# Popular Science Monthly/Volume 83/August 1913/The Size of Organisms and of their Constituent Parts in Relation to Longevity, Senescence and Rejuvenescence

(1913)
The Size of Organisms and of their Constituent Parts in Relation to Longevity, Senescence and Rejuvenescence by Edwin Grant Conklin

 THE SIZE OF ORGANISMS AND OF THEIR CONSTITUENT PARTS IN RELATION TO LONGEVITY, SENESCENCE AND REJUVENESCENCE[1]

By Professor EDWIN G. CONKLIN

PRINCETON UNIVERSITY

I. Body Size

BODY size is one of the most variable properties of organism; the smallest living things are probably invisible to the highest powers of the microscope, the largest are gigantic beasts weighing many tons. Within the same class, and in animals equally complex in structure, variations in size are enormous, as, for example, in the elephant and the mouse. Within the same species, where structural differences are insignificant, size differences may be very great. In some species there are great differences of size between males and females; in extreme cases males may be minute and rudimentary forms, without mouths and alimentary canals, and capable of living for only a few hours, as in certain rotifers, worms and arthropods, whereas the females are relatively large and perfect individuals capable of an extended existence.

In Crepidula a genus of marine gasteropod which I have studied and to which I must particularly direct your attention, I have found[2] great differences of body size in the mature individuals of different species and also in different individuals of the same species. The volume of the average adult male of C. fornicata is 125 times that of the average male of C. convexa; the volume of the female of the former species in 33 times that of the latter. In these gasteropods the males are always much smaller than the females; the volume of the average female of C. plana is about 15 times that of the average male. All mature animals of this genus are sedentary, and many of them live in or on dead shells which are the homes of hermit crabs. In the species C. plana I have found an interesting class of dwarfs; the animals of usual size live in large shells inhabited by a species of large hermit crabs (Pagurus hernhardus); the dwarfs live in small shells occupied by a species of little hermits (Pagurus longicarpus). The dwarfs are sexually mature and, unless forcibly removed, live their whole life long in the small shells, where they attain an average size only one thirteenth that of the normal forms but if the dwarfs are forcibly taken out of the small shells and put into larger ones they may grow up to be as large as animals of typical size. These dwarfs are, therefore, only a physiological variety, produced by environmental conditions.

What are the causes of such differences in size of animals of the same species? What explanation can be offered for the enormous difference in size between an elephant and a mouse? What are the factors generally involved in determining size?

1. There is plainly an inherited factor in all specific differences of this kind. Every species of animal and plant has a more or less characteristic body size which may be said to constitute the norm of that species. This norm may be altered to a certain extent by environmental conditions, but such possible modifications are relatively slight; no amount of environmental influence could ever make a mouse grow to the size of an elephant. The limits of body size of a race or species are as certainly inherited as are any other characteristics; their causes, whatever they may be, are intrinsic in the constitution of the germinal protoplasm.

What is the nature of this inherited factor which determines the possible size of organisms? Undoubtedly it is found in the power of growth as contrasted with limitations to growth, with the rate and duration of assimilation as contrasted with dissimilation. Increase in size may be due to mere imbibition of water, or to an actual increase in the quantity of protoplasm, and secondarily of formed products, in the body. In this discussion the latter process alone will be termed growth. As long as assimilation exceeds dissimilation organisms grow, when the one balances the other they remain unchanged in size, when dissimilation exceeds assimilation they dwindle. The large-sized Crepidulæ continue to grow for a much longer time than the small-sized ones. A mouse achieves its full growth after 60 days and may live approximately 60 months; an elephant continues to grow for about 24 years and may live approximately 150 years.

What it is which keeps up this process of growth so much longer in one species than in another we do not know—and as so often happens, it is precisely this which we most desire to know, for length of life as well as size of body depends primarily upon the rate and duration of assimilation. It may be that there is some peculiar secretion or enzyme which stimulates growth and varying quantities of which cause one species to continue to live and grow for a much longer time than another species; it may be that some substance is formed in the course of development which limits growth and that it appears earlier in some species than in others. Since assimilation and dissimilation are chemical processes it is very probable that the factors which determine rate and duration of growth, and consequently body size and length of life, are of a chemical nature. This is a subject upon which there has been much speculation and but little work and to which experimental investigation might well be directed with promise of important results.

2. Another supposed factor which is not precisely hereditary nor yet strictly environmental is the size of the germ cells, of the "Ausgangszellen," from which an animal develops. Morgan[3] and Chambers[4] found that small eggs of the frog give rise to smaller tadpoles and to smaller frogs than do large eggs. Popoff[5] maintains that spermatozoa as well as ova vary in size, owing to slight inequalities of division during the genesis of these cells, and he supposes that when a large egg is fertilized by a large spermatozoon a large individual results, whereas if the sex cells are smaller than usual the individual developing from them will also be smaller. In favor of this hypothesis may be cited the fact that small eggs of Rotifera, Phylloxera and Dinophilus give rise to small and rudimentary males, whereas the larger eggs give rise to relatively large females. Within the same species, where the mode of development is the same for all individuals, egg size may be a factor in determining body size, but it is a relatively unimportant factor, since the size of an animal depends not merely upon its initial size, but chiefly upon the rate and duration of its growth. In many cases the smaller egg continues to grow for a longer period than does the larger one and in the end gives rise to a larger adult. This is strikingly shown in different species of Crepidula, where species with small eggs give rise to large animals and those with large eggs give rise to small animals. The large eggs produce large embryos, and the small eggs small embryos, but the latter continue to grow for a much longer period than the former and in the end give rise to animals of much larger body size than those which come from the large eggs. An egg of C. fornicata is about one quarter the volume of one of C. convexa, but the adult female of the former species is about 32 times the volume of one of the latter species, while the males of the former species are 125 times the volume of those of the latter species. Other cases of a similar sort are known and they show that in different species egg size can not be correlated with body size, and even within the same species it is a relatively unimportant factor in determining size.

