A text-book of experimental cytology (1931) 8

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Gray J. A text-book of experimental cytology. (1931) Cambridge University Press, London.

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Chapter Eight Mitosis

The first observations of the living nucleus in a dividing cell were made, about 1850, on the oocytes of invertebrates. In the immature condition, these cells are characterised by very large conspicuous nuclei, which can easily be seen under low magnifications. As soon as maturation begins, the outline of the nucleus disappears and is not again visible until segmentation into two daughter cells has occurred, when a small definitive nucleus can be seen in each cell. Jlost of the earlier observers were agreed that, prior to each cell dhision, the nucleus disappeared in toto and a new nucleus was reformed de novo after each segmentation. When, however, it became possible to preserve the eggs with osmic acid or other fixative, and to apply a differential method of staining, the apparent disappearance of the nucleus, prior to cell di\dsion, was traced to a profound reorganisation of the nucleus rather than to its complete incorporation into the cytoplasm. As the methods of fixation and staining became more and more effective, the whole series of changes undergone by a dividing nucleus was found to be essentially the same in all plants and animals.

Within more recent times, however, the validity of the results obtained from fixed preparations have been questioned, so that it becomes important to know how far the phenomena of mitosis, as usually described, conform to what is known to occur within the living cell. It has already been mentioned that a ‘resting’ or interkinetic nucleus in the living condition is usually optically homogeneous with the exception of definitive nucleoli, whereas in the preserved state the nucleus appears to be divided into at least two phases, only one of which has an affinity for basic stains and is distributed throughout the nucleus as a series of granules or filaments. It would thus appear that such preparations are a very doubtful guide to intranuclear structure. Nevertheless, it will be shown later that as far as the chromatic constituents of the kinetic nucleus are concerned, fixed preparations give a remarkably faithful picture of the events which occur during life. It is only in respect to the aehromatic portions of the dividing nucleus that real misinterpretation may be caused by fixation.

As studied in permanent preparations, the main outlines of typical mitosis are as follows. First, there appear on the surface of the nucleus two small areas or granules which pass to opposite poles of the nucleus, and wliich may or may not be surrounded by a series of diverging rays. These bodies are known as the eentrosomes and the ravs, if present, are called astral rays. Soon after the eentrosomes have reached the poles of the nucleus, the latter elongates along the polar axis and its cliromatic constituents become incorporated into a definite number of cliromosomes ; this is the end of the proplme. Quite suddenly the outline of the nucleus disappears and the chromosomes are seen lying as an irregular group midway between the eentrosomes, and the area formerly outlined by the nucleus is occupied bv a series of fibrils which converge to each centrosome. The fibrillar area is the spindle. At the close of this metaphase condition the chromosomes cease to be scattered over the centre of the spindle, but are organised on a flat plate across its equator. The anaphase follows ; each chromosome segregates into two longitudinal halves, one of which passes towards each centrosome. Finally during the telophase, each group of daughter chromosomes forms an irregular mass of interfolding chromosomes or (in other cases) forms a mass of swollen vesicles which, losing their affinity for stains, are gradually built up into a new nucleus.

The forces involved in mitosis have long been the subject of speculation, and many unfruitful theories have been based on the assumption that every detail seen in a coagulated cell is a true picture of the living nucleus. Within recent years, however, it has proved possible to see every stage of dhdsion in a living nucleus, and the chances of misinterpreting preserved material is thereby greatly diminished. The following description is taken from that given by Strange ways (1922) who observed the choroidal cells of the chick in vitro. When about to divide, the cells withdraw their characteristic pseudopodia and assume an oval or romided shape (fig. 77). The first observable change in the nucleus is the conversion of the organised nucleoli into hazy granules ; these are then transformed into definite chromosomes which can be seen lying within the nucleus as a number of fine threads, often writhing about Tike eels in a box’. At this stage, the outhne of the nucleus suddenly disappears and the chromosomes rapidly arrange themselves at riaht angles to the spindle which, ^dth the centrosomes, can now be seen It is interesting to note that no trace of the spindle can be recognised until the outline of the nucleus has disappeared. About five to ten minutes after the first appearance of the spindle, the chromosomes begin their anaphasic movement toward the centrosomes and axe clearly seen as finger-like processes; at this stage the cell is oval in form. Having reached the poles, the chromosomes seem to fold in upon themselves and form a single faint irregular body, which is later surrounded by a clear zone unmistakably differentiated off from the cytoplasm. Finally a definitive nucleus with a nucleolus becomes visible in each daughter ceil.

Pig. 45. Effect of fixation and staining on the dividing nuclei of spermatoe\i;es {Chorihippiis). a, d, g, living cells ; 6, e, li, fixed with osmie acid vapour and Flemming’s solution; c,/, i, stained preparations. (From Belar.)

Fig. 46. Diagrammatic figures of typical mitosis. (From Wilson, 1928.)

Fig. 47. Nine observations at lO-minute intervals of connective tissue ceil of a rat (34° C.). Note withdrawal of pseudopodia during cleavage. (From Lambert and Hanes.)

From this account and others (BSlar, 1929) it can be realised that the classical accounts of mitosis are undoubtedly correct in their main outlines except in so far as the achromatic parts of the nucleus are concerned. Particularly convincing in this respect are the observations of Martens (1922) and (1929), who have carefully compared the effect of fixation on plant and animal cells respectively at different stages of mitosis. Even the finest structures seen in the living nucleus appear unchanged after an adequate process of fixation (figs. 40, 45).

