Popular Science Monthly/Volume 85/August 1914/The Cellular Basis of Heredity and Development III

THE

POPULAR SCIENCE

MONTHLY

AUGUST, 1914

 THE CELLULAR BASIS OF HEREDITY AND DEVELOPMENT[1]
By Professor EDWIN GRANT CONKLIN

PRINCETON UNIVERSITY

A. Introductory

HEREDITY is to-day the central problem of biology. This problem may be approached from many sides—that of the observer, the statistician, the practical breeder, the experimenter, the embryologist, the cytologist—but these different aspects of the subject may be reduced to three general methods of study, (1) the observational and statistical, (2) the experimental, (3) the cytological and embryological. Before taking up these different aspects of heredity it is important that we should have clear definitions of the terms employed and a fairly accurate conception of the processes involved.

1. Definitions

Heredity originally meant heirship, or the transmission of property from parents to children, and in the field of biology it has been defined erroneously as "the transmission of qualities or characteristics, mental or physical, from parents to offspring" (Century Dictionary). The colloquial meaning of the word has led to much confusion in biology, for it carries with it the idea of the transmission from one generation to the next of ownership in property. A son may inherit a house from his father and a farm from his mother, the house and farm remaining the same though the ownership has passed from parents to son. And when it is said that a son inherits his stature from his father and his complexion from his mother, the stature and complexion are usually thought of only in their developed condition, while the great fact of development is temporarily forgotten. Of course there are no "qualities" or "characteristics" which are "transmitted" as such from one generation to the next. Such terms are not without fault when used merely as figures of speech, but when interpreted literally, as they frequently are, they are altogether misleading; they are the result of reasoning about names rather than facts, of getting far from phenomena and philosophizing about them. The comparison of heredity to the transmission of property from parents to children has produced confusion in the scientific as well as in the popular mind. It is only necessary to recall the most elementary facts about development to recognize that in a literal sense parental characteristics are never transmitted to children.

2. The Transmission Hypothesis

And yet the idea that the characteristics of adult persons are transmitted from one generation to the next is a very ancient one and was universally held until the most recent times. Before the details of development were known it was natural to suppose, as Hippocrates did, that white-flowered plants gave rise to white-flowered seeds and that blue-eyed parents produced blue-eyed germs, without attempting to define what was meant by white-flowered seed or blue-eyed germs. And even after the facts of development were fairly well known it was generally held that the germ cells were produced by the adult animal or plant and that the characteristics of the adult were in some way carried over to the germ cells; but the manner in which this supposed transmission took place remained undefined until Darwin attempted to explain it by his "provisional hypothesis of pangenesis." Darwin assumed that minute particles or "gemmules" were given off by every cell of the body, at every stage of development, and that these gemmules then collected in the germ cells which thus became storehouses of little germs from all parts of the body. Afterwards, in the development of these germ cells, the gemmules, or little germs, developed into cells and organs similar to those from which they came.

3. Germinal Continuity and Somatic Discontinuity

Many ingenious hypotheses have been devised to explain things which are not true, and this is one of them. The doctrine that adult organisms manufacture germ cells and transmit their characters to them is known to be erroneous. Neither germ cells nor any other kind of cells are formed by the body as a whole, but every cell in the body comes from a preceding cell by a process of division, and germ cells are formed, not by contributions from all parts of the body, but by division of preceding cells which are derived ultimately from the fertilized egg (Fig. 23). The hen does not produce the egg, but the egg produces the hen and also other eggs. Individual traits are not transmitted from the hen to the egg, but they develop out of germinal factors which are carried along from cell to cell, and thus from generation to generation.

There is a continuity of germinal substance, and usually of germinal cells, from one generation to the next. In some animals the germ cells are set apart at a very early stage of development, sometimes in the early cleavage stages of the egg. In other cases the germ cells are first recognizable at later stages, but in practically every case they arise from germinal or embryonic cells which have not differentiated into somatic tissues. Germinal continuity and somatic discontinuity of successive generations in sexually produced organisms is not a theory but an established fact. In general, germ cells do not come from differentiated somatic cells, but only from undifferentiated germinal cells, and if in a few cases differentiated cells may reverse the process of development and become embryonic cells and even germ cells it does not destroy this principle of germinal continuity and somatic discontinuity.

Thus the problem which faces the student of heredity and development has been cut in two; he no longer inquires how the body produces the germ cells, for this does not happen, but merely how the latter produce the body and other germ cells. The germ is the undeveloped organism which forms the bond between successive generations; the person is the developed organism which arises from the germ under the influence of environmental conditions. The person develops and dies in each generation; the germ plasm is the continuous stream of living substance which connects all generations. The person nourishes and protects the germ, and in this sense the person is merely the carrier of the germ plasm, the mortal trustee of an immortal substance.

This contrast of the germ and the person, of the undeveloped and the developed organism, is fundamental in all modern work on heredity. It was especially emphasized by Weismann in his germ plasm theory and recently it has been given prominence by Johannsen under the terms genotype and phenotype; the genotype is the fundamental hereditary constitution of an organism—it is the germinal type; the phenotype is the developed organism with all of its visible characters—it is the somatic type.

But important as this distinction is between germ and soma it has sometimes been over-emphasized. This is one of the chief faults of Weismann's theory. The germ and the soma are generically alike, but specifically different. Both germ cells and somatic cells have come from the same oosperm, but have differentiated in different ways; the tissue cells have lost certain things which the germ cells retain and have developed other things which remain undeveloped in the germ cells. But the germ cells do not remain undifferentiated; both egg and sperm are differentiated, the former for receiving the sperm and for the nourishment of the embryo, the latter for locomotion and for penetration into the egg. But while the differentiations of tissue cells are usually irreversible, so that they do not again become germinal cells, the differentiations of the sex cells are reversible, so that these cells, after their union, again become germinal cells.