3. It is well known that many extrinsic factors influence the cliaracter, rate and duration of metabolism, and consequently the size of organisms. Among these extrinsic factors I shall mention only a few which are known to be important, viz., (a) quantity and quality of food, (b) secretions of certain glands, particularly the sex glands, thymus, thyroid and hypophysis, (c) various chemical substances, such as ether, alcohol, tobacco, lecithin, etc., (d) temperature, (e) oxygen, (f) presence or absence of waste products, (g) conditions of normal or abnormal stimulation and irritability. These extrinsic factors which influence growth have been studied by many investigators, but owing to lack of time I shall pass over all of them except the last named. In the case of the dwarf Crepidulæ which are found in the small shells with the small hermit crabs there is practically no evidence that any of the other factors except the last named, are involved in this dwarfing. These animals live in open shells on sandy sea beaches along with the giant forms; so far as I can determine, the food supply is superabundant, while the conditions of temperature, aeration and freedom from waste products are-identically the same for dwarfs and giants. The only difference which I have been able to detect is the size of the shells to which the animals are attached; those which are attached to the small shells of Nassa or Litorina live and die as dwarfs, reaching only about one thirteenth the volume of those which are attached to the larger shells of Natica; however, if they are removed from the smaller shells and placed on the larger ones they may grow to typical size. The dwarfs, however, are continually hampered by their limited quarters; they are unable fully to expand the foot or the mantle, and they are more frequently irritated by the movements of the hermit crabs than are those in the larger shells. Under these circumstances they probably take less food than those in larger quarters, and although they become perfectly differentiated and sexually mature they are dwarfed in size. Similarly I have found that Paramecium confined in capillary tubes never grows nor divides, though it may live indefinitely, and although precautions may be taken to change the medium frequently and thus to remove waste products and to supply abundant food and oxygen. In such tubes Paramecium is continually irritated and presumably takes less food than when in unconfined spaces.

II. Body Size, Cell Size and Cell Number

Is the size of an organism due to the size of its constitutent parts, or to the number of those parts, or to both of these causes combined? Evidently different organisms differ in this regard. In many plants and lower animals the number of constituent parts is directly correlated with the body size; branches and leaves, segments and organs may increase in number indefinitely with the growth of the organism. In tapeworms and many annelids the number of segments, with their characteristic organs, increases throughout life; but in more highly differentiated forms the number of body segments and organs is constant, and does not increase in number after embryonic stages. In spite of the information occasionally conveyed by examination papers, the number of bones or other organs in the human body does not depend upon the size of the man.

In animals in which the number of organs is constant the constituent parts of such organs may vary in number with the size of the organs. Thus in a large Crepidula plana the gill is composed of more than two hundred large filaments, in a dwarf it consists of only fifty or sixty small ones. The liver, sex glands and salivary glands are composed of a larger number of lobules in large animals than in small ones, and the size of each lobule is also larger. Evidently the number of such body parts, whether segments, organs, filaments or lobules, depends upon the power of growth and subdivision of each of these parts. In general the more complex any part becomes the less capable it is of subdivision, and so in all highly differentiated animals we find the body parts and organs are constant in number, though variable in size; whereas in lower animals the number of body parts as well as their individual size may vary with the size of the body as a whole.

Cells are generally recognized to be the ultimate independent units of organic structure and function; the causes of growth and differentiation, of assimilation and dissimilation, of longevity, senescence and rejuvenescence are to be looked for in cells. What is the relation of body size to cell size and cell number? A large number of investigators have studied this problem in a wide range of animals and plants, and with apparently conflicting results; nevertheless enough is now known I think to permit a general answer to this question. Just as in the case of body parts and organs, so also with cells, complexity of differentiation and power of division are generally in inverse ratio. In many animals and plants certain types of cells continue to divide throughout life, where other types cease to divide at an early age. In both plants and animals those cells which continue to divide throughout the growing period become more numerous in large organisms than in small ones, but not individually larger; on the other hand cells which cease to divide at an early stage in the life cycle become individually larger in large animals than in small ones, though in closely related forms their number may remain the same. In short, the size of cells is directly proportional to the rate and duration of growth and inversely proportional to the rate of division. It is well known that muscle cells and nerve cells cease to divide at a relatively early age, whereas epithelial and gland cells, mesenchyme, blood and sex cells continue to divide for a longer period, if not throughout life; accordingly, one would expect to find that muscle cells and nerve cells are larger in giants than in dwarfs, but that the other types of cells named would differ in number but not in size—and this is the general result reached by most of the investigators who have worked on this subject (Donaldson, Levi, Boveri, Conklin,[6] et al.). In the most highly differentiated cells {e. g., muscle, nerve) growth takes place independently of cell division; in less highly differentiated cells (e. g., epithelium, mesenchyme) the two processes go hand in hand.

It is an important fact that growth in size and growth in complexity arc separable processes, for although they are usually coincident during embryonic development they are not causally united. Just as growth in body size may, or may not, be accompanied by growth in complexity, so cell division may, or may not, be accompanied by differentiation. Cell divisions may thus be classified as differential and non-differential; the former are associated with growth in complexity as well as in size, the latter with growth in size only; the former are relatively constant in number for a given species, the latter vary in number with the size of the individual. The earlier cleavages of the egg are more generally differential than are the later ones, and within the same genus and even in related genera and phyla the number and character of differential cleavages is very constant. Thus in all annelids and mollusks, with the exception of cephalopods, the ectoderm comes from three quartets of cells which are cut off, one after another, at the animal pole of the egg, and in all cases each of these quartets gives rise to homologous regions of the larvæ of the different forms; the left posterior member of the fourth quartet (4d) is the mesentoblast and in all annelids and mollusks (except cephalopods) it gives rise to the mesodermal bands and to the posterior part of the intestine; and in general homologous portions of larval or adult animals come from homologous portions of the eggs of these animals through the medium of homologous differential cleavages.