Velocity of mitosis

In order that the events of mitosis should be seen in true perspective, it is of value to consider, at this early stage, the length of time occupied by each phase of the process. It is only by observing the living nucleus that the velocity of mitosis can be observed with any accuracy. The velocity of mitosis means the period of time which elapses between the onset of the prophase and the completion of the telophase. This must be carefully distinguished from the frequency of mitosis which means the number of nuclear divisions per unit of time. The distinction is clearly seen in the mitotic di^dsions of cells arown in vitro. Strangeways (1922), Fischer (1925 b) and others have shown that at about 38“ C. fibroblasts and other cells divide once in every forty-eight hours, although only a small fraction of this time is occupied bv actual mitosis ; on the other hand, the mitoses seen in the early stages of the segmentation of an egg follow each other without anv period of interkaryokinetic rest. At 17° C. a nuclear division of an egg of Echinus miliaris occupies about thirty-four minutes and is immediately followed by the next division (Gray, 1927).

Table XXIII

Mitotic cycles in EchinibS miliaris (17° C.). (Gray, 1927)

j 1st to 2nd cleavage

33 min.

i 2nd „ 3rd „

32 „

1 3rd „ 4th „

36 „

! 4th }j oth >}

35 ,,

1 5th „ 6th „

35 „

6th „ 7th „ 33 „

It is curious to find that the mitotic cycle of a large echinoderm egg is completed in practically the same time as that of the much smaller nuclei of the rat (Lambert, 1913) and of the chick (Wright, 1923), although its temperature is much lower. According to Lambert (1913) the connective tissue cells of the rat, when grown in vitro, require for each mitotic cycle at 38° C. about 21 to 29 minutes and at 35° C. from 35 to 50 minutes.

The period of time occupied by the different phases of division is not easy to determine and the observed figures show some variation from each other. Direct observations of living vertebrate cells have been made by Levd (1916) and by Lewis and Lewis (1917). With one exception the time tables of these authors are in agreement and may be illustrated by the time table given by Lewis and Lewis, who observed the mesenchyme cells of chick embryos; the cells were observed in Locke’s solution at 39° C.

In a fairly comprehensive series of observations, Lewis and Lewis found that very considerable variation may occur in the velocity of each individual phase (see Table XXIV). The only significant difference between the observations of Lewis and Levds and those of Levi is that the former allow from 30 to 60 minutes as the normal period of the prophase, whereas Levi restricts the same phase to a period of 5 to 20 minutes. It is obviously difficult to determine in the living cell the precise moment at which the prophase begins, but as pointed out by Wright (1925) the period can be accurately determined from

Table XXIV

Prophase ...

Average time in minutes

30-~60

Metaphase

2-10

Anaphase ...

2-3

Telophase

3-12

Reconstruction of nucleus

30-120

2-3 hours

Table XXV (from Wright)

i

Early

prophase

Spireme

Meta phase

Ana phase

Telo phase

Recon struction

of

nucleus

No. of mitoses found

140

115

91

66

91

S6

% of total found, equalling % of total time required for whole cycle

24

19

15

12

15

15

Time in minutes for each phase

Total time; 34 min.

8

5 1

4

!

5

5

fixed preparations if the duration of any other phase of the di^■ision is accurately known. Thus, assuming that the period of the telophase is 5 minutes, then by observing the relative numbers of prophases and telophases seen in a culture, the period of the prophase can be calculated, since the number of each phase present must be proportional to the time occupied for the completion of that particular phase.

The above table shows the results obtained by Wright from cells of the chick’s heart incubated at 37° to 38° C. It would appear that the whole cycle occupies about 34 minutes and that the time allowed bv Le^Tis and Lewis for the prophase is too long.

In echinoderm eggs Eplirussi (1926) gives the following time table

for PciTdcentTOtus Uvidus*

Table XXVI

1

Time in minutes after fertilisation

i

(

At 18-5°

At 26°

' Formation of the amphiaster

! Disappearance of nuclear membrane

i Metaphase plate

Division of chromosomes

Beginning of anaphase

End of anaphase Reconstruction of nucleus ...

18

48

52

56

59

65

74

16

28

32

36

39

43

50

in all investigation of the velocity of mitosis it is important to remember that the velocity of mitosis is very much reduced if the conditions of incubation are at all unfavourable. The presence of even slight traces of CO, are sufficient to reduce the velocity in Echinus^ggs (Gray, 1927).

We may now, perhaps, consider how far experimental methods have thrown light on the various phases of mitosis in the hope that it may be possible to form some conception of the types of mechanisms involved. It is convenient to start by considering each phase as a separate entity.