In many theories of heredity it is assumed that there is a specific "inheritance material," distinct from the general protoplasm whose function is the "transmission" of hereditary properties from generation to generation, and whose characteristics, as compared with the general protoplasm, are great stability, independence and continuity. This is the idioplasm of Nägeli, the germ-plasm of Weismann. But thtre is no reason to suppose that "germ-plasm" is anything other than germinal protoplasm, which is found in all cells in early stages of development but which becomes limited in quantity or altered in quality in tissue cells. A "germ-plasm" which is absolutely distinct from and independent of the general protoplasm is a mere fiction which finds no justification in reality.

4. The Units of Living Matter

The entire cell, nucleus and cytoplasm, is the ultimate unit of living matter which is capable of independent existence. Neither the nucleus nor the cytoplasm can for long live independently of each other, but the entire cell can perform all the fundamental vital processes. It transforms food into its own living material, it grows and divides, it is capable of responding to many kinds of stimuli. But while the parts of a cell are not capable of independent existence they may perform certain of these vital processes.

Not only is the cell as a whole capable of assimilation, growth and division, but every living part of the cell has this power. The nucleus builds foreign substances into its own substance, and after it has grown to a certain size it divides into two; the cytoplasm does the same, and this process of assimilation, growth and division occurs in many parts of the nucleus and cytoplasm, such as the chromosomes, chromomeres, centrosomes, plastosomes, etc. In all cases cells come from cells, nuclei from nuclei, chromosomes from chromosomes, centrosomes from centrosomes, and probably plastosomes from plastosomes, etc.

Indeed, the manner in which all living matter grows indicates that every minute particle of protoplasm has this power of taking in food substance and of dividing into two particles when it has grown to maximum size. Presumably this power of assimilation, growth and division is possessed by particles of protoplasm which are invisible with the highest powers of our microscopes, though it is probable that these particles are much larger than the largest molecules known to chemistry. The smallest particle which can be seen with the most powerful microscope in ordinary light is about 250μμ (millionths of a millimeter) in diameter. The largest molecules are probably about 10μμ in diameter. Between these invisible molecules and the just visible particles of protoplasm there may be other units of organization. These hypothetical particles of protoplasm have been supposed by many authors to be the ultimate units of assimilation, growth and division. In so far as these units are supposed to be different in different species, or with respect to different hereditary characters, they are known also as inheritance units.

It is assumed in practically all theories of heredity that the " inheritance material," or more correctly the germinal protoplasm, is composed of ultra-microscopical units which have the power of individual growth and division and which are capable of undergoing many combinations and dissociations during the course of development, by which combinations and dissociations they are transformed into the structures of the adult. Various names have been given to such units by different authors; they are the physiological units of Herbert Spencer, the gemmules of Darwin, the plastidules of Elsberg and Haeckel, the pangenes of de Vries, the plasomes of Wiesner, the idioblasts of Hertwig, the biophores and determinants of Weismann.

With the publication of Weismann's work on the germ-plasm in 1892 speculation with regard to these ultra-microscopic units of life and of heredity reached a climax and began to decline, owing to the highly speculative character of the evidence as to the existence, nature and activities of such units. But with the rediscovery of Mendel's principles of heredity the necessity of assuming the existence of inheritance units of some kind once more became evident, and, without attempting to define what such units are or how they behave modern students of heredity invariably accept their existence. They are now called determiners or factors or genes, and are usually thought of as elements or units of the germ cells which condition the characters of the developed organism, and which are in a measure independent of one another; though of course neither they nor any other parts of a cell are really independent in the sense that they can exist apart from one another. They are to be thought of as we think of certain chemical radicals which exist only in combination with other chemical elements in the form of molecules, and yet may preserve their identity in many different combinations.

If there are inheritance units, such as determiners or genes, as practically all students of heredity maintain, they must be contained in the germ cells, and it becomes one of the fundamental problems of biology to find out where and what these units are. But whether we assume the existence of these units or not we know that the germ cells are exceedingly complex, that they contain many visible units such as chromosomes, chromomeres, plastosomes and microsomes, and that with every great improvement in the microscope and in microscopical technique other structures are made visible which were invisible before, and whether the particular hypothetical units just named are present or not seems to be a matter of no great importance, seeing that, so far as the analysis of the microscope is able to go, there are in all protoplasm differentiated units which are combined into a system—in short, there is organization.

5. Heredity and Development

The germ cells are individual entities and after the fertilization of the egg the new individual thus formed remains distinct from every other individual. Furthermore, from its earliest to its latest stage of development it is one and the same organism; the egg is not one being and the embryo another and the adult a third, but the egg of a human being is a human being in the one-celled stage of development, and the characteristics of the adult develop out of the egg and are not in some mysterious way grafted upon it or transmitted to it.

Parents do not transmit their characters, but their germ cells, to their offspring, which germ cells in the course of long development give rise to adult characters similar to those of the parents. The thing which persists more or less completely from generation to generation is the organization of the germ cells which differentiate in similar ways in successive generations if the extrinsic factors of development remain similar.

In short, heredity may he defined as the particular germinal organization which is transmitted from one generation to the next: inheritance or heritage is the sum of all those qualities which are determined or caused by this germinal organization. Development is progressive and coordinated differentiation of this germinal organization, by which it is transformed into the adult organization. Differentiation is the formation and localization of many different kinds of substances out of the germinal substances, of many different structures and functions out of the relatively simple structures and functions of the oosperm.

This germinal organization influences not merely adult characters, but also the character of every stage from the egg to the adult condition. For every inherited character, whether embryonic or adult, there is some germinal basis. We receive from our parents germ cells of a particular kind and constitution and under given conditions of environment these cells undergo regular transformations and differentiations in the course of development which differentiations lead to particular adult characteristics. In the last analysis the causes of heredity and development are problems of cell structures and functions—problems of the formation of particular kinds of germ cells, of the fusion of these cells in fertilization, and of the subsequent formation of the various types of somatic cells from the fertilized egg cell.

