ABSTRACT
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 a2, b2, c2, d2 and the macromeres by a1,b1,c1,d1. The members of the other tier of four cells divide equally into two mesomeres A1, A2, B1, 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 simultaeously. The following chart indicates the process of segmentation as observed ; the last column of the chart is inserted from the account given by MacBride.
Cleavage chart of Echinus miliaris
The velocity of segmentation is shown in Table I. These observations were made 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were kept in beakers containing about 300 c.c. sea-water which was kept agitated, except when a sample of eggs was withdrawn for examination.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.
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 17° 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.
In 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 inconveniently small it is essential that they should be kept agitated in the water ; if they are allowed to settle to the bottom and thus crowd together, the velocity of segmentation is rapidly reduced and tends to vary greatly in individual eggs. If kept agitated and the 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 Salmo fario 1 gm. of embryo consumes the same amount of oxygen per hour independently of its age.
For young fish embryos it is clear that the oxygen consumed per hour in a 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 of living material in an echinoderm egg at different phases of its development. It has 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 17° 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.
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 development in cold-blooded animals, although in warm-blooded animals, such as the chick, the percentage 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 good. If, however, we may assume that only a definite fraction of the original respiration of the egg is associated with growing elements then the observed figures follow the same law as that applicable to growth in vitro.
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 secondary nature. Such a conclusion is supported by the fact that a sudden increase of respiration 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.
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 than as one of cell-reproduction is familiar to embryologists and is in harmony with 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 with the reduplication of cells grown in vitro, or with the process of reproduction 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-urchin is, by cleavage, subdivided into portions of convenient size for differentiation, 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 derm cell at a later stage) is presumably smaller than the critical size and so does not divide 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
Successive cleavages of the egg of Echinus miliaris are separated by equal intervals of time.
The time required for the complete cleavage cycle is independent of the size of the cell.
The rate of cell-division bears no obvious relationship to the rate of metabolism.
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.