On the other hand, non-differential cleavages are relatively inconstant in number, position and character; they vary greatly in number in different species, or even in different individuals of the same species, depending upon the size of the egg or embryo. Thus in different species of the genus Crepidula the differential cleavages are almost precisely the same in all, though the relative volumes of the eggs of different species vary from 1 to 27, but the non-differential cleavages are much more numerous in the large eggs than in the small ones. It is the fact that the earlier cleavages of eggs are so generally differential that makes possible the study of cell lineage; if such cleavages were generally non-differential they would be relatively inconstant and lacking in significance.

In animals with determinate cleavage of the egg the number and nature of the cells at any given stage of differentiation is, under normal conditions, absolutely constant for each species, and it may be a constant number even for different species of a genus, especially if the eggs of the different species do not differ greatly in size. In various ascidians (Styela, Ciona, Molgula, Phallusia, Ascidia) there is a close correspondence in the character and number of the cleavage cells present at corresponding stages of development, even up to advanced stages. For example in all these genera there are 118 cells present when the cupshaped gastrula is first formed and the prospective fate of each of these cells is indicated herewith: 10 will give rise to endoderm cells, 12 to muscle cells, 16 to mesenchyme cells, 8 to chorda cells, 8 to neural plate cells, 64 to ectodermal epithelium.

At the stage when the gastrula begins to elongate there are 218 cells distributed as follows: 26 endoderm cells, 12 muscle cells, 20 mesenchyme cells, 16 chorda cells, 40 neural plate cells, 104 ectodermal epithelial cells.

Each of these cells is characteristic in position, structure, size and potency, and this is true of all species and genera of simple ascidians hitherto studied with respect to this matter.

In a number of species of small body size Martini[7][8][9] has determined that there is a high degree of constancy in the number of cells in the adult body. In the appendicularian Fritellaria pellucida the number of cells is constant in the following organs: 28 flattened epithelial cells of body, 446 oikoplasts (columnar epithelial cells of body), 10 large gland cells in the tail, 7 flattened epithelial cells of the pharynx, 10 large cells of the endostyle, 24 small cells of the endostyle, 4 branchial gland cells, 7 branchial cells, 6 ciliated funnel cells, 19 epithelial cells in the stomach, 10 epithelial cells in the pyloris, 17 epithelial cells in the small intestine, 12 epithelial cells in the large intestine, 6 or 7 epithelial cells in the rectum, 39 cells in the brain, 25 cells in the chief caudal ganglion, 23 cells in the remaining nerve cord, 8 nuclei in heart and pericardium, 20 muscle cells, 12 large chorda cells, 4 small chorda cells.

In different individuals of this species there is a high degree of constancy in the number of these cells, the only variation being in the occasional presence or absence of a single subdivision of a cell.

Also in the rotifer Hydatina senia he finds that there are all together just 959 cells, or rather nuclei, in the entire body of the adult, and that each organ consists of a perfectly characteristic number of cells. Even in different species of rotifers the number of cells in many homologous organs is the same; thus there are generally 6 cells in the anterior part of the œsophagus, 6 pairs of cells in the excretory tubules, and 13 cells in the cingulum, one of which is on the dorsal mid line.

In the nematode Ascaris megalocephala Goldschmidt[10] found 162 cells in the nervous system, while Martini[11] finds 65 muscle cells in Oxyuris, and 87 muscle cells in Sclerostoma, the latter being derived from 65 cells of an earlier stage.

A similar constancy of cell number has been found by Woltereck[12] in Polygordius larvæ, by Apathy in the central nervous system of Hirudinea, by Gaule and Donaldson[13] in spinal ganglia of frogs, and by many investigators in small but highly differentiated parts, such as the ommatidia of compound eyes, the lens fibers of vertebrate eyes, the nurse cells of certain arthropod and annelid ova, etc. Such cases of cell constancy are, as Martini remarks, "the crowning fact of determinate development." In all such cases the definite number of cells in the entire body or in a particular organ must be determined by a definite number of cell divisions which proceed from the egg, or from the protoblast of the organ, and this limitation in the number of cell divisions must in some way be determined by heredity. Since increase of differentiation is associated with decrease of cell division, the latter being stopped altogether when differentiation has reached a certain stage, it seems probable that all cases of cell constancy are due to constancy of differentiation.

Where the number of cells in an organ or in an animal is very large it is not possible to prove that the cell number is constant, but in many cases where cell division ceases in embryonic stages the cell number is constant. In such cases cell division does not continue after differentiation is complete, though cell growth does. To all such cases in which there is cell constancy Martini gives the name "Eutelie."

On the other hand, there are many animals in which the number of cells in any particular organ is not constant but is proportional to the size of the organ. In Crepidula the number of egg cells within the ovary and the number laid in any season varies with the size of the animal, but the size of individual eggs remains constant for each species; the same is also true of epithelial cells, gland cells and blood cells. The divisions by which such cells are formed are in general non-differential, and since both growth and division in such cases continue throughout life the size of any given type of cell is fairly uniform whatever the body size may be. In differential cell divisions, or in highly differentiated cells which do not continue to divide throughout life, the size of cells varies directly with the body size and with the infrequency of division.

III. Cell Size and Nucleae Size

In a series of recent papers Richard Hertwig[14][15] and several of his pupils have maintained that there is a definite ratio between the size of the nucleus and the size of the cell; this is the "Kernplasmarelation," or the nucleus-plasma ratio. When this ratio is altered by the greater growth of the nucleus, Hertwig thinks that it leads to a "tension," which brings about division, and thus the normal nucleus-plasma ratio is restored. This ratio is supposed to be a constant one under normal conditions, and if at any time it is altered it is capable of self regulation.

On the other hand, I[16] have found that this ratio varies greatly in different cells of an animal, and indeed within the same cell at different stages of the division cycle, that it may be experimentally altered, and that it is a result, rather than a cause, of the frequency of cell division.