B. The Germ Cells

Observations and experiments on developed animals and plants have furnished us with a knowledge of the finished products of inheritance, but the actual stages and causes of inheritance, the real mechanisms of heredity, are to be found only in a study of the germ cells and of their development. Although many phenomena of inheritance have been discovered in the absence of any definite knowledge of the mechanism of heredity, a scientific explanation of these phenomena must wait upon the knowledge of their causes. In the absence of such knowledge it has been necessary to formulate theories of heredity to account for the facts, but these theories are only temporary scaffolding to bridge the gaps in our knowledge, and if we knew all that could be known about the germ cells and their development we should have little need of theories. In the first lecture we looked at the germ cells and their development from the outside, as it were; let us now look inside these cells and study their minuter structures and functions.

Only a beginning has been made in this minute study of the germ cells and of their transformation into the developed animal, and it seems probable that it may engage the attention of many future

Fig. 22. Diagram Showing tub "Cell Lineage" of the Body Cells and Germ Cells in a Worm or Mollusk. The lineage of the germ cells ("germ track") is shown in black, of ectoderm in white, and of endoderm and mesoderm in shaded circles. The whole course of spermatogenesis and oogenesis is shown in the lower right of the figure beginning with the primitive sex cells (Prim. Sex Cells) and ending with the gametes, the genesis of the spermatozoa being shown on the left and of the ova on the right.

generations of biologists, but nevertheless we have come far since that day, only about thirty-five years ago, when Oscar Hertwig first saw the approach and union of the egg and sperm nuclei within the fertilized egg. Indeed so rapid has been the advance of knowledge in this field that many of the pioneers in this work are still active in research.

1. Fertilization

The development of the individual may be said to begin with the fertilization of the egg, though it is evident that both egg and sperm must have had a more remote beginning, and that they also have undergone a process of development by which their peculiar characteristics of structure and function have arisen—a subject to which we shall return later. But the developmental processes which lead to the formation of fully developed ova and spermatozoa come to a full stop before fertilization and they do not usually begin again until a spermatozoon has entered an ovum, or until the latter has been stimulated by some other outside means. In some animals and plants eggs may develop regularly without fertilization, the stimulus to development being supplied by

Fig. 23. Diagrams of the Maturation and Fertilization of the Egg of a Mollusk (Crepidula). A, B. First maturation division (1st Mat. Sp.) C. Second maturation division (2d Mat. Sp.) and first polar body (1st PB) resulting from first division. ♂N, sperm nucleus, ♂C, sperm centrosome. D. Approach of sperm nucleus (♂N) and sperm (♂S) to egg nucleus (♀N) and sphere (♀S); the second polar body (2dPB) has been formed and the forest has divided (1stPB). E. Meeting of egg and sperm nuclei and origin of cleavage centrosomes. F. First cleavage of egg showing direction of currents in the cell.

certain external or internal conditions; in other cases, as Loeb discovered, eggs which would never develop if left to themselves may be experimentally stimulated by physical or chemical changes in the environment, so that they undergo regular development. The development of an egg without previous fertilization is known as parthenogenesis or virgin reproduction; if it occurs in nature it is natural parthenogenesis, if in experiments it is artificial parthenogenesis. Natural parthenogenesis is relatively rare and in the vast majority of animals and plants the egg does not begin to develop until a spermatozoon has entered it.

But the spermatozoon not only stimulates the egg to develop, as environmental conditions may also do, but it carries into the egg living substances which are of great significance in heredity. Usually only the head of the spermatozoon enters the egg (Figs. 4-7) and this consists almost entirely of nuclear material which has a strong chemical affinity for certain dyes, and hence is called chromatin (Figs. 23 A and B); when the egg has matured and is ready to be fertilized its nucleus also consists of a small mass of chromatin (Fig. 23 C). Both of these condensed chromatic nuclei then grow in size and become less chromatic by absorbing from the egg a substance which is not easily stained by dyes and hence is called achromatin (Fig. 23 D and E). The chromatin then becomes scattered through each nucleus in the form of granules or threads which are embedded in the achromatin; this is the condition of a typical "resting" nucleus. The spermatozoon also brings into the egg a centrosome, or division center, around which an aster appears consisting of radiating lines in the protoplasm of the egg (Fig 23 B).

The moment that the spermatozoon touches the surface of the egg the latter throws out at the point touched a prominence, or reception cone (Fig. 4), and as soon as the head of the sperm has entered this cone some of the superficial protoplasm of the egg flows to this point and then turns into the interior of the egg in a kind of vortex current. Probably as a result of this current the sperm nucleus and centrosome are carried deeper into the egg and finally are brought near to the egg nucleus (Fig. 23 D and E). In the movements of egg and sperm nuclei toward each other it is evident that they are passively carried about by currents in the cytoplasm; the entrance of the sperm serves as a stimulus to the egg cytoplasm which moves according to its pre-established organization.