Prophase

The first sign of approaching division in a living nucleus consists in a loss of the normal homogeneity of the nuclear contents owing to the formation of threads or granules which have a high affinity for basic stains. As far as we can tell, this process must represent the segregation of the nucleic acid in the nucleus from the more basic constituents. As long as nucleic acid is associated with strong basic radicles or is in the form of a nucleo-protein it has no affinity for basic stains, for this property’- is peculiar to the free acid. Since chromatin is, in all probability, nucleic acid, we may infer that when chromosomes are formed inside the nucleus they contain free nucleic acid, w’hereas the more basic portions of the nucleo-proteins remain elsewhere or are in some way destroyed. Apart from this one conclusion, the chemistry of the dividing nucleus is entirely unknown, and vhen we remember that the nucleic acid derived from all animal cells, irrespective of group, genus, or species, is identically the same in composition, it becomes obvious how a purely chemical conception of living matter fails at present to give even a reasonable picture of biological facts (see p. 128). Genetically a chromosome must possess a higlily complex constitution which is specific to each species of organism; all we know, chemically, is that one invariable constituent is nucleic acid. Perhaps the nearest picture we can get is that of Mathews (1921, p. 175), wherein the nucleic acid is regarded as a gelatinised matrix in which are concealed those highly specific entities known as genes.

Fig. 48. Metaphase chromosomes of Dissosteim being torn by needles. (From Chambers, 1924.)

Fig. 49. Metaphase chromosomes in pollen mother cell of Tradescantia showing spiral structure. (From Sakamura.)

The chromosomes of animal cells are undoubtedly solid bodies in that they possess individuality of form; the same is true of plant chromosomes, whose consistency can be demonstrated by microdissection (Chambers and Sands, 1923). For the direct investigation of plant chromosomes Chambers and Sands (1923) used the pollen mother cells of Tradescantia, and by means of fine hooked needles were able to seize a single chromosome by each end and subject it to a longitudinal pull (see also fig. 48). By such methods they concluded that these particular chromosomes are elastic ‘jelly-like’ cylinders whose cortex differs from its central less refractive core.

The actual segregation of organised chromosomes within a homogeneous nucleus has been observed in at least three different tj’pes of cell ; in the chick cells grown in vitro by Strangeways (1922), in the oocvtes of ^istericts, and the spermatocytes of the grasshopper Dissosteira (Chambers, 1 924). Chambers’ observations on Dissosieira are of peculiar interest for they shoAv that the opening phases of

Fig. 50. Showing effect of puncture on a living spermatocyte of Dissosteira. a, Normal ceil ; 6, four minutes after puncture : the cytoplasm has degenerated and fine filaments have appeared in the nucleus; c, nine minutes after puncture: note formation of •chromosomes’. (From Chambers, 1924.)

normal nuclear activity can be induced by localised injury. Within a minute of being punctured, fine granular streaks can be seen within the spermatocyte nucleus and these soon grow into distinct filaments on whose surface is arranged a series of refringent granules. The granules groiv in size and each thread thickens. Ten minutes later, the whole nucleus contracts somewhat, and the granules on the

MITOSIS

151

filaments fuse together, thus converting the latter into a number of homogeneous hyaline bodies. There can be little doubt that these bodies represent true chromosomes, and as Chambers points out, their origin from hyaline axial threads Avith surface granules had preAUOUsly been described by Martens in 1922 in Paris quadrifolia.

In some spermatocytes , on the other hand, nuclear puncture results in the formation, not of typical prophasic chromosomes, but in the direct formation of bodies which strangely resemble chromosomes of the metaphase (fig. 51). Apparently the result of puncture depends on the stage to which the nucleus has normally progressed towards mitosis before the operation ; in each case puncture simply accelerates the normal process of development. An interesting observation was also made by puncturing a nucleus in which the chromosomes could already be detected. In this case the nuclear membrane disappeared, but the network of chromosomes persisted. On seizing a loop of the network, it was possible to draw it out as an elastic thread. On releasing it, it regained its original form (fig. 52).

Fig. 51. Chromosome formation within a hyaline nucleus of Dissosieira, following puncture. (Fronl Chambers, 1924.)

The opening stages of the normal mitotic prophase in Ihdng plant cells has been carefully studied by Martens (1927). In these cells the chromatic elements can be seen prior to the prophase, and the chromosomes (during the prophase) are deliminated by a rupture of the interchromosomal junctions which are characteristic of the interkinetic nucleus ; no spireme is formed, but each condensing chromosome becomes orientated parallel to its mates on the short axis of the nucleus: the whole prophase occupies from 36 to 45 minutes ( (fig. 40).

Fig. 52. Intranuclear filament seized by needle, a, Fragment of filament before stretching ; 5, after stretching. (Prom Chambers, 1924.)

The metaphase

Soon after the differentiation of the intranuclear chromosomes, the outline of the nucleus disappears. How far this is due to the dissolution of a definite nuclear membrane is uncertain, although a membrane appears to be present at the surface of the nucleus of an Asterias oocyte (Chambers, 1921). At this stage the prophase ends and the metaphase begins. The chromosomes now become arranged in a flat equatorial plate across the centre of the spindle. It seems clear that the force which compels the chromosomes to move to the equator of the spindle is located at the poles of the spindle or in the polar asters. This conclusion follows from the observation of F. R. Lillie (1912), who showed that in monastral Nereis eggs the chromosomes remain scattered at the conclusion of the prophase and showed no tendency to form a metaphase plate. Similarly, if the eggs of echiiioderms are subjected to ether before fertilisation, the male proiiucleus often fails to fuse with the female pronucleus; in such cases a bipolar aster may develop in association with the male nucleus, but only a single aster develops near the female nucleus. In these circumstances (fig. 53) the male chromosomes form a metaphase plate, w^hereas those of the female remain scattered (Wilson, 1902). That the arrangement of the chromosomes on a metaphase plate is not haphazard was shown by R. S. Lillie (1905) and later by Cannon (1923). Both of these authors have shown that the chromosomes on the metaphase plate arrange themselves in the same pattern as a series of ni&gnets, 3-11 orient3t6<l in sncli 3 wny 3S to repel one nnotlier in a strong externnl field (fig. 54). Such 3 sj'stem can be arranged as follows (Cannon). A number of corks, through each of which is feed a similar small rod magnet, are floated in a vessel containing water in such a way that all the magnets are pointing vertically upwards and all with the same pole uppermost; the north pole for instance. Since similar magnetic poles repel each other, these floating magnets vdll also repel each other and so collect at the sides of the vessel. If now a strong south pole is brought over the water on which the magnets are floating the latter, while still exerting a