Within the same cell the size of the nucleus varies greatly at different stages of the division cycle, while the volume of the cell as a whole remains relatively constant. The nucleus is smallest during the anaphase, or later stages of division, when it consists of a compact plate of condensed chromosomes; it is largest immediately before the nuclear membrane dissolves at the prophase of the next division. In the cleavage of the egg of Crepidula plana the nucleus-plasma ratio in identically the same cell varies from approximately 1:6 whto the nucleus is largest, to 1:286 when it is smallest; that is, the volume of the nucleus increases nearly 50 times.during the resting period between the previous anaphase and the subsequent prophase; during this same time the volume of the cell remains practically unchanged.

Even when measured at the same phase of the division cycle the nucleus-plasma ratio differs greatly in different cleavage cells; at maximum nuclear size the volume of the nucleus of certain cells of Crepidula ${\displaystyle (4A-4C)}$ may be 3 times that of the protoplasm, whereas in other cells ${\displaystyle (1A-1D)}$ the volume of the protoplasm may be 14.5 times that of the nucleus. At minimum nuclear size the nucleus-plasma ratio may vary from 1:29 in the cells ${\displaystyle 1a^{2}-1d^{2}}$, to 1: 285.5 in the cells ${\displaystyle 1A-1D}$.

The growth of the nucleus between successive divisions is due to the absorption from the cell body of a particular kind of cell substance, which constitutes the achromatin of the nucleus; at the beginning of this growth the nucleus is composed of compact chromosomes, at its end it consists of a large vesicle of achromatic substance in which the chromatin usually exists as scattered granules. At the next division some of these granules form chromosomes and all the rest of the nuclear content is liberated into the cell body, to be again absorbed by the daughter nuclei during the succeeding rest period. There is thus a sort of diastole and systole of the nuclear vesicle during every division cycle of a cell, achromatin being taken up by the nucleus during its growth and liberated again into the cell body during its division.

In different cleavage cells of Crepidula plana, when the yolk is eliminated from consideration, the nucleus-plasma ratio varies from 1:0.37 to 1:14.5; that is, the volume of the actual protoplasm in certain cells may be only one third the volume of the nucleus, or in other cells it may be fourteen times that volume, depending largely upon the length of the resting period.

In general the size of a nucleus is directly proportional to the volume of the general protoplasm in the cell, to the length of the resting period, and in cases of abnormal or irregular distribution of chromosomes, to the number and volume of the initial chromosomes which go to form the nucleus. The inciting cause of cell division is not to be found in departures from a normal nucleus-plasma ratio, which is a result rather than a cause of the rate of cell division, but rather in the coincidence of certain metabolic phases in nucleus, centrosome and protoplasm.

If the growth period of the nucleus is very long, the greater part of the protoplasm may be taken into the nucleus, as in those cleavage cells in which the nuclear volume is about three times as great as that of the protoplasm outside of the nucleus; if the growth period of the nucleus is short, the nucleus remains correspondingly small. If nuclear division is prevented by hypertonic solutions or by decreased oxygen tension, the nuclei may grow to an enormous size until they contain the greater part of the cell protoplasm.[17]

In certain stages of the division cycle it is possible by the use of hypertonic solutions to prevent the daughter chromosomes from absorbing achromatin, and in such cases these chromosomes form small, densely chromatic nuclei, while the achromatin may be gathered into one or many vesicles. In other cases, the chromatin may be caused to contract and to squeeze out the achromatin. The latter case is similar to that which takes place normally in the formation of a spermatozoon from a spermatid, where there is a condensation of the chromatin of the spermatid nucleus and a squeezing out of the achromatin; this diminution of the nucleus is coincident with the transformation of the protoplasm of the spermatid into differentiation products. A similar thing happens in superficial epithelial cells which are undergoing keratinization; up to a certain stage, the nuclei of such cells shrink in size and become more densely chromatic in proportion as the cell protoplasm is converted into metaplasm. The same thing is true of gland cells, muscle cells, fiber cells and fat cells in which the general protoplasm is progressively being changed into differentiation products, and coincidently the individual nuclei shrink in size and become more densely chromatic.

In no case do metaplasmic substances or differentiated structures of the cell enter into the nucleus during its growth, and the relative quantities of general protoplasm and of differentiated products in a cell can be determined by the size to which the nucleus will grow during interkinesis, under given conditions of time, temperature, etc. By subjecting eggs to centrifugal force, the quantities of protoplasm and yolk in the cleavage cells may be greatly changed, and under such circumstances the size of a nucleus is always proportional to the volume of the protoplasm in which it lies; the heavier yolk which segregates at the peripheral pole, and the lighter watery or oily substance which gathers at the central pole of the centrifuged egg do not contribute to nuclear growth, only the clear protoplasm which lies in the middle zone enters the nucleus or contributes to its growth. In muscle cells with small nuclei, the quantity of general protoplasm (sarcoplasm) which may enter into the nucleus or contribute to its growth is small; in nerve cells, it is evidently larger, since the nuclei of such cells are relatively large, but the substance which may enter the nucleus of a nerve cell is by no means as great in quantity as in germ cells and blastomeres, thus indicating that much of the substance of a nerve cell is too highly differentiated to enter into the nucleus. In epithelial and gland cells, the size of nuclei is limited not only by the presence of metabolic products in the cells, but also by the occurrence of cell division and the consequent limitation of the growing period of the nucleus.

The following table gives the cell diameter and nuclear diameter at maximum size, the corresponding nuclear volume, the cell volume less the nuclear volume, and the nucleus-cell ratio, in a number of different kinds of cells in adult individuals of Crepidula plana:

The nucleus-cell ratio of these cells varies from 1:1.3 to 1:88.6, depending primarily upon the quantity of formed substance in the cells. The nuclei arc relatively largest in germ cells before the formation of yolk, and in embryonic cells in which there is relatively little formed substance; in such cases a relatively great part of the protoplasm may enter the nucleus. The nuclei are relatively smallest in those cells in which the protoplasm has been most completely transformed into products of metabolism or differentiation, such as gland cells filled with secretion, red blood cells of mammals in which the nuclei completely disappear, egg cells filled with yolk, and spermatozoa in which most of the protoplasm has been transformed into the contractile substance of the flagellum.