2. Cleavage and Differentiation

When the sperm nucleus has come close to the egg nucleus the sperm centrosome usually divides into two minute granules, the daughter centrosomes, which move apart forming a spindle with the centrosomes at its poles and with astral radiations running out from these into the cytoplasm (Fig. 23 F). At the same time the chromatin granules and threads in the egg and sperm nuclei run together into a smooth thick thread, the spireme, which is coiled within the nucleus. At this stage it is sometimes possible to see that the spireme is composed of a series of granules, like beads on a string; these granules are the chromomeres. The spireme then breaks up into a number of pieces in the form of short threads or rods (Fig. 24 C and D); these are the chromosomes. The

Fig. 24. Fertilization of the Egg of the Nematode Worm Ascaris;N, egg nucleus; ♂N, sperm nucleus; Arch, archiplasm; C, centrosome; A, B, approach of germ nuclei; C, D, formation of two chromosomes in each germ nucleus E, F, stages in the division of the chromosomes which are split in E and are separating in F; only three chromosomes are shown in F. (From Wilson after Boveri.)

number of these chromosomes is constant for every species and race, though the number may vary in different species. In the thread worm, Ascaris, there are usually two chromosomes in the egg nucleus and two in the sperm nucleus (Fig. 24 D). In the gastropod, Crepidula, there are about thirty chromosomes in each germ nucleus and sixty in the two. Then the spindle and asters grow larger and the nuclear membrane

Fig. 25. Maturation and Fertilization of the Egg of the Mouse. A, first polar body and second maturation spindle; B, second polar body and maturation spindle; C, entrance of the spermatozoon into the egg; D-G, successive stages in the approach of egg and sperm nuclei; H, formation of chromosomes in each germ nucleus; I, first cleavage spindle showing chromosomes from egg and sperm on opposite sides of spindle. (After Sobotta.)

grows thinner and finally disappears altogether, leaving the chromosomes in the equator of the spindle (Figs. 23 F, 24 E and F, 25 I).

Each of the chromosomes then splits lengthwise into two equal parts, and in the splitting of the chromosomes it is sometimes possible to see that each chromomere divides through its middle. The daughter chromosomes then separate and move to opposite poles of the spindle, where they form the daughter nuclei, and at the same time the cell body begins to divide by a constriction which pinches the cell in two

Fig. 26. Successive Stages in the Cleavage or the Egg of Mollusk (Crepidula), showing the separateness of the male and female chromosomes (♂ ch,ch) and the male and female halves of each nucleus (♂N,N).

in the plane which passes through the equator of the spindle (Fig. 26 B). Finally the daughter nuclei grow in size by the absorption of achromatin from the cell body and the substance of the chromosomes is again distributed through the achromatin in the form of threads and granules and thus the daughter nuclei come back to a "resting" stage similar to that with which the division began, thus completing the "division cycle" of the cell.

During the whole division cycle it is possible in a few instances to distinguish the chromosomes of the egg from those of the sperm, and in every instance where this can be done it is perfectly clear that these chromosomes do not fuse together nor lose their identity, but that every chromosome splits lengthwise and its halves separate and go into the two daughter cells where they form the daughter nuclei. Each of these cells therefore receives half of its chromosomes from the egg and half from the sperm. Even in cases where the individual chromosomes are lost to view in the daughter nuclei those nuclei may be clearly double, one half of each having come from the egg chromosomes and the other half from the sperm chromosomes (Fig. 26 B).

At every subsequent cleavage of the egg the chromosomes divide in exactly the same way as has been described for the first cleavage. Every cell of the developing animal receives one half of its chromosomes from the egg and the other half from the sperm, and if the chromosomes of the egg differ in shape or in size from those of the sperm, as is sometimes the case when different races or species are crossed, these two groups of chromosomes may still be distinguished at advanced stages of development. Where the egg and sperm chromosomes are not thus distinguishable it may still be possible to recognize the half of the nucleus which comes from the egg and the half which comes from the sperm even up to an advanced stage of the cleavage (Fig. 26).

At the same time that the maternal and paternal chromosomes are being distributed with such precise equality to all the cells of the developing organism, the different substances in the cell body outside of the nucleus may be distributed very unequally to the cleavage cells. The movements of the cytoplasm of the egg which began with the flowing of the surface layer to the point of entrance of the sperm, lead to the segregation of different kinds of plasms in different parts of the egg and to the unequal distribution of these substances to different cells.

One of the most striking cases of this is found in the ascidian, Styela, in which there are four or five different kinds of substance in the egg which differ in color, so that their distribution to different regions of the egg and to different cleavage cells may be easily followed, and even photographed while in the living condition. The peripheral layer of protoplasm is yellow and when it gathers at the lower pole of the egg where the sperm enters it forms a yellow cap. This yellow substance then moves, following the sperm nucleus, up to the equator of the egg on the posterior side and there forms a yellow crescent extending around the posterior side of the egg just below the equator. On the anterior side of the egg a gray crescent is formed in a somewhat similar manner and at the lower pole between these two crescents is a slate blue substance, while at the upper pole is an area of colorless protoplasm. The yellow crescent goes into cleavage cells which become muscle and mesoderm, the gray crescent into cells which become nervous system and notochord, the slate-blue substance into endoderm cells and the colorless substance into ectoderm cells.

before or immediately after the first cleavage, the anterior and posterior, dorsal and ventral, right and left poles are clearly distinguishable, and

Fig. 29. Sections of the Egg of Styela, showing maturation, fertilization and early cleavage; 1 P. S., first polar spindle, p. b., polar bodies, ♂N, sperm nucleus, ♀N, egg nucleus, p. l., peripheral layer of yellow protoplasm, Cr, crescent of yellow protoplasm, A3, A3, anterior cells, B3, B3, posterior cells of the 4-cell stage. In 1 the sperm nucleus and centrosome are at the lower pole near the point of entrance; in 2 and 3 they have moved up to the equator on the posterior side of the egg; in 4 the egg and sperm nuclei have come together and the sperm centrosome has divided and formed the cleavage spindle; in 5 the egg is dividing into right and left halves in 6 it is dividing into anterior and posterior halves.

the substances which will give rise to ectoderm, endoderm, mesoderm, muscles, notochord and nervous system are plainly visible in their characteristic positions.

