stage. Of great interest also is the question about the nature of substances
responsible for synchronization of cell divisions. The presence of these substances
in the supernatant of homogenized mature eggs and their capacity to shift the
onset of desynchromzation, offer new possibilities for the investigation of their
nature.
SUMMARY
1. The time and character of the transition from synchronous to asynchronous
cleavage divisions were studied in the animal region and marginal zone in
sturgeon and frog embryos at different temperatures. The effect of the mature
egg cytoplasm on this process was also studied.
2. The transitional period, termed as the period of desynchronization, was
found to occur between synchronous and asynchronous cleavage divisions. It
differs from the synchronous period by desynchronization of mitotic phases in
nuclei of different blastomeres and from the asynchronous period by the absence of the decrease in the mitotic index. After complete desynchronization, the
relative number of nuclei at different mitotic phases corresponds on the average
with the duration of these phases during the synchronous period. During the
period of desynchronization nuclei still have karyomere structure.
3. Desynchronization usually begins with the appearance of single desynchronized nuclei. At the next division a considerable proportion of nuclei divide
asynchronously. The onset of desynchronization depends on temperature: at
extreme temperatures the differences in the time of the onset of desynchronization may amount to 2 T0. The end of desynchronization is characterized by a
sharp decrease in the mitotic index and does not depend on temperature. Both in
sturgeons and frogs it occurs at the same stage: at 15 T0.
4. The onset of the asynchronous period is characterized by a sharp decrease
in the mitotic index within one cell division and accordingly by an increase of the
percentage of interphase cells. Interphase nuclei at this time do not have karyomere structure and do contain nucleoli. A sharp decrease in the mitotic index in
sturgeon and frog embryos in the course of cleavage coincides in time with the
onset of morphogenetic nuclear function. The duration of the asynchronous
period is constant at the optimum temperatures but varies at extreme temperatures, owing to a shift of the onset of gastrulation to earlier cleavage stages (at
high temperatures) or to later ones (at low temperatures).
5. In embryos injected with cytoplasm (3-5 % of the total egg volume) from
mature unfertilized eggs, desynchronization starts one division later as compared with normal untreated embryos; desynchronization starts two divisions
later if embryos are injected with the supernatant fluid from homogenized
mature unfertilized eggs. However, these injections have no effect on the time of
the sharp decrease in the mitotic index and the onset of gastrulation.
24-2
372
E. V. CHULITSKAIA
Pe3hOMe
1. H a 3apo,AbiLLiax oceTpa (Acipenser güldenstädti colchicus V. Marti) H jiflryiuKH
(Rana temporaria) M3yHajiH BpeMA M x a p a r r e p nepexojia OT CHHxpoHHbix ^ejieHMÎi
^pOÖJieHHH K aCMHXpOHHblM B ÖJiaCTOMepaX aHHMaJIbHOH OOJiaCTM H KpaeBOH 3 0 H b l
npH pa3Hbix TeMnepaTypax. Mccue^OBajiH TaKwe BJIHAHHC Ha 3TOT npouecc HHTOnjia3Mbi 3pejioro flfiu,a.
2. Y 3apOAbiujefi H3yMeHHbix BHAOB MOK^y CHHXPOHHMM H acHHxpoHHbiM nepnoAaiviM ApoöJieHHfl JIOKHT nepHOfl ,necHHxpoHH3au,HH. O T nepno^a CHHxpoHHbix
fleJieHHH
OH OTJIHHaeTCH ÄeCHHXpOHH3aiIHeH (|)a3 M H T 0 3 a B
flßpax
p a 3 H b l X ÖJiaCTO-
MepoB, a OT acHHXpoHHoro nepHOzia-OTcyTCTBHeM yjioBHMoro HauiHM MCTOAOM
nazieHHH MHTOTHnecKoro HH^eKca (MM). Flocjie HacrynjieHHfl nojiHon ^ecHHxpOHH3au,HH B AejieHHH pa3Hbix H^ep nacTOTa, c KOTopofi BCTpenaioTCfl flflpa Ha pa3Hbix
4>a3ax MHT03a B KjieTKax o^Horo 3apoflbiuia B cpedueM 6üH3Ka eme K OTHOCHTejibHOH
npOflOJDKHTeJIbHOCTW 3THX (j)a3 BO BpeMfl U,HKJia OflHOTO ACJieHHfl B nepWOfl CHH-
xpOHHbix aeneHMvt .apoÔJieHHfl. B nepHO,n ,necHHxpoHH3auHH flxupa wvietoT eme KapnoMepnoe cTpoeHHe.