I have not been able to measure the volume of muscle cells in Crepidula, but such measurements have been made by Eycleshymer[18] for the striated muscle cells of Necturus. From the measurements given by Eycleshymer, I have calculated the nucleus-plasma ratio in the usual manner, i. e., by determining the ratio of the nuclear volume to the cell volume less the nuclear volume; and I find that in the 7 mm. and 8 mm. embryos this ratio is about 1 :11, whereas in the 23 cm. adult it is about 1 :73. The increase of cell substance is therefore less than seven times, instead of twenty or thirty times, that of the nucleus, as he states.

Ratio of Nuclear Volume to Cell Volume in Adult Individuals of Crepidula plana[19]

It is important to note that Eycleshymer found that as the fibrillæ are progressively formed out of the protoplasm of the cell, the nuclei are crowded out of the center of the cell toward its periphery; that the nuclei become more densely chromatic, and especially so on the side of the nucleus toward the fibrillæ; and that possibly the nuclei may disintegrate and their chromatin go to form the dark bands of the striated muscle fiber. These facts seem to me to justify the conclusion which I reached in a former paper:[20]

It is probable that the contractile substance which makes up the larger part of the muscle cell does not contribute to the growth of the nucleus as does the protoplasm of embryonic cells—that so far as the growth of the nucleus is concerned it acts as does yolk, oil, membranes, fibers or other products of metabolism and differentiation. If only the sarcoplasm of the muscle cell and not its contractile substance is able to contribute to the growth of the nucleus, the small volume of the nuclei as compared with the entire cell would find a ready explanation. There can be no doubt that the plasma is the chief seat of differentiation, as Minot has emphasized, and that highly differentiated cells, such as muscle, nerve, and some kinds of connective tissue, have a larger amount of plasma and its products, relative to the nucleus, than have embryonic cells. In the case of fiber cells, fat cells and probably muscle cells, the cell body becomes filled with the products of differentiation and metabolism, which like the yolk in egg cells, or the secretion products in liver cells can not enter the nucleus and consequently do not influence its size. In such tissue cells the cell body is relatively much greater as compared with the nucleus, than in purely protoplasmic cells, but I have been unable to find any evidence that the ratio of protoplasm (using this term in its usual sense) to the nucleus is greater in tissue cells of Crepidula than in the blastomeres.

Just as the size of a nucleus in any given species is proportional to the volume of the general protoplasm, so the volume of its chromosomes is proportional to the volume of the nucleus. The number of chromosomes and their relative sizes are characteristic for each species, but the absolute size of chromosomes depends upon the size of the nucleus from which they come. Throughout the period of cleavage, as the cells and muclei grow smaller, the chromosomes also diminish in size. The view of Boveri[21] that the chromosomes divide when they have grown to their original size before division, and that thereby a definite specific size of ihe chromosomes is maintained, finds no confirmation in the work of Erdmann,[22][23] Schleip[24] or myself; while the view of Koehler[25] that the autonomy of the chromosomes may be extended to their growth, which is supposed to be independent of that of other cell constituents, is flatly contradicted by the facts.

During the cleavage stages at least, neither the nuclei as a whole nor the chromosomes double in volume at each successive division as is so often assumed. The total volume of the nuclei at the 70-cell stage of Crepidula plana is only 2.25 times their volume at the 2-cell stage. The volume of the protoplasm more than doubles, at the expense of the yolk, between the 1-cell and the 24-cell stages, while the total nuclear volume increases less than 1.5 times during this period. Jennings[26] has shown that the rate of growth is numerically greater than I had stated if one compares any stage with its immediately preceding stage, but of course this criticism does not apply to the total actual growth of nuclear material during any given period of development. It is often said that there is a "colossal increase of nuclear mass" but no increase in the protoplasm during the cleavage stages of the egg; and correspondingly there is said to be a great increase in the ratio of nucleus to plasma in the cleavage period. Upon this supposed increase in the nuclear material as compared with the plasma, Minot and Hertwig have based their hypotheses that the cleavage of the egg represents a period of rejuvenescence. However, in Crepidula and Fulgur among the gastropods and in Styela among ascidians there is no great change in the nucleusplasma ratio during cleavage, and I believe that this will be found to be generally true for other animals. On the other hand, there is a considerable increase in the plasma at the expense of the yolk, during the cleavage period in these animals, and in this fact, rather than in sji increase of nuclear substance, is to be found the cause of such rejuvenescence as may occur in these stages.

IV. Longevity, Senescence and Rejuvenescence

Apart from accidental causes of death, longevity is determined by the duration of the excess of anabolism over katabolism. If destructive metabolic changes gain ascendency over constructive ones at an early period the organism is short lived; if constructive processes are indefinitely in the ascendent the organism is potentially immortal. Such a condition is shown in Paramecium where Woodruff[27] has reared more than 3,000 generations without conjugation and without loss of vitality. These and other similar experiments have demonstrated the essential truth of Weismann's doctrine that Protozoa are potentially immortal. Woodruff found that the most important factors for maintaining vigor are proper food and freedom from the poisonous effects of waste products. In higher animals there is no doubt that both of these environmental factors are important, but there are also other important factors which influence length of life which are not entirely environmental.

Duration of assimilation conditions not merely body size, but also length of life. Very large animals are long lived and small ones are apt to be short lived, though the latter is by no means universally true—length of life being conditioned by duration of the ascendency of assimilation over dissimilation, whereas size is conditioned also by rate of assimilation as contrasted with dissimilation.

Weismann has pointed out a relation between longevity and the rate of reproduction—animals in which there is a slow rate of reproduction being in general long lived, while those in which the rate of reproduction is rapid are generally short lived. Numerous exceptions to this rule may be cited, though in many cases it is undoubtedly true; but Weismann has not proved that length of life is the result of slow reproduction. It may well be that both length of life and rate of reproduction are dependent upon the duration and rate of assimilation and dissimilation in somatic and germinal cells.