At the first cleavage of the egg each of these substances is divided into right and left halves (Fig. 29, 5). The second cleavage cuts off two anterior cells containing the gray crescent from two posterior ones containing the yellow crescent (Fig. 29, 6 and Fig. 30, 1). The third cleavage separates the colorless protoplasm in the upper hemisphere from the slate-blue in the lower (Fig. 30, 2). And at every successive cleavage the cytoplasmic substances are segregated and isolated in particular cells,—and in this way the cytoplasm of the different cells comes to be unlike (Figs. 30 and 31). When once partition walls have been

Fig. 30. Cleavage of the Egg of Styela, showing distribution of the yellow protoplasm (stippled) and of the clear and gray protoplasm to the various cells, each of which bears a definite letter and number.

formed between cells they permanently separate the substances in the different cells so that they can no longer commingle.

What is true of Styela in this regard is equally true of many other ascidians, as well as of Amphioxus and of the frog, though the segregation of substances and the differentiation of cells is not so evident in the last named animals because these substances are not so strikingly colored. Indeed the segregation and isolation of different protoplasmic substances in different cleavage cells occurs during the cleavage of the egg in all animals, though such differentiations are much more marked in some cases than in others.

Fig. 31. Gastrula and Larva of Styela, showing the cell lineage of various organs, and the distribution of the different kinds of protoplasm to these organs. Muscle cells are shaded by vertical lines, mesenchyme by horizontal lines, nervous system and chorda by stiples.

This same type of cell division, with equal division of the chromosomes and more or less unequal division of the cell body continues long after the cleavage stages—indeed throughout the entire period of embryonic development. Sometimes the division of the cell body is equal, the daughter cells being alike; sometimes it is unequal or differential— but always the division of the chromosomes is equal and non-differential. When once the various tissues have been differentiated the further divisions in these tissue cells are usually non-differential even in the case of the cell bodies.

There can be no doubt that this remarkably complicated process of cell division has some deep significance; why should a nucleus divide in this peculiarly indirect manner instead of merely pinching in two as was once supposed to be the case? What is the relation of cell division to embryonic differentiation? In this process of mitosis, or indirect cell division, two important things take place: (1) Each chromosome, chromomere and centrosome is divided exactly into two equal parts so that each daughter structure is at the time of its formation quantitatively one half the size and qualitatively precisely like its mother structure. (2) Accompanying the formation of radiations, which go out from the centrosomes into the cell body, diffusion currents are set up in the cytoplasm which lead to the localization of different parts of the cytoplasm in definite regions of the cell, and this cytoplasmic localization is sometimes of such a sort that one of the daughter cells may contain one kind of cell substance and the other another kind. Thus while mitosis brings about a scrupulously equal division of the elements of the nucleus, it may lead to a very unequal and dissimilar division of the cytoplasm. In this is found the significance of mitosis and it suggests at once that the nucleus contains undifferentiating material, viz., the idioplasm or germplasm, which is characteristic of the race and is carried on from cell to cell and from generation to generation; whereas the cell body contains the differentiating substance, the personal plasm or somatoplasm which gives rise to all the differentiations of cells, tissues and organs in the course of ontogeny.

Weismann supposed that the mitotic division of the chromosomes during development was of a differential character, the daughter chromosomes differing from each other at every differential division in some constant and characteristic way, and that these differentiations of the chromosomes produced the characteristic differentiations of the cytoplasm which occur during development. But there is not a particle of evidence that the division of chromosomes is ever differential; on the contrary, there is the most complete evidence that their division is always remarkably equal both quantitatively and qualitatively. If daughter chromosomes and nuclei ever become unlike, as they sometimes do, this unlikeness occurs long after division and is probably the result of the action of different kinds of cytoplasm upon the nuclei, as is true, for example, in the differentiation of the chromosomes in the somatic cells as contrasted with the germ cells of Ascaris (Fig. 32). But while the chromosomes invariably divide equally, other portions of the nucleus may not do so. Achromatin and oxychromatin, like the cytoplasm, may divide unequally and differentially, and this is probably a prime factor in development.

On the other hand, the differential division of the cytoplasm is a regular and characteristic feature of ontogeny—indeed the segregation and isolation of different kinds of cytoplasm in different cells is the most important function of cell division in development. Thus we find in the division apparatus of the cell a mechanism for the preservation

Fig. 32. Differentiation of Germ Cells and Somatic Cells in the Egg of Ascaris. A and B, second cleavage division showing that the chromosomes remain entire in the lower cell, which is in the line of descent of the sex cells ("germ track"), but that they throw off their ends and break up into small granules in the upper cells, which become somatic cells. C, 4-cell stage, the nuclei in the upper somatic cells being small and the ends of the chromosomes remaining as chromatic masses in the cell body outside of the nuclei, while the nuclei in the lower cells are much larger and contain all the chromatin. D, third nuclear division, showing the somatic differentiation of the chromosomes in all the cells except the lower right one, which alone is in the germ track and will ultimately give rise to sex cells. (After Boveri.)

in unaltered form of the species plasm, idioplasm or germ-plasm of the nucleus, and for the progressive differentiation of the personal plasm or somatoplasm of the cell body.

3. The Origin of the Sex Cells

The sex cells are the latest of all cells of a developing organism to reach maturity, and yet they may be among the earliest to make their appearance. Every sex cell, like every other type of cell, is a lineal descendant of the fertilized egg (Fig. 22), but the period at which the sex cells become visibly different from other cells varies from the first cleavage of the egg in some species to a relatively advanced stage of development in others.

(a). The Division Period. Oogonia and Spermatogonia

When the primitive sex cells are first distinguishable they differ from other cells only in the fact that they are less differentiated; they have relatively larger nuclei and smaller cell bodies—a condition which is indicative of little differentiation of the cell body since the products of differentiation such as fibres, secretions, etc., swell the size of the cell body, but do not contribute to the growth of the nucleus. These primitive sex cells or gonia divide repeatedly, but the oogonia grow more rapidly and divide less frequently than the spermatogonia. As a result of this difference in the rate of growth and division the spermatogonia become much smaller and immensely more numerous than the oogonia. This period in the genesis of the sex cells is known as the division period (Fig. 22).