3. riepexoA OT CHHxpoHHbix AejieHHiï ^.poÖJieHHH K ,zi,ecHHxpoHH3au,Mn HanHHaeTCfl
KaK npaBHJio c HapyweHHfl CHHXPOHHOCTH Renemm B eji,HHH4Hbix flApax, a Ha cjiejiyiomeM ^ejieHHH y x e 3HaMMTejibHaH nacTb a^ep HaHHHaeT ^ejiHTbca acHHxpoHHO.
Hanajio nepnoflaflecMHxpOHH3au,HHnpn pa3Hbix TeMnepaTypax 3aivieTH0 BapbMpyeT;
npH KpaHHHX TeMnepaTypax pa3JiHHHfl B HacTynjieHHH ^ecHHxp0HH3au,HH n3MepflK>TCfl
2 T 0 . KoHeit nepno/ia flecHHxpoHH3au,HH onpeztenfleTCfl pe3KHM na^eHHeM M H M He
3aBHCnT OT TeMnepaTypbi: KaK y oceTpa, TaK n y jiflryuiKW OH npnyponeH Bcer^a K
OAHOMy H TOMy 7KQflCTieHHK)— CTaßHH 15 T0.
4. Hanajio acHHXpoHHoro nepHO^a xapaKTepn3yeTCH pe3KHM na,o,eHneM MM B
TeneHHe o^Horo ztejieHHfl M cooTBeTCTByjomuM B03pacTaHneM npoueHTa KJICTOK B
COCTOAHHH HHTep(j)a3br. HHTepcj)a3Hbie flflpa B STO BpeMfl He MMeioT y>Ke KapnoMepHoro CTpoeHHH H co,n,ep>KaT njyphuuKH. Pe3Koe na^eHHe MM y 3apoAbTUjeH oceTpa M
jiaryujKH Ha CTaxu-iH apoôJieHHfl coBna^aeT co CTa^new Hanajia MopchoreHeTHHecKon
(J)yHKU,HH fl,nep. npoflOJDKHTejibHOCTb acHHXpoHHoro nep^o^a npw onTMMajibHbix
TeMnepaTypax nocTOAHHa H MO>KCT M3MeHflTbCfl TOJTbKO npw KpaHHMx TeMnepaTypax
3a cneT cMemeHMfl Hanajia racTpyjifliinn Ha HeMHoro 6ojTee paHHne cTa/inn zipoöjieHHa (npw AeiïcTBHH BHCOKHX TeMnepaTyp) HJTW Ha 6ojiee no3flHne cTa^nw (npw
^eftcTBHH HH3KHX TeMnepaTyp).
5. npH HHteKUHH B Apo6fluj,HHC5i 3apoAbiin Heóojibujoro KOJiHMecTBa(3-5 % oóteMa
noua) i;HTonjia3Mbi H3 3pejioro HeonjiOAOTBOpeHHoro afiua A6CHHXpOHH3auHH B
fleJieHHH XRçp HaHMHaeTCfl Ha O^HO ziejreHHe no3/i,Hee no cpaBHeHHio c HopMajibHbiMH
HeonepnpoBaHHbiMH 3apozii>niiaMH, a npn ^o6aBJieHHn Hafloca^OMHoft JKHAKOCTH
roMoreHH3HpoBaHHbix 3pejibix HeonjioAOTBopeHHbix HHII, — Ha ^Ba ,a,ejieHHH no3AHee.
B TO 7KQ BpeM5i Ha CTaAHK) pe3Koro nafl,eHMfl MM H HanajTO racTpyjiflUHH noAOÔHbie
MH^eKLIHH He OKa3bIBaK)T BJlHflHHfl.
I am grateful to Professor T. A. Dettlaff for helpful discussion at all stages of this
investigation.
Desynchronizatlon of cleavage divisions
373
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(Manuscript received 17 October 1968,
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