THE MECHANISM OF CELL-DIVISION
III. THE RELATIONSHIP BETWEEN CELL-DIVISION AND
GROWTH IN SEGMENTING EGGS
BY J. GRAY,
King's College, Cambridge.
(From the Marine Station, Millport, N.B.)
(Received ist September 1926)
(With One Chart and One Text-figure.)
THE fact that dividing cells are usually only found in actively growing tissues does
not necessarily prove that growth and cell-division are two closely correlated phenomena. Growth may be defined as an increase in the amount of organised material
which is present in the cell and which is essentially part of the living machine.
Cell-division, on the other hand, only implies the cleavage of the cell into two or
more parts. The purpose of this work is to enquire into the relationship between
the two phenomena when they are taking place together. The material used was
the segmenting eggs of Echinus miliaris.
I. THE VELOCITY OF CELL-DIVISION.
The course of segmentation has been briefly described by MacBride (1914)
and is as follows. The first three divisions divide the egg into eight equal blastomeres arranged in two tiers of four cells each. These can be denoted by the letters
A, B, C, D and a, b, c, d respectively. The 4th cleavage gives the 16-celled stage
by a division of each of the members of one tier into a small cell below, known as
a micromere, and a larger cell above, known as a macromere. The micromeres can
be denoted by the letters a%, b2, c2, d2 and the macromeres by alt blt clt d1. The
members of the other tier of four cells divide equally into two mesomeres Ax, A2,
By, B2, etc. The 5th cleavage involves the unequal division of the micromeres into
smaller and larger micromeres; each of the other cells divides almost equally,
giving a total of 32 cells. Before the blastula stage is reached each of the smaller
micromeres divides only once, each larger micromere divides three times, and each
mesomere and macromere divides five times (MacBride, 1914).
All the cells can readily be observed up to the 32-celled stage, but in the 6th
and 7th cleavages only the mesomeres and macromeres were carefully observed.
During all these cleavages the different types of blastomere divide practically
sj^Htaneously. The following chart indicates the process of segmentation as
observed; the last column of the chart is inserted from the account given by
MacBride.
J. GRAY
Cleavage chart of Echinus miliaris
The Mechanism of Cell-division
315
velocity of segmentation is shown in Table I. These observations were
^ P at a temperature of 17-0° (± 0-5°) C. The cultures were kept in beakers
containing about 300 c.c. sea-water which was kept agitated, except when a sample
of eggs was withdrawn for examination. The method of timing the cleavage was as
follows. The time required for the first appearance of cleavage furrows was noted,
then the time required for 50 per cent, of the eggs to show signs of cleavage, and
finally the time for 90 per cent, of the eggs to show complete cleavage. The average
of the first and last observations was found to agree to within 2 or 3 minutes with
the second observation, and this was taken as the average value of the time required
for cleavage. By this method it is possible to compensate for the fact that the time
elapsing between the first appearance of the cleavage furrow and the completion
of the cleavage decreases as the sizes of the cells decrease with successive cleavages.
Table I.
Time in minutes
9
A
A
B
C
D
E
E
a
b
c
c
d
e
Average
From
From
From
From
From
From
From
fert. to 1st 1st to 2nd 2nd to 3rd 3rd to 4th 4th to sth Sth to 6th 6th to 7th
cleavage
cleavage
cleavage
cleavage
cleavage
cleavage
cleavage
60
72
35
3O
40
69
69
65
65
28
28
34
34
38
33
67
33
32
35
37
35
35
35
34
35
33
33
37
37
33
34
34
3i
3i
31
33
35
35
35
38
38
32
. 36
35
35
33
32
33
33
The figures show clearly that if allowance is made for the time required for the
fusion of the male and female pronuclei (roughly 30 minutes at 170 C.) each of the
first seven cleavages occurs in a regular sequence and the time required for a complete cleavage cycle is the same in each case although the size of most of the cells
has decreased almost in geometrical progression: 1, -5, -25, -125, -06, -03.
As far as I know, the only comparable observations are those of Erdmann (1908)
for Strongylocentrotus lividus whose results differ fundamentally from those given above.
Erdmann's figures are given below.