(b). The Growth Period. Oocytes and Spermatocytes

This period of rapid cell divisions is followed by a period of growth without division during which the developing sex cells are called primary oocytes or spermatocytes. This growth period may be very long in the case of the oocytes, lasting, for example, in the human female from the time of birth to the end of the reproductive period; during this long time the oocytes in the ovary probably never divide—there are as many of them at birth as at any later time; during this period of growth the ovarian egg becomes relatively large, in some animals, e.g., birds, the largest of all cells. The growth period of a spermatocyte lasts for a briefer time than does that of an oocyte so that the former remains relatively small (Fig. 22).

All of the cell divisions which take place during the division period are of the usual kind, in which every chromosome splits lengthwise into two and the two halves then separate and move to opposite poles of the spindle where they break up into threads and granules and form the daughter nuclei, as is shown in Fig. 24. But during the growth period of the oocytes and spermatocytes the chromosomes form a closely wound coil of long chromatin threads (Fig. 33), and when these threads uncoil later it is seen that the chromosomes have united in pairs (Figs. 33 D and E, 34 B, 35 B); this process is known as synapsis, or the conjugation of the chromosomes, and there is evidence that one member of each synaptic pair is derived from the father, and the other from the mother. The union of these chromosomes is probably not so close that they lose their identity, though there may possibly be some interchange of substance between them. By this union of the chromosomes into pairs the number of separate chromosomes is reduced to half the normal number; if there are usually 4 chromosomes, as in Ascaris, they are reduced to 2 pairs; if 48 chromosomes, as in man, there are 24 of these pairs.

Fig. 33. Different Stages in the Development of the Egg of the Rabbit. A, at the beginning of the growth period showing slender chromatic threads in the nucleus; B, later stage in which these threads ball up and parallel threads conjugate forming the shorter, thicker thread shown in C; D and E, segmentation of the long thread into chromosomes (?) each of which shows its double nature; F, later stage in which the distinctions of the chromosomes is temporarily lost. (After Winiwarter.)

In the conjugation of the chromosomes it is plain that, generally speaking, those chromosomes which are similar in shape and size unite; big chromosomes unite with big ones, little ones with little ones, and those of peculiar shape with others of similar shape (Figs. 34 B, 35 B). It is probable that the two members of a pair of conjugating chromosomes are homologous not merely in shape and size but also in function, though this homology does not amount to identity.

In some instances it can be proved that one member of each conjugating pair of chromosomes comes from one parent and the other from the other parent, and it is probable that this is always true. In every cell of every individual which has developed from a fertilized egg there are two full sets of chromosomes, one of which came from the sperm and the other from the egg; but when this individual in its turn produces germ cells homologous chromosomes of each set unite in pairs during the growth period.

These synaptic pairs are the bivalent chromosomes, and in addition to showing the line of junction by which they are united they frequently show a longitudinal split through the middle of each chromosome and

Fig. 34. Spermatogenesis of a Nematode Worm (Ancyracanthus). A, chromosomes of sperm mother cell, 11 in number, before their union into pairs; B, early stage of first division; 10 of the chromosomes have united into 5 pairs and each of these has split lengthwise; 1 chromosome remains unpaired; C, first maturation division after the 5 pairs of chromosomes have pulled apart; the unpaired chromosome is going entire to one pole of the spindle; D, two cells resulting from this division, one containing 5 and the other 6 chromosomes; E, four cells resulting from the division of two cells like B, in which each chromosome has split into two so that changing into spermatozoa, one containing 5 and the other 6 chromosomes. (After two of the cells contain 5 and two contain 6 chromosomes; F, two of these cells Mulsow.)

at right angles to the line of junction. It thus happens that these bivalent chromosomes are frequently four-parted and such four-parted chromosomes are known as tetrads (Figs. 34 B, 35 B).

Fig. 35. Oogenesis of a Nematode Worm (Ancyracanthus). A, egg mother cell containing 12 chromosomes before their union into pairs; B, early stage of first maturation division; all the chromosomes have united into 6 pairs, each of which has split in two so that the pairs are really four-parted (tetrads); C, the six tetrads in the first maturation division; D, egg containing 6 chromosomes, after both first and second maturation divisions; the eliminated chromosomes are shown as the polar bodies at the margin of the egg; E and F, eggs after fertilization; the egg nucleus is above and contains 6 chromosomes, the sperm nucleus below and contains 5 chromosomes in one case and 6 in the other; the former becomes a male with 11 chromosomes, the latter a female with 12 chromosomes.

(c). The Maturation Period

Finally at the close of the growth period both oocyte and spermatocyte undergo two peculiar divisions one following immediately after the other, which are unlike any other cell divisions. These are known as the first and second maturation divisions and they are the last divisions which take place in the formation of the egg and sperm. In one or the other of these two maturation divisions the pairs of chromosomes separate along the line of junction, one member of each pair going to one pole of the spindle and the other to the other pole, so that in each of the daughter cells thus formed only a single set of chromosomes is present (Fig. 34 C and D); but since the position of the pairs of chromosomes in the spindle is a matter of chance it rarely happens that all the paternal chromosomes go to one pole and all the maternal ones to the other; thus each of the sex cells comes to contain a complete set of chromosomes though particular individual chromosomes may have come from the father while others have come from the mother. There is reason to believe that homologous chromosomes show general resemblances but individual differences, and consequently when the members of each pair separate and go into the sex cells, these cells differ among themselves because the individual chromosomes in different cells are not the same.