Fig. 53. Fertilised egg of Toxopneustes after treatment with ether. The male chromosomes are arranged normally at the equator of a bipolar spindle. The female nucleus has only one aster; there is no spindle, the chromosomes remain scattered and do not form a metaphase plate. (From Wilson.)

repellent action on each other, are attracted towards the south pole and they now arrange themselves in equilibrium in a definite order. It is found that any number up to five will arrange themselves at the corners of a regular figure. If, however, there are six magnets, five are arranged at the corners of a pentagon, whilst the sixth passes to the centre of the figure. With ten magnets on the other hand, two magnets lie within an octagon formed by the remainder; with fifteen magnets there are five within the outer ring. The actual arrangements of chromosomes on the equatorial plate being identical with that occupied by the magnetic model (see fig. 54), it seems reasonable to assume that the chromosomes are orientated by two forces, one of which is exerted by the poles of the spindle. It should, however, not be forgotten that both Strangeways (1922) and Chambers (1924) have observed spontaneous movements by the chromosomes themselves. Strangeways describes the motion as ‘ eel-like , whereas

Fig. 54. Illustrating figures of equatorial plates, in each of which all the chromosomes arrpraetically of the same size and shape. Under each plate is the name of the genus from which it is taken and in front of this name is figured the predicted arrangement of the chromosomes. (From Cannon.)

Chambers’ description is that of an amoeboid type of movement, in which one part of a chromosome expands at the expense of the rest. The close of the metaphase of mitosis is usually marked by the longitudinal cleavage of each chromosome. Concerning this process almost nothing is kirown beyond the fact that it may occur at a much earlier so that on arrival at the equator of the spindle each chromosome is made up of two longitudinal halves. The process by which these halves are formed as separate entities is independent of a bipolar spindle, since it occurs in monastral systems (Nereis).

The achromatic mechanism

From a kinetic point of view, the anaphase is the most interesting phase of mitosis, for it is at this period that the daughter chromosomes migrate relatively rapidly to the poles of the spindle. Before attempting to evaluate the numerous theories which have been put forward to elucidate this phenomenon, it is essential to consider in (Treater detail the nature of the spindle in which the chromosomes move.

In every mitotic division of a nucleus, there exists a clear spindleshaped body at each pole of which there can usually be detected a granule or area known as the centrosome. In the cells of most of the higher plants, these structures compose the whole of the achromatic apparatus of the dividing nucleus, but in many animal cells there exist round each centrosome a series of diverging rays known as the aster. There are therefore two types of achromatic figure, (i) an anastral type, and (ii) an astral type. Since the kinetic phenomena of mitosis are alike in the two cases, one may conclude that however necessary the asters are during the process of cell division (see Chapter IX) they play no essential role in the division of the nucleus ; the essential structure for nuclear division appears to be the spindle. On the other hand, much of the evidence suggests that the formation of a spindle only occurs when the centrosomes are present, and that these bodies give rise to both spindle and asters. Unfortunately, the centrosomes are always extremely small and may be visible in the living cell. Whenever two asters come into contact with each other a spindle is formed between them and the resultant figure is known as an amphiaster. In view of the diversity of opinion concerning the origin of centrosomes and other parts of the achromatic figure it is difficult to give a logical account which will cover all the known facts.

In preserved material the mitotic spindle is almost universally represented by a series of fibres converging at the two poles (spindle fibres) ; at the metaphase some of these fibres are closely applied to the chromosomes. Spindle fibres have never been seen in the living cell, for during life the spindle appears as a homogeneous hyaline body enclosing the chromosomes. That spindle fibres are the artificial products of fixation is confirmed by the work of M. R. Lewis (1923), who showed that they only appear in living cells if the medium is sufficiently acid in reaction. In a medium of pH 4*6 fibres become visible in the spindles of cells dividing in vitro: on removing the acid the fibres disappear, and this phenomenon can be repeated several times vithont killing the cells. Since nearly all cjdcdogical fixatives contain considerable quantities of acid, there can be htt le doubt that visible spindle fibres must be regarded as artefacts. At the same time the fibres seen in fixed or coagulated cells are probably indications of a field of force between the poles of the spindle. Hardy (1899) showed that if a immoovcicous film of albumen is coagulated whilst in a state of tension, definite fibrillae are formed in a plane parallel

Fi- 55 LiwiK. primarv spermatocUe of Dissosteira. 1 , Note the chromosomes on metaphase plate and the clear spindle surrounded by rmtachondna ; ... late anaphase showins the two polar graphs of chromosomes and the linear airangement of mitachondria. (From Chambers. 1924.)