No. of cleavage
Time in minutes required for cleavage
cycle at room temperature
Series A
ISt
2nd
3rd
4th
5th
6th
100
40
5°
74
107
115
Series B
99
36
56
72
107
my experience an apparent reduction in the velocity of segmentation is the result
of inappropriate experimental conditions; unfortunately Erdmann does not appear to
describe her experiments in any detail. Unless the number of eggs used in a culture is
3
J. GRAY
i6
inconveniently small it is essential that they should be kept agitated in the water; if J
are allowed to settle to the bottom and thus crowd together, the velocity of segment
is rapidly reduced and tends to vary greatly in individual eggs. If kept agitated andThe
water changed periodically, quite dense suspensions develop quite normally at a uniform
rate.
As far as Echinus miliaris is concerned, Table I establishes the fact that the
velocity of cell-division is independent of the size of the cell since the small micromeres at the vegetative pole of the egg divide at the same rate as the larger
macromeres. It has already been shown that the active agents which effect the
division of the cells are the asters (Gray, 1925) and that the size of the resultant cells
of a cleavage depends on the volume of the fully developed asters. The simplest
conception of the whole process is that the size of the cell is proportional to the size
of the fully formed cleavage aster and that the rate of division depends on the rate
of formation of the aster. In this way the rate of cleavage is independent of the size
of the cell. It should be remembered that although the actual cleavage of these cells
is a discontinuous process occurring about every 35 minutes, yet the whole of this
period is occupied by the steady development of the cleavage mechanism, viz. the
asters. There is no period of quiescence from cleavage; this is of importance when
comparison is made with other types of cell.
Table I also shows that the velocity of cleavage of different batches of eggs is
approximately the same. It was also found to be independent of the sperm used,
even if the latter had been mixed with sea-water for about 2 hours.
II. THE VELOCITY OF GROWTH.
By the growth of an egg is meant the conversion of yolky material into elements
which are essentially parts of the living organism. In the case of an echinoderm egg
this process can only be followed by the adoption of some more or less arbitrary
standard by which to measure the amount of living material present. In the case
of a fish embryo it is possible to measure the rate of conversion of yolk into living
embryo by mechanical separation and by weighing. The following table shows
that for a considerable period during the early stages of development of Salmofario
1 gm. of embryo consumes the same amount of oxygen per hour independently
of its age.
Age in days
Wet weight of
embryo in mg.
c.c. O2 per gm.
per hour
55
58
60
64
68
32
39
43
55
70
'62
•67
•65
•66
•64
For young fish embryos it is clear that the oxygen consumed per hour i
given time is a measure of the amount of living embryonic tissue present.
^
It therefore seems reasonable to apply the same test to tissues of a different type.
In other words, the level of respiration can be used as an index of the amount
The Mechanism of Cell-division
317
oLliving material in an echinoderm egg at different phases of its development.
^Pnas been shown by Meyerhof (1911) and by Warburg (1918) that the heat production and the O2 consumption increases during development to a level considerably higher than that of the newly fertilised egg. These results are usually
expressed in such a form as to suggest that each successive cleavage produces
a definite increase in the amount of heat evolved or in the amount of O2 consumed.
Meyerhof's original data and the observations published in a previous paper
(Gray, 1925 a) show clearly, however, that the act of cleavage does not in itself
materially affect metabolism. The following experiments constitute an attempt
to determine whether or not the metabolic activity of the cells determines the
moment at which cleavage occurs. The technique of the experiments has
already been described (1925 a) and I have to thank the staff of the Marine
Biological Station, Millport, N.B., for the assistance which made it possible to
carry out the work.
Fig. 1 shows the course of the respiration during the first 10 hours of development at 170 C. Five more or less complete experiments were performed, the details
of which are recorded in Table II, whereas in Fig. 1 the average results are recorded.
Table II. Echinus miliaris.
T i m e after
fertilisation
Relative amount of O 2 consumed per hour
A
B
C
D
E
Average
IOO
102
IOO
102
102
IOO
IOI
IOO
102
III
113
118
122
126
133
IOO
103
105
112
I2O
13O
142
IOO
104
108
119
122
122
140
140
IOO
102
104
h. m.