In this way the number of chromosomes in the mature egg or sperm comes to be one half the number present in other kinds of cells, and when the egg and sperm unite in fertilization the whole number is again restored. The double set of chromosomes is known as the diploid number, the single set as the haploid number, and the maturation division in which this reduction from the double to the single set takes place is the reduction division. It is generally held that this reduction takes place in the first of the two maturation divisions (Fig. 34, C, D), and that the second of these divisions is like an ordinary mitosis in that each chromosome splits into two and the halves move apart, such a division being known as an equation division (Fig. 34 E), but it is possible that some chromosome pairs undergo an equation division in the first maturation mitosis and a reduction division in the second, while other chromosome pairs may reverse this order.

It is an interesting fact that long before the reduction of chromosomes had been actually seen Weismann maintained on theoretical grounds that such a reduction must occur, otherwise the number of chromosomes would double in every generation, and he held that this reduction must take place in one of the maturation divisions; this hypothesis of Weismann's is now an established fact.

As the result of these two maturation divisions four cells are formed from each cell (spermatocyte or oocyte) of the growth period. In the spermatogenesis each of these four cells is transformed into a functional spermatozoon (Fig. 34 E), by the condensation of the nucleus into the sperm head and the outgrowth of the centrosome and cytoplasm to form the tail. In the oogenesis only one of these four cells becomes a functional egg while the other three are small rudimentary eggs which are called polar bodies and which take no further part in development (Fig. 23, D-F). The fertilization of the egg usually takes place coincidently with the formation of the polar bodies—and so we come back once more to the stage from which we started, thus completing the life cycle.

4. Sex Determination

In the formation of the sex cells one can frequently distinguish at an early stage, differences between the larger oogonia and the smaller and more numerous spermatogonia; this difference is the first visible distinction in the development of the two sexes. In the case of the human embryo this distinction can be made as early as the fifth week, and it is evident that the real causes of this difference must be at a still earlier period of development.

The cause of sex has been a favorite subject of speculation for thousands of years. Hundreds of hypotheses have been advanced to explain this perennially interesting phenomenon. The causes of sex determination have been ascribed to almost every possible external or internal influence and the world is full of people who think they have discovered by personal experience just how sex is determined. Unfortunately these hypotheses and rules are generally founded upon a few observations of selected cases. Since there are only two sexes the chances are that any hypothesis will be right half the time, and if only one forgets the failures of a rule and remembers the times when it holds good it is possible to believe in the influence of food or temperature or age, of war or peace or education on the relative number of the sexes, or on almost any other thing. By statistics it has been shown that each of these things influences the sex ratio, and by more extensive statistics it has been proved that they do not.

This was the condition regarding the causes of sex determination which prevailed up to the year 1902. Immediately preceding that year it had been found that the kinds of spermatozoa were formed in equal numbers in certain insects; one of these kinds contained a peculiar "accessory" chromosome, and the other lacked it. The manner in which these two types of spermatozoa were formed had been carefully worked out by several investigators without any suspicion of the real significance of the facts. It was shown that an uneven number of chromosomes might be present in the spermatogonia of certain insects and that when maternal and paternal chromosomes united in pairs in synapsis one "odd" chromosome was left without a mate (Fig. 34 B). Later, in the reduction division, when the synaptic pairs separated, the odd chromosome went entire into one of the daughter cells, and the spermatozoa formed from this cell contained one chromosome more than those formed from the other daughter cell (Fig. 34 C and D).

Chiefly because these two kinds of spermatozoa occur in equal numbers McClung in 1902 concluded that this accessory chromosome was a sex determinant. In 1905 Wilson discovered in a number of bugs that while there were two types of spermatozoa, one of which contained, and

Fig. 36. Diagrams of Sex Differentiation in the Bug, Protenor. The oocyte contains 6 chromosomes and the spermatocyte 5 chromosomes which are not yet united into synaptic pairs; the "sex" chromosomes are shown in black and white, two are present in the oocyte, but one is present in the spermatocyte. In the reduction division the synaptic pairs separate, giving rise to two types of spermatozoa, one of which has the sex chromosome and the other lacks it; all ova are alike in this regard. If an egg is fertilized by a sperm without the sex chromosome a male results; if fertilized by a sperm containing the sex chromosome a female results. (After Wilson with modifications.)

Fig. 37. Diagrams of Sex Differentiation in the Beetle, Tenebrio, showing 5 synaptic pairs of chromosomes (there are actually 10 pairs); in the oocyte all pairs are equal in size; in the spermatocyte one pair is unequal. These pairs separate in the reduction division giving rise to two types of spermatozoa and one type of ova; eggs fertilized by one type of sperm give rise to females, those fertilized by the other type give rise to males. (After Stevens with modifications.)

the other lacked, the accessory chromosome, there was only one type of egg, since every egg contained the accessory chromosome, and he pointed out that if an egg were fertilized by a sperm containing an accessory, two accessories would be present in the zygote, this being the condition of the female, while if it were fertilized by a sperm without an accessory there would be present in the zygote only the accessory derived from the egg (Fig. 35 E and F, Fig. 36).

In other cases Miss Stevens as well as Wilson discovered that two accessory chromosomes, differing in size, might be present in the male whereas in the female they are of equal size (Fig. 37). In such cases two types of spermatozoa are produced in equal numbers, one containing a large and the other a small accessory chromosome, whereas every egg contains one large accessory chromosome. If such an egg is fertilized

Fig. 38. Diagrams of Sex Differentiation in the Thread Worm, Ascaris. The sex chromosomes are here joined to ordinary chromosomes, there being two in the egg mother cell and one in the sperm mother cell. All eggs contain one of these sex chromosomes, while half of the spermatozoa have it and half do not. Eggs fertilized by one type of sperm produce females, those fertilized by the other type produce males. (From Wilson.)

by a sperm containing a large accessory (the X chromosome) it gives rise to a female, if by a sperm containing a small accessory (the Y chromosome) it gives rise to a male (Fig. 37).