There is also an undoubted relation between longevity and adaptability, or the power of regulation. If life is continuous adjustment of internal conditions to external conditions, length of life may be said to depend upon the duration and perfection of such adjustment. The power of regulation is much less perfect in some animals than in others, and at certain stages of the life cycle than at other stages. But in all animals this power is greatest where the relative proportion of protoplasm to metaplasm, or differentiation products, is greatest, and where the protoplasm is most labile. In Protozoa this power of regulation is shown at every division and it suffers no abatement in successive generations; in Metazoa generally the power of regulation is greatest in early stages of development and in tissues in which protoplasm is abundant, and it diminishes as life advances and as the products of differentiation more and more replace the protoplasm. In the fission of a Paramecium there is a certain amount of dedifferentiation preceding division and of redifferentiation succeeding it, and as a result of this the two halves of the original Paramecium become alike; furthermore, in successive generations, there is no accumulation of the products of differentiation. In the division of the eggs of Metazoa the cleavage cells sooner or later become unlike, owing to the differentiations present in the mother cell and the failure of complete regulation in the daughter cells. This progressive differentiation is accompanied by a progressive loss of the power of regulation, and when the general protoplasm is so completely transformed into differentiation products that the power of regulation is completely lost, the organism as a whole must lose the power of adjustment to external conditions, and hence of indefinitely continued life.

Many different hypotheses have been advanced to account for the running down of the vital machine. That death is not a necessary corollary of life is evidenced by the potential immortality of Protozoa and of the germ cells of Metazoa. Senescence, like all other processes occurring in organisms, is primarily a cellular phenomenon. The decline and degeneration of cells begins in the earliest stages of individual development; in many cases large numbers of germ cells regularly undergo degeneration, apparently as the result of intrinsic rather than of extrinsic causes. The polar bodies which are formed during the maturation of the egg are at the same time the smallest and the shortest lived cells in the entire life cycle; they rarely last beyond the cleavage period and do not grow at all. Evidently their degeneration is due to lack of the power of assimilation, rather than to the accumulation of waste products, or to the increase of formed material. This lack of the power of constructive metabolism is evidently not due to lack of chromatin, for at the time of their formation they contain as much chromatin as the egg cell itself; they usually contain very little protoplasm, but even when the' quantity of protoplasm in them is very greatly increased, through the effects of pressure or centrifugal force at the time of their formation, they still lack the power of assimilation and differentiation. Such a large polar body resembles an unfertilized egg, and like it is incapable of development unless stimulated by the entrance of a spermatozoon or by some artificial means.

In many cases certain cleavage cells run through their development quickly and then degenerate and disappear, while other neighboring cells live as long as the organism itself. Many larval or fetal organs have a very short life; the cells of which they are composed grow and divide rapidly for a time and then dissimilation exceeds assimilation and they dwindle and disappear. Throughout the mature life of any metazoan many cells are continually growing old and dying, while others take their places. Even in the oldest organisms certain types of cells are still young enough to grow and divide, and there is no reason to doubt that such cells are potentially immortal and, if saved from the general death of the organism by isolation, might live indefinitely. Cells which continue to grow and divide throughout life apparently never grow old. It is customary to speak of the germ plasm as potentially immortal, but it is not generally recognized that other kinds of plasm may also be immortal. Indeed all kinds of protoplasm may be regarded as potentially immortal, except when processes of constructive metabolism are prevented in one way or another. In most cases the power of cell division is lost before that of growth, and the presence or absence of cell division is therefore indicative of youthful or of senile conditions in the cells concerned. Measured by this standard, certain cells grow old at a very early stage in the life cycle, whereas others remain young until overwhelmed by the general death of the organism. Senescence then is not a uniform process for the entire organism; it begins in certain cells at a very early stage of development, while it may not appear at all in other cells.

The possible causes of senescence and rejuvenescence may be classified as structural and functional, though these two should not be regarded as mutually exclusive. Indeed it is practically certain that both structure and function are involved in these processes as in most other vital phenomena. However, different students of this subject have placed emphasis more or less exclusively upon either the structural or the functional causes of senescence and rejuvenescence.

Under the structural causes may be cited Minot's hypothesis that senescence is caused by an increase in the amount of protoplasm as compared with the nucleus. In 1890 he[28] summarized his views on this subject in the following words:

We have then to state, as the general result of the studies which we har© just made, that the most characteristic peculiarity of advancing age, of increasing development, is the growth of protoplasm; the possession of a large relative quantity of protoplasm is a sign of age. . . . We see that there is a certain antithesis, we might almost say a struggle for supremacy, between the nucleus and protoplasm.

In several subsequent papers and books,[29] Minot has developed this idea at length. In his book on "Age, Growth and Death,"[30] he concludes that

Rejuvenescence depends on the increase of the nuclei, senescence depends on the increase of the protoplasm and on the differentiation of the cells.

R. Hertwig's[31] views are apparently diametrically opposed to those of Minot, He finds that senescence or rather "depression" and "physiological degeneration," in Actinospherium and Infusoria are accompanied by an enormous growth of the nucleus. He regards the immature egg cell with its great nucleus as in a condition of depression similar to that found in the protozoa named. By the processes of maturation and fertilization this nuclear material is greatly reduced and thus the cells are brought back to a normal condition.

As opposed to the hypotheses of Minot and Hertwig, it may be pointed out that the larger part of a resting nucleus is composed of achromatin which has been absorbed from the cell body, and that the size of a nucleus depends chiefly upon the quantity of general protoplasm in a cell and upon the length of the resting period during which the nucleus is absorbing this protoplasm. So far from there being an antithesis between nucleus and general protoplasm, we find that the general protoplasm is common to both; small nuclei occur only in cells with a small amount of such protoplasm, while large nuclei occur only in cells with a large amount. It is not the increase in the general protoplasm which causes the nuclei to become relatively small, but rather the increase in the differentiation products and the corresponding decrease in the general protoplasm.