0
30
1
0
1 3°
2
2
0
30
3 °
3 3°
4
0
4 3°
5
0
5 3°
6 0
6 30
7 0
7 30
8 0
8 30
9 0
10
0
i°3
III
116
122
127
I2
Z
136
137
145
162
172
2OO
2IO
219
234
234
235
no
"9
"9
126
132
141
153
161
173
204
229
249
249
258
256
135
153
I7O
193
2OI
206
148
162
177
199
2l8
243
255
2
|5
260
260
264
147
155
174
183
200
218
255
270
279
272
in
118
121
131
134
142
151
163
178
198
219
242
248
258
257
If the whole of the respiration of the newly fertilised egg were due to the activity of
the system taking part in growth, it would be very difficult to harmonise the above results
with what is known of other growing tissues. In the case of cells growing in vitro the
amount of organised tissue formed by a unit mass in unit time is a constant quantity; in
the case of growing organisms the same law appears to apply to the early stages of developiM^t in cold-blooded animals, although in warm-blooded animals, such as the chick, the
jSreentage growth-rate may show a decline from an early stage. If the increase in respiration for each successive half-hour in Table II be expressed as a fraction of the respiratory
level at the beginning of the period it will be found that neither of the above rules holds
J. GRAY
good. If, however, we may assume that only a definite fraction of the original l t O p
of the egg is associated with growing elements then the observed figures follow the
law as that applicable to growth in vitro.
260
240
220
200
.180
160
B/astulae beginning
to swim
120
100
80
6040
20
cleavages
^r
gnd yd ^fh gfh gfh '
lUntitt.
i
2
3 4
5 6
7
10
Hours after Fertilisation
Fig. I.
If i?0 be the total rate of respiration of the newly fertilised egg, and if A be the respiration due to the non-growing elements in the egg then (Rg — A) represents the respiration
due to the growing elements. If the rate of respiration is proportional to the amount of
this latter type of substance then
f^HR-A)
where R is the total rate of respiration at time t. That this assumption fits the observed
figures is shown in Table III.
It is possibly simply a coincidence that the value of
is of the same order as
the ratio which exists between the respiration of the egg before fertilisation to the value it
settles down to soon after fertilisation. If, on the other hand, the level of the respiration
prior to fertilisation is really an index of the amount of growing substance in the cell, then
the sudden increase which occurs on fertilisation must be regarded as of a secoi^By
nature. Such a conclusion is supported by the fact that a sudden increase of respir^ron
on fertilisation is not shown by eggs other than those of the sea-urchin; but conclusions
based solely on the efficiency of a convenient formula should not be given much weight.
The Mechanism of Cell-division
3*9
Table III. A=
(R-A)
R
R
h. m.
(R-A)
obs.
calc.
obs.
calc.
0 30
1 0
1 30
2
0
2 30
3
0
3 3°
4
0
IS
IS
18
100
102
100
17
19
26
T
4 3°
5
0
5 3°
6
6
7
0
30
0
33
36
46
49
57
66
78
25
29
34
121
4i
131
134
142
151
163
114
119
126
134
142
152
164
178
178
198
219
195
217
49
57
67
113
79
93
no
'34
132
93
103
106
104
in
118
21
no
Fig. 1 shows that, from the time of fertilisation until just before the blastula
is ready to swim, the respiratory level has risen to about 2-6 times its initial value;
in other words, it requires about 6-5 hours for the living material in the egg to
double its mass. Even if we accept the view expressed by Table III the time
required to double the respiratory level of the growing organism is about 2-5 hours.
In both cases it is clear that cleavage occurs long before each resultant blastomere
contains as much living material as the initial undivided cell. Both the velocity
of metabolism and the total metabolism between successive cleavages is different,
and we must conclude that although the velocity of growth and the velocity of
cleavage may depend on some common factor, yet they are not dependent on one
another.
The sudden rise in the velocity of respiration which occurs when the blastula
begins to swim may reasonably be associated with the expenditure of mechanical
energy. At present no reason can be assigned for the fall in the acceleration which
precedes the hatching of the blastula. It may indicate that the blastula represents
the end of a definite growth cycle; if this be the case, it is noticeable that the cycle
is asymmetrical in nature.
III. DISCUSSION.
The facts described in this paper suggest that considerable caution is necessary
in using the phenomenon of cell-division as an index of growth. The lack of any
obvious correlation between the rate of cleavage and the rate of growth of a segmenting egg indicates that segmentation is to be regarded as a process of subdivision
of the organism into parts convenient in size for subsequent differentiation into
the various tissues. This conception of cleavage as a process of subdivision rather
as one of cell-reproduction is familiar to embryologists and is in harmony
the fact that the same larval structure can consist of a varying number of cells
*
according to the conditions of development. This point of view is often neglected,
however, when comparisons are made between the segmentation of an egg either
320
J. GRAY
with the reduplication of cells grown in vitro, or with the process of reproduc|jMi
in the Protozoa.