A.11 recent evidence supports the view that the spindle is a comparatively rigid structure. This was first shown by Morgan (1910), who found that it was possible to move the spindle bodily by centrifuo’al force through the matrix of an echinoderm egg; Shearer (1910) found that the spindle of Histriobdella was also a rigid struck capable of isolation from the cell. The later work of Chambers (191 1 ) (see fig. 56) shows quite clearly that the spindle is capable of considerable mechanical distortion and manipulation; it is an elastic gel and not in any way comparable to a fluid with low viscosity. Similarly in plant cells (Chambers and Sands, 1923), the spindles in the pollen mother cells of Tradescantia can be dissected out as a whole and are highly elastic.

It is of importance to note that the length of the major axis of tlie spindle determines the distance during the anaphase of mitosis. If an ecliinoderm egg is allowed to segment in sea water containing a small trace of ether (0-05 per cent.), the spindle is abnormally small and it is found that the two daughter nuclei, although perfectly normal, are abnormally close together.

The origin of the spindle appears to differ in different types of cell. In some plants and in some protozoa it is formed entirely from the nucleus, since it is well developed inside that body before the nuclear membrane disappears at the end of the prophase. In the higher plants, on the other hand, the spindle is formed from polar caps lying outside the nucleus; in most animal cells it arises outside the nucleus although in close association with it.

The formation of a spindle is closely associated with the presence of the two polar centrosomes, but it is useful to remember that the facts can be considered from two distinct points of view. From the first of these, the spindle is regarded as a definite cell organ formed from detiiiite material known as archiplasm, which may be located inside or outside the nucleus. From the other point of view, the spindle is an entirely transient structure which is the result of the mutual effect of two centrosomes on material of either nuclear or cytoplasmic origin. If the centrosomes lie within the nucleus the spindle will be formed of nuclear material; if on the other hand the centrosomes lie outside the nucleus, the spindle may be formed from the cytoplasm. In cases (e.g. the ecliinoderm egg) where the centrosomes lie very close to the surface of the nucleus, it seems almost certain that the spindle is composed of the achromatic parts of the nucleus. Immediately before the dissolution of the nuclear membrane, the nucleus is elongated between the two centrosomes, the nuclear boundary suddenly disappears and the area which it enclosed is then clearly defined as the spindle.

Fig. 56. Metaphase spindle of Dissosteira dissected out of the cell: note the extensile nature of the spindle when drawn out by a needle. (From Chambers, 1924.)

The polar asters

In many embryonic animal cells the poles of the mitotic spindle are marked by well-defined asters^ whose rays pass outward from the pole and extend into the cytoplasm of the cell. In the case of an ecHnoderm egg the rays of the aster appear to be marked out by the radial arrangement of granules which, elsewhere in the cell, are more or less evenly distributed throughout the C5rtoplasm (see fie 57) Durine the* metaphase of nuclear division these asters are very conspicuous objects and are seen to consist of a large number of ravs each composed of a hyaline homogeneous substance radiating out from a central clear area of protoplasm (centrosphere). According to Chambers (1917). the ravs are broadest at their base and gradually t«e7along their length milil they are indistinguishable from He aeneral hvaline matrix of the protoplasm. It is interesting to note tiiat the smaller the aster the more clearly defined are the rays ; as the aster grows in size, so the rays become longer but less distinct. Iccordinff to Chambers, the tongues of granular cytoplasm lying between the ravs are rigid, whereas the rays and the centrosphere "insist of a liquid of relatively low viscosity. The rigidity of the astral cytoplasm renders the whole aster rigid, so that it can be moved about niechanicallv in the cell, and can be distorted in form by means of needles (see fig. 57). An aster can be tivisted into a spiral and on being released from distortion it may or may not resume its normal shape. If the above description applies to asters in general, it would seem that an apparent absence of asters in some cells may be due to an absence of cytoplasmic granules, since it is these structures which make it possible to differentiate optically between the asters and the surrounding cytoplasm.

Fig. 57. a. Astral rays r, astral rays bent by greatly enlarged; tip of ray bent by needle; insertion of needle. (From Chambers, 1917.)

It seems probable that we ought to regard the asters not as transient cell organs but as the expression of a specific dynamic activitv. Thus, if a dhdding cell is subjected to a low temperature, or to such an anaesthetic as ether, the whole of the rays instantly disappear leaving the centrosphere as a clearly defined homogeneous mass. This phenomenon occurs whenever a dividing cell is subjected to any form of inhibition, e.g. tearing the asters by means of needles, or mechanical pressure.