In most respects I am in hearty accord with Minot's latest formulation of the causes of senescence.[32] In this work he particularly emphasizes the effect of differentiation in causing senescence. Indeed he concludes, "dass die Differenzierung als die wesentliche Ursache des Altwerdens zu betrachten ist." Nevertheless, he still holds that the greater growth of the protoplasm, relative to the nucleus, is the essential basis of differentiation; and that we may distinguish in development an earlier and shorter period, which is characterized by the preponderating growth of the nucleus, from a second and longer one characterized by growth and differentiation of the protoplasm—the former being the period of rejuvenescence, the latter the period of senescence. In Crepidula, as I have shown,[33] the growth of nuclear material during early cleavage is not greater than that of the protoplasm, and in general the size of a nucleus is directly proportional to the quantity of general protoplasm and to the length of the resting period, because general protoplasm is absorbed by the nucleus during interkinesis, whereas products of differentiation do not enter the nucleus. A causal explanation is thus given of the relation between nuclear size and cell size at different stages of development; and in the fact that differentiation products can not enter the nucleus we have, I believe, a causal explanation of the relation between differentiation and senescence.

The principal objection to Minot's formulation of the cause of senescence is that it overemphasizes the antithesis between nucleus and protoplasm and does not with sufficient clearness distinguish between the general protoplasm and its differentiation products. It is undoubtedly true that with advancing age and differentiation there is an increase of cellular as compared with nuclear substance, but the significant thing here is the fact that this cellular increase is not so much in the protoplasm as in the products which are formed from it and which can not enter into the nucleus.

By all odds the most important structural peculiarity of senescence is the increase of metaplasm or differentiation products at the expense of the general protoplasm. This change of general protoplasm into products of differentiation and of metabolism is an essential feature of embryonic differentiation and it continues in many types of cells until the entire cell is almost filled with such products. Since nuclei depend upon the general protoplasm for their growth, they also become small in such cells. If this process of the transformation of protoplasm into differentiation products continues long enough it necessarily leads to the death of the cell, since the continued life of the cell depends upon the interaction between the general protoplasm and the nucleus. In cells laden with the products of differentiation, the power of regulation is first lost, then the power of division, and finally the power of assimilation; and this is normally followed by the senescence and death of the cells.

In some cases the progressive transformation of protoplasm into metaplasm may be reversed; in some manner the formed material is dissolved and converted into general protoplasm, the protoplasm and nuclei increase in size, the cells begin to divide and may become capable of regulation. In short, this reversal of the differentiation process leads to the rejuvenescence of senile cells. Minot[34] holds that differentiated cells do not become undifferentiated—but at least it must be admitted they may lose their products of differentiation and metabolism; gland cells lose their secretion granules, egg cells their yolk, spermatozoa within the egg their flagella, injured muscle cells their fibrillge, etc. In such cases differentiation products are either eliminated from the cell or are transformed into a more labile and more general form of protoplasm, though the latter is probably not undifferentiated. I have used the term dedifferentiation for this process.

Among functional causes of senescence may be mentioned the well-known opinion of Metschnikoff, that the organism is slowly poisoned by its own waste products. Metschnikoff especially emphasizes the effects of intestinal fermentation and putrefaction in producing old age. Zoologists are familiar with the fact that, in certain Polyzoa and Tunicata which lack kidneys or efficient means of eliminating urea, or other nitrogenous waste, the tissues gradually become laden with such waste substances and the animal becomes senile and finally dies, but before this happens it may give off one or more buds which are relatively free from these waste products and which continue the life of the colony. It is a general phenomenon both in plants and animals that buds are composed of protoplasm which is not laden with products of differentiation or metabolism, and hence they exhibit youthful characteristics although the body from which they come may be senile.

Another functional cause of senescence is to be found in a decrease in the power of constructive metabolism. This factor has been recently advocated by Child[35] in a very valuable paper, in which he concludes that anything which decreases the rate of metabolism, such as "decrease in permeability, increase in density, accumulation of relatively inactive substances, etc.," will lead to senescence. On the other hand, "Rejuvenescence consists physiologically in an increase in the rate of metabolism and is brought about in nature by the removal in one way or another of structural obstacles to metabolism."

It is well known that constructive metabolism can not take place in the absence of a nucleus, and I have elsewhere[36] shown that the interchange between the nucleus and the protoplasm is a condition of assimilation. I have likewise shown that only the general protoplasm can enter the nucleus and that the products of differentiation are excluded from it. The progressive increase of such products and corresponding decrease in the general protoplasm lessen this interchange between nucleus and cell body and thus decrease the power of constructive metabolism.

In conclusion it may be said that there are several factors which produce senescence, but that the chief of these is the progressive differentiation of the protoplasm. As Minot has well said "Old age and death are the price which we pay for our differentiation." If we could find means by which this progressive differentiation could be stopped or reversed when it has gone too far, we might hope to attain potential immortality. That the possibility of this is not a mere delusion is shown by the fact that there are many animals which either in whole or in part are capable of rejuvenescence. In Protozoa the dedifferentiation which usually precedes or accompanies division is a process of rejuvenescence, and where such dedifferentiation and division are long delayed even protozoans show signs of old age. The same is true of germ cells; the mature egg and sperm are senile cells not because the one has a very large nucleus and the other a very small one, but because both are loaded with products of differentiation which interfere with constructive metabolism. When the sperm enters the egg and either leaves behind its old cell body or dissolves it, and its nucleus gets a new protoplasmic body, it is rejuvenated; likewise when the egg begins to dissolve the yolk and other products of differentiation with which it has been loaded it begins to live anew.

Similarly any adult animal or plant which is capable of dedifferentiation is also capable of renewing its youth. It has long been known that encystment and accompanying loss of differentiation lead to rejuvenescence. Jacobs,[37] working under my direction, found that when the rotifer, Philodina, becomes senescent, it may be rejuvenated if it is completely dried up and afterwards put back into water; in this treatment it evidently undergoes dedifferentiation.