^^
In the case of the Protozoa, yeast, or bacteria, a definite correlation appears to
exist between the velocity of growth and the velocity of cleavage. Perhaps the most
striking example of this is provided by the experiments of Slator (1913). This
author showed that the specific rate of increase in the number of yeast cells in
a nutritive medium can be measured by an estimation of the specific rate of increase
in the fermentative powers of the culture; the results of his experiments show that
the fermentative power of a single yeast cell has the same average value throughout
a prolonged period of cell-proliferation; in other words, a yeast cell grows to a definite
average size before proliferating. A similar state of affairs probably applies to
bacteria, Protozoa, and vertebrate tissues grown in vitro. The average size of
individual cells of this type remains fairly constant even after prolonged celldivision, showing that before division occurs growth has compensated for the
reduction in size effected by a previous division. It must be remembered, however,
that division in these cases is probably of a discontinuous nature, and, unlike the
segmenting egg, there are distinct pauses between the completion of one cleavage
cycle and the initiation of another.
Even in unicellular forms it is not difficult to upset the correlation between celldivision and growth. Henrici (1924) found considerable variations in the size of
individual Bacterium coli during the life of his cultures. Again, if for any cause
a culture of Protozoa becomes unhealthy the process of cleavage is less affected
than that of growth and the average size of individual organisms show a marked
reduction.
It has been shown that during the first seven cleavages of the egg of E. miliaris
the velocity of division is constant at the same value for all the cells irrespective
of their size. At a later stage, however, other factors intervene, particularly in
the case of the smaller cells, which cease to divide before the blastula stage is
reached. This is a well-known phenomenon and shows that at various times single
cells or groups of cells may cease to divide or may acquire a cleavage rhythm
different from that of their neighbours. Unfortunately, nothing is known at present of
the growth processes accompanying such changes. How far changes in the rate of
cell-division are related to cell-differentiation cannot be considered in any detail.
Different types of cell differ in this respect. Nerve cells and muscle fibres do not
divide when once differentiation has begun but they grow very actively during the
whole growth cycle of the animal. Epithelial cells, however, appear to divide as
soon as they have grown to some critical size.
From the evidence at our disposal it may be concluded that there is probably
no fundamental association between growth and cell-division. On the other hand,
each type of cell appears to have some characteristic size which is reached by
a balance between growth and cleavage. The large fertilised egg of the sea-urofaui
is, by cleavage, subdivided into portions of convenient size for differential^,
and the cleavage mechanism proceeds to effect this critical size without any pause
between the divisions. A newly divided Protozoon or epithelial cell (or an echino
The Mechanism of Cell-division
321
derm cell at a later stage) is presumably smaller than the critical size and so does
^ftdivide until this size is reached by growth; during this period the cleavage
mechanism remains inert. From this point of view cell-division and growth are
the two processes whereby a growing tissue maintains its cells at an appropriate
size. They are, however, two very distinct phenomena.
SUMMARY.
1. Successive cleavages of the egg of Echinus miliaris are separated by equal
intervals of time.
2. The time required for the complete cleavage cycle is independent of the size
of the cell.
3. The rate of cell-division bears no obvious relationship to the rate of metabolism.
4. Cell-division and growth are the factors which determine the size of individual cells. In some cases these factors may establish a well-defined equilibrium,
but in segmenting eggs they are probably quite independent of each other.
BIBLIOGRAPHY.
ERDMANN, R. (1908). Arch./. Zellforsch. 2, 76.
GRAY, J. (1925). Proc. Comb. Phil. Soc. (Biol. Set.), 1, 164.
(1925 a). Proc. Camb. Phil. Soc. (Biol. Sd.), 1, 225.
HENRICI, A. T. (1924). Proc. Soc. Exp. Biol. and Med. 21, 345.
MACBRIDE, E. W. (1914). Text of Embryology, 1. London.
MEYERHOF, O. (1911). Biochem. Zeit. 35, 246.
MURRAY, H. A. (1926). Journ. Gen. Physiol. 9, 1.
SLATOR, A. (1913). Biochem. Journ. 7, 197.
WARBURG, O. (1918). Pfluger's Arch. 160, 324.
BjEB-iviv
22