The origin of the asters has been sought by a variety of methods and is still uncertain. In some cases it seems quite clear that they are formed at or near the surface of the nucleus. In echinoderm eggs

Fig. 58. Destruction of asters by ether or by mechanical disturbance, a, Norma! esg 45 minutes after fertilisation. The course and extension of the astral rays were drawn with all possible accuracy; h, same egg exposed to ether, note disappearance of astral rays leaving clearly defined astrospheres ; c, normal egg; d, one aster destroyed by mechanical disturbance with a needle, (a and b from Wilson; c and d from Chambers.)

the first aster to appear arises from a centre near the middle piece of the spermatozoon soon after the latter has entered the egg. This aster rapidly enlarges in size as the male pronucleus moves towards the egg nucleus, so that when fusion occurs the whole egg is filled by its radiations. The rigidity of this aster is shown by the fact that it is able to distort the spherical form of the cell (Gray, 1924), and by the fact that it can be meclianically pushed and rolled about in the c\’toplasm without losing its form (Chambers, 1917). As the astral rays increase in length, the centrosphere grows in size, so that there appears to be a centripetal flow of fluid towards the centre. According to Chambers, the male nucleus is only in equilibrium when it is lying near the centre of the aster, since if the nucleus be displaced it always tends to resume its central position. Siniilarlv as long as the female pronucleus lies outside the confines of the astral rays it remains stationary, but as soon as the rays reach it, the female nucleus moves with increasing velocity towards the centre of the aster, and is thus brought into contact with the nucleus of the spermatozoon. Not only the nuclei, but even oil drops move towards the centre of the aster, so that there appears to be a force directed towards the centre of the aster which acts thi’oughout that region of the cell traversed by the rays. As soon as the male and female pronuclei have fused together, the large male aster gradually fades away and is replaced by the small amphiaster which will be described below. According to Chambers (1917) the disappearance of the monastral rays is attended by a loss of rigidity of the cytoplasm, since.a needle can now be drawn through the egg without disturbing the structures hung on each side of the needle.

Fig. 59. a. Normal egg; h, the aster has been destroyed in the lower cell: note the compression of the latter cell against the more rigid cell containing the aster. (From Chambers, 1924.)

Additional e^ddence to show the rigidity of an aster is illustrated in fig. 59 b (after Chambers), which shows the compression of a blastomere whose aster has been mechanically destroyed; the second blastomere which contains an aster remains spherical, although both blastomeres are subjected to the same pressure from the hyaline membrane of the cells (see Chapter IX).

The existence of a single aster is of very considerable theoretical importance; it shows that the amphiaster is not the expression of a bipolar force of the type of electricity or magnetism. Large monasters can be produced in egg cells by a variety of methods; drugs, such as strychnine or chloral hydrate (Hertvdg, 1887), hypertonic sea water (Morgan, 1896;

Wilson, 1901 ; Herlant, 1918), and by mechanical agitation (Boveri, 1903; Painter, 1915). Wilson found that when the eggs of Toxopneustes were Fig- 60. Monastral egg of exposed to hypertonic sea water for a short period, large monasters developed subsequently in normal water. Under these conditions the whole of the normal cycle of nuclear changes occurred (including the division of the chromosomes) but the daughter chromosomes failed to move apart, and the cell did not divide. Such monastral cycles of nuclear activity occurred rhythmically in the same egg for as many as six times, after which normal bipolar cleavages might occur (Wilson, 1901).

More than one observer (Boveri, 1903 ; Painter, 1915) has observed that towards the end of the monastral cycle the large monaster moves towards the cell periphery, thereby flattening the centrosphere into a curved disc parallel to the surface of the egg; meanwhile the surface of the egg furthest from the centrosphere exhibits extensive cytoplasmic activity. This centrifugal movement of the aster is of importance when we come to consider the role of the asters during cleavage of the cell.

As mentioned above, the large male aster of echinoderms appears to have its origin at the originally posterior end of the sperm nucleus, and in certain cases there is strong evidence to support the vievr that the aster actually arises from the ‘ centrosome ’ lying in the middle piece of the spermatozoon. Boveri believed that the middle piece of the sperm alone was capable of producing an aster, and that the nucleus itself was not directly involved. The origin of the sperm aster from the middle piece of the spermatozoon is, however, contradicted by the work of Lillie (1912) on Nereis^ where the middle piece of the spermatozoon does not enter the egg and where the sperm aster arises at the proximal pole of the sperm nucleus. Lillie further showed that the aster can arise at any fractured surface of the male proiiucleus. This he demonstrated by centrifuging the fertilised egg in such a way as to break the male pronucleus into two portions, only one of which entered into the egg. In every case a male aster developed at the proximal surface of the nuclear fragment The size of the male aster appeared to be proportional to the size of the male pronucleus. Lillie’s observations suggest that the aster arises from a constituent of the nucleus, and that the so-called "centrosome’ is essentially of nuclear origin and is not derived from a permanently extra-nuclear source. It looks as though both centrosome and aster are the result of changes in or near the surface of a nucleus which has reached a definite phase of nuclear activity.

The a7nphiaster

In all typical cases of astral mitosis, two asters arise near the surface of the nucleus. At first they are small, although the ravs are very distinct. As the intervening spindle increases in size, the centrospheres of the asters grow and the rays project further and further into the cytoplasm of the cell. No rays are visible inside the spindle. When the cell dmdes into two halves, each daughter cell contains one aster which fades away soon after division is complete; at the next mitosis two small asters again appear at the surface of each daughter nucleus.

If we regard an aster as the product of a definite cell organ, then it would appear that the latter is capable of regular subdivision just as is the case of a chromosome. This view was accepted by Boveri who gave the name arckiplasm to the specific material giving rise to both asters and spindle. Similarly where the centre of each aster is occupied by a definite centrosome it seems reasonable to beheve that each daughter centrosome is derived as a cleavage product from a pre-existing centrosome.