Child[38] found that after planarians in a condition of apparent extreme senility had been starved for some time, they afterward became young within a few hours or days. Evidently the starving served to use up a part of the structural substance which prevented rapid metabolism. Similar conditions of renewed vigor are shown by many animals after long hibernation. The great breeding activity of many animals, such as frogs, so soon after their winter sleep, may find a physiological explanation in this using up of metabolic products during hibernation and the subsequent increase in vitality.

In similar manner it is known that the new tissue which is formed in regeneration comes from undifferentiated (epithelial or lymphoid) or from dedifferentiated cells (e. g., muscle cells of amphibia, etc.). In the latter case also there is a rejuvenescence, due to the loss of differentiation products. In this case dedifferentiation is evidently due, in the first instance, to the injury. It is at least possible that the failure to regenerate lost parts, which many animals show, is due to the inability of the cells to undergo dedifferentiation and subsequent rejuvenation.

In conclusion, we find that the life of a cell is dependent upon the continued interaction of nucleus and protoplasm; that as the protoplasm is transformed into products of differentiation this interaction of nucleus and protoplasm is reduced and constructive metabolism is diminished; that when the quantity of protoplasm present has been reduced beyond a certain point, either by its transformation into metaplasm, or by other means, constructive processes fail to compensate for destructive ones, and the cell grows old and finally dies. On the other hand, processes which lead to the increase of the general protoplasm in a cell, either by the growth of the protoplasm already present or by the conversion of metaplasm into protoplasm, lead also to the growth of the nucleus, to increased interchange between nucleus and protoplasm, and hence to increased powers of assimilation, cell division and regulation. Anything which decreases the interchange between nucleus and protoplasm leads to senility; anything which decreases this interchange renews youth.

1. Lecture before the Harvey Society, New York, March 7, 1913.
2. Conklin, "Body Size and Cell Size," Jour. Morph., 12, 1912.
3. Morgan, "Relation between Normal and Abnormal Development, etc.," Arch. Entw. Mech., 18, 1904.
4. Chambers, "Einfluss der Eigrösse, etc.," Arch, mil. Anat., 72, 1908.
5. Popoff, "Experimentelle Zellenstudien," Arch. Zellforschung, 1, 1908.
6. Conklin, "The Organization and Cell Lineage of the Ascidian Egg," Jour. Acad. Nat. Sci. Phila., 13, 1905.
7. Martini, "Die Konstanz histologische Elemente bei Nematoden, etc.," Verh. Anat. Gesell, 22, 1908.
8. "Darwinismus und Zellkonstanz," Sitz. u. Abh. naturforsch. Gescll. Rostock, 1, 1909.
9. "Studien über die Konstanz histologischer Elemente," I., II., III., Zeit. wiss. Zool, 92, 94, 1909; 102, 1912.
10. Goldschmidt, "Das Nervensystem von Ascaris, etc.," Zeit. mss. Zool., 90, 1908.
11. Martini, "Die Konstanz histologische Elemente bei Nematoden, etc.," Verh. Anat. Gesell, 22, 1908.
12. Woltereck, "Beiträge zur praktischen Analyse der Polygordiusentwicklung," Arch. Entw. Mech., 18, 1904.
13. Donaldson, "The Growth of the Brain," Scribners, New York, 1895.
14. Hertwig, R., "Ueber Korrelation von Zell- und Kerngrösse, etc.," Biol. Centralb., 22, 1903.
15. Hertwig, R., "Ueber neue Probleme der Zellenlehre," Archiv Zellfors., 1908.
16. Conklin, "Cell Size and Nuclear Size," Jour. Exp. Zool., 12, 1912.
17. Conklin, "Experimental Studies on Nuclear and Cell Division," Jour. Acad. Nat. Sci. Phila., 15, 1912.
18. Eycleshymer, "The Cytoplasmic and Nuclear Changes in the Striated Muscle Cells of Necturus," Am. Jour. Anat., 3, 1904.
19. Nucleus shrunken and irregular in shape.
20. Conklin, "Cell Size and Nuclear Size," Jour. Exp. Zool., 12, 1912.
21. Boveri, Zellenstudien V., Jena, 1905.
22. Erdmann, "Experimentelle Untersuchungen der Massenverhältnisse, etc.," Arch. Zellforsch., 2, 1908.
23. Erdmann, "Qualitative Analyse der Zellbestandteile, etc.," Ergeb. Anat. Entw., 20, 1912.
24. Schleip, "Das Verhalten des Chromatins, etc.," Arch. Zellforsch., 7, 1911.
25. Koehler, "Ueber die Abhängigkeit der Kernplasmarelation, etc.," Arch. Zellforsch., 8, 1912.
26. Jennings, "Nuclear Growth during Early Development," Am. Nat., 46, 1912.
27. Woodruff, "Dreitausend und dreihundert Generationen von Paramecium, etc.," Biol. Centralb., 33, 1913.
28. Minot, "On Certain Phenomena of Growing Old," Proc. Am. Ass'n Adv. Sci., 29, 1890.
29. Minot, "Ueber Vererbung und Verjüngung," Biol. Centralb., 15, 1895.
30. Minot, "Age, Growth and Death," Putnams, New York, 1908.
31. Hertwig, R., "Ueber die Kernkonjugation der Infusorien," Abh. Bayer. Akad. Wiss., II. Kl., 17, 1889.
32. Minot, "Moderne Probleme der Biologie," Fischer, Jena, 1913.
33. Conklin, "Cell Size and Nuclear Size," Jour. Exp. Zool.,
34. Minot, "Moderne Probleme der Biologie," Fischer, Jena, 1913.
35. Child, "A Study of Senescence and Rejuvenescence, etc.," Arch. Eniw. Mech., 31, 1911.
36. Conklin, "Cell Size and Nuclear Size," Jour. Exp. Zool., 12, 1912.
37. Jacobs, "The Effects of Desiccation on the Rotifer Philodina roseola, Jour. Exp. Zool., 6, 1909.
38. "A Study of Senescence and Rejuvenescence, etc.," Arch. Eniw. Mech., 31, 1911.