In other animals one may not be able to distinguish separate X or Y chromosomes and yet such structures may be joined to one or two ordinary chromosomes. This is the case in the thread worm, Ascaris (Fig. 38), where two such accessory elements are present in the female, each being joined to the end of an ordinary chromosome, whereas in the male only one such element is present. Here also two classes of spermatozoa are found one with and the other without the accessory element, whereas all ova have this element, and in this case also sex is probably determined by the type of spermatozoon which enters the egg (Fig. 38).

Even in man sex is determined in the same manner according to several recent investigators. There are in the spermatogonia of man 47 chromosomes, accordingly to Winiwarter, one of which is the X or accessory chromosome (Fig. 39 A). These unite in synapsis into 23 pairs, leaving the X chromosome unpaired (Fig. 39, B) and in the reduction division the pairs separate, while the X chromosome goes entire into one of the daughter cells, which consequently contains 23 + X chromosomes, whereas the other daughter cell contains 23 chromosomes (Fig. 39 C and D). The former gives rise to spermatozoa with 24 chromosomes, the latter to spermatozoa with 23 chromosomes. In the female there are probably 48 chromosomes, according to Winiwarter, there being two X chromosomes, one from each parent, and after the reduction divisions every egg contains 24 chromosomes. If an egg is fertilized by a sperm containing 24 chromosomes an individual with 48 chromosomes, or a female, is produced; if fertilized by a sperm with 23 chromosomes an individual with 47 chromosomes, or a male, results (Fig. 39).

Fig. 39. Diagrams of Sex Differentiation in Man. A, spermatogonium with 47 chromosomes one of which is the "sex" chromosome. B, spermatocyte showing 23 synaptic pairs and a single unpaired "sex" chromosome. C, reduction division in which the synaptic pairs separate while the sex chromosome does not divide, consequently the second spermatocytes D and D′ contain respectively 23 ${\displaystyle {{\ce {+}}}}$ X and 23 chromosomes. E and E′, second maturation division in which every chromosome divides, giving rise to two equal classes of spermatids and spermatozoa, one of which has 24 chromosomes and the other 23. If an egg containing 24 chromosomes is fertilized by a sperm with 24, a female with 48 chromosomes is produced; if an egg with 24 chromosomes is fertilized by a sperm with 23, a male results. (From Morgan after Winiwarter.)

It must be said that other investigators, notably Guyer and Montgomery, have not found 47 chromosomes in the spermatogonia of man, but 23. Since both the latter investigators worked on negroes whereas Winiwarter worked on white men it has been suggested quite recently by Morgan and Guyer that there may be twice as many chromosomes in the white race as in the black. A similar condition in which one race has twice as many chromosomes as another race of the same species is found in two races of the thread worm, Ascaris megalocephala, but it is still too soon to affirm that this is true of white and black races of man, though the facts seem to point in that direction.

Similar correlations between chromosomes and sex have been observed in more than one hundred species of animals belonging to widely different phyla. In a few classes of animals, particularly echinoderms and birds, the evidence while not entirely convincing seems to point to the fact that two types of ova are produced and but one type of spermatozoa; but the general principle that sex is determined by the chance union of male-producing or female-producing gametes is not changed by such cases.

On the other hand, there are many observations which seem to indicate that the sex ratio may be changed by environmental conditions acting before or after fertilization and that therefore sex is determined by extrinsic rather than by intrinsic causes. Most of these observations, as already remarked, are now known to be erroneous or misleading, since they do not prove what they were once supposed to demonstrate. But there remain a few cases which can not at present be explained away in this manner. Perhaps the best attested of these are the observations of R. Hertwig and some of his pupils on the effects of the time of fertilization on the determination of sex. If frog's eggs, which are always fertilized after they are laid, are kept for some hours before spermatozoa are mixed with them, or if the female is prevented for two or three days from laying the eggs after they have entered the oviducts, the proportion of males to females is enormously increased. Hertwig attempts to explain this extremely interesting and important observation as due to the relative size of nucleus and cytoplasm of the egg; but in general this nucleus-plasm ratio may vary greatly irrespective of sex and there is no clear evidence that it is a cause of sex determination.

Miss King, also working on frog's eggs, has increased the proportion of males by slightly drying the eggs or by withdrawing water from them by placing them in solutions of salts, acids, sugar, etc., but the manner in which drying increases the proportion of males is wholly unknown.

Extensive statistics show that in many animals including man more males are born than females, whereas according to the chromosome theory of sex determination as many female-producing sperm are formed as male-producing. It is possible to explain such departure from the 1:1 ratio of males and females in conformity with the chromosome theory if one class of spermatozoa are more active or have greater vitality than the other class, or if after fertilization one sex is more likely to live than the other. In the human species it is known that mortality is greater in male babies before and after birth than in female babies, and if before fertilization the activity or vitality of male-producing spermatozoa is greater than that of female-producing ones it would explain the greater number of males than of females. In certain insects it is known that females only develop from fertilized eggs, and in one of these cases, viz., Phylloxera, Morgan has discovered that this is due to the fact that all the male-producing spermatozoa degenerate and that only female-producing spermatozoa become functional. Possibly experimental alterations of the sex ratio, such as Hertwig, King and others have brought about, may be explained in a similar way. At least the chromosomal theory of sex determination is so well established in so many cases and has been found to be true in so many instances where at first it seemed impossible of application, that it ought not be abandoned until unmistakable evidence can be adduced against it. For the present at least we are justified in concluding that sex is irrevocably determined at the time of fertilization.

(To be concluded.)

1. Second of the Norman W. Harris Lectures for 1914 at Northwestern University on "Heredity and Environment in the Development of Men," to be published by the Princeton University Press.