There is, unfortimately, a considerable amount of confusion involved by the word centrosome. As Wilson (1925, p. 6T5) points out, it is used in at least four different senses. The most convenient usage is that the

Fig. 61. Aniphiaster of FcAiwaryg. (From Chambers.)

c 6 TitT 0 S 0 Tti 6 deiiotes that structure (at the centre of the astral rays) which does not disappear when the rays disappear. This permanent structure may or may not contain a small highly staining granule, the ceniriole.

In a few cases the centriole appears to be present during the nondividing phases of the cell’s existence, although this is by no means clearly established. It must be remembered that the centriole or definitive centrosome is very minute and can only be recognised during mitosis from the fact that it lies at the focus of the astral rays where there are no other cell granules. During the interkinetic period it is not easy to differentiate clearl}^ between a centriole and other cell inclusions having similar staining properties.

The main support of morphological cytology is afforded to the view that the asters are formed from permanent cell organs which are usually located on the outer surface of the nucleus. At the same time it must be admitted that during the interkinetic phases no trace can often be found of centrosomes or centrioles. The evidence from experimental enquiry gives, however, more support to the view that the centrosomes and their subsequent asters are essentially transitory organs which can arise de novo and independently of any pre-existing centrosome. In 1896 Morgan described the origin of asters in the cytoplasm of echinoderm eggs after treatment with hypertonic sea water. These asters are commonly known as c\i:asters to distinguish them from those w^hich normally arise near the surface of the nucleus. These C 5 d:asters were carefully examined by Wilson (1901) who showed, by examination of the living eggs of Toxopneusies, that they were capable of division and capable of deforming the surface of the cell just as is the case with normal asters (fig. 85). Since c\i:asters appeared to develop in different regions of the cytoplasm simultaneously, it seemed extremely probable that they arose de novo, although at the centre of each was a centriole comparable to that of a normal aster. On the other hand, the exposure of the egg to hypertonic sea water undoubtedly leads to a precocious activity of the normal nuclear asters, leading to the formation of multipolar spindles and it is conceivable that the c}d:asters w^ere derived by division from normal asters. Wilson attempted to analyse the position further by shaking eggs into fragments and exposing the enucleate fragments to hypertonic sea water. In such fragments cvTasters always developed. Unfortunately mechanical agitation undoubtedly upsets the normal asters (see above), and although this objection appeared to have been met by the work of McClendon (1908) on Asierias eggs, Yatsii (1908) later showed that cytasters will only develop in enucleated fragments of Cerebratulus eggs if the original germinal vesicle had broken down prior to enucleation.

All attempt to form an adequate theory of the origin of asters was made by Conklin (1902), who suggested that asters can develop in the cytoplasm wherever there is present an essential derivative of the nucleus, which need not necessarily take the form of a definitive granule. This view seems to cover most of the facts : we may imagine that the nucleus can so affect the neighbouring cytoplasm that an aster is formed. In a normal cell this action is greatest at the surface of the nucleus: on the other hand, if we so treat the egg that this iiiicleo-cytoplasniic reaction occurs much more readily, it seems reasonable to suppose that the products of the germinal vesicle having probably been distributed all over the cell, asters will develop here and there in the peripheral cytoplasm, and at the same time there will be an abnormal number of asters formed at the surface of the nucleus itself.

The real interest afforded by asters lies in the fact that they are almost certainly the active agents of cell division, although they are not directly concerned ^riih nuclear division. The role of the asters ill cell division is discussed in the follovdng chapter, but it is interesting to note that derivatives of centrosomes are undoubtedly concerned in other types of cell activity. The Henneguy-Lenhossek theory (see Gray, 1928) postulates that the basal granule of a cilium is homologous wdth the nuclear centrosome, and there can be no possible doubt that the two bodies are derived from a common source. Jordan and Helvestine (1922) claim that the ciliated cells in the epididymis of the rat di\dde amitotically because the division centres of the cells are functioning as basal granules. The recent work of Kindred (1926) indicates that in frog’s epithelia there is still left near or in the nuclei the potentiality of forming centrosomes after the original centrosomes have become basal granules of cilia. It would seem that, in this case, centrosomes can be regenerated by a nucleus just as occurs in the sperm head of Nereis. As long as it was reasonable to regard spindle fibres as real and contractile elements in the cell, the change in function from a centrosome to a basal granule was of very great theoretical importance, for it suggested that the forces exerted by the asters or in the spindle were comparable to those which move a cilium. This position is, however, no longer tenable, and the centrosomal force remains obscure.

The anaphase

Having considered the nature of the achromatic spindle in some detail, we may return to a consideration of the movements seen during normal mitosis. It is obvious that during the anaphase there is a force which moves the daughter chromosomes towards the poles of the achromatic spindle, and it is generally assumed that the energy for movement is derived from the spindle or its poles, and not from the chromosomes themselves. The nature of this force remains unknown, although it has been the subject of much ingenious speculation. Among the earliest theories of mitotic movement were those of Klein (1878) and van Beneden (1888), who looked upon the fibres seen in the preserved spindle as contractile fibrillae comparable to muscle fibres. Some of these fibrillae were assumed to be attached at one end to the pole of the spindle and at the other end to the chromosomes. Since the pole of the spindle was rigidly fixed in position, the daughter chromosonies moved to the poles when the fibrillae attached to them under^vent the process of contraction. This theory was accepted and elaborated by Boveri (1887) who showed that, during the fertilisation of Ascaris, the chromosomes appeared to be drawn on to the spindle by the activity of their attached fibrils ; these fibrils became shorter and thicker during the process of contraction, just as is the case in a muscle fibre. To some extent Boveri differed from van Beneden, since he believed that the astral rays and spindle fibres were formed anew during each mitotic cycle from the archiplasm of the cell, whereas van Beneden and his contemporaries looked upon the rays and fibrils as permanent ceil organs which, although orientated to form asters and spindlefibres during mitosis, were present in an unorganised manner during the interkinetic periods. At a later date, Heidenhain (1894, 1896) constructed his well-known models of anaphasic movement in which the fibrillae were represented by elastic strands of rubber. Heidenhain’s belief in the contractile properties of the mitotic fibrillae was strengthened by his discovery of a large permanent aster in the motile leucocytes of Salamandra; similar fibrillae were observed in the pigment cells of fish by Solger (1891) and Zimmermann (1898). These facts, together with the homology of the mitotic centrosome with the basal granule of a contractile cilium, suggested that in each case the fibrillae arising from the basal bodies are contractile in nature.

From time to time objections were urged against the contractile theory of mitosis, and numerous alternatives were suggested. The fact that spindle fibres are not visible in the living cell means, however, that unless it is very extensively modified the W'hole theory is of very little value. The fibres described by Boveri and Heidenhain are 'undoubtedb' due to a field of force between the centrosomes the chromosomes, but they throw no light on its nature, and cannot possibly be regarded as permanent structures comparable to muscle fibres. At the same time it is conceivable that the force which moves the chromosomes is of the same nature as that which causes a change in the shape of a muscle. It is w^ell known that contraction is not dependent on the presence of fibrillae (amoeboid movement, contraction of cardiac muscle cells), so that some form of active contraction along definite lines may be possible -wfithin the mitotic spindle. If such be the case, one would expect that any active "through a vdscous spindle during the anaphase would be marked by an increase in the energy expended by the whole cell, and since mitosis ceases in the absence of oxygen, it might be possible to associate the movement of the chromosomes with an increase in the oxygen consumption of the cells. The experimental difficulties attending such inv'estigations are considerable, but are not insuperable, and it has been showm (Gray, 1925) that there is no detectible change in the oxidative activity of the cell during mitosis. These observations were made on the segmenting eggs of Echinus which form peculiarly suitable material for investigation since, vith proper precautions, it is not difficult to ensure that all the eggs are in the same mitotic phase at any particular time. At the same time it must be remembered that the anaphase only lasts for about five minutes, and unless the amount of oxygen involved was significantly large compared to that required for other cell purposes it would not be easv to measure. We must at present conclude, how^ever, that there is little hope of estimating the energy changes involved during anaphasic movement, since if such changes exist they are too small to be estimated by the methods at present available; this means that one hopeful line of attack remains closed.

The conception of the asters and spindle as a field of force dates from 1873, when Fol pointed out their similarity to the field between two unlike magnetic poles . If the achromatic structures of a dividing nucleus are regarded as transient structures and not as a rearrangement of pre-existing material, a reasonable explanation is forthcoming for the ease with which the astral rays can he made to disappear in the presence of anaesthetics and other substances. Whenever the niitotic forces cease to exist, the field of force disappears just as the field of an electromagnet disappears when the current ceases to flow. Unfortunately the dynamic conception of mitosis does not carry us verv far. Some of the earlier hypotheses are obviously untenable, and' have been summarised by Meves (1896, 1898) and by Prenant <1910). The more modern theories are as yet incapable of experimental analysis. Before considering any of these in detail it is important to stress the fact that, so far, there is no direct evidence to show that the existence or activity of an achromatic system involves an expenditure of energy by the cell, and for this reason it is impossible to form any idea of the magnitude of the forces involved. Were it possible to detect an increased evolution of heat, or an increased consumption of oxygen whenever a nucleus is dividing mitotically, it would be possible to form a reasonable conception of the whole process. This is, however, not the case: there are no appreciable changes in heat production (Meyerhof, 1911) or of oxygen consumption (Gray, 1925) during mitosis.

Most of the earlier dynamical theories of mitosis involved the conception of an electrical or magnetic field of force and a full description of these theories will be found in the works of Prenant (1910), Meek (1913), and Wilson (1925). Apart from the difficulty of explaining the existence of monasters, all the electrical theories of mitosis are unacceptable in that they are purely speculative. The available experimental evidence is, in fact, definitely against the view that the chromosomes move to the poles of the spindle because there exists an electrostatic field of a particular type. It is true that the behawour of a nucleus in an electric field shows that the chromatin bears a negative charge relative to the rest of the nucleus, but McClendon (1910) has shown that to upset the normal orientation of chromosomes -svithin a spindle it is necessary to apply currents far more powerful than those which are likely to exist in living cells. McClendon exposed the groAving root tip of hyacinth or onion bulbs to a current of 110 volts for varying periods by application of copper sulphate electrodes to the surface of the tissue. In most of his experiments the current was passed for 30 minutes and A^aried from 0-00001 to 0-01 amperes. As soon as the current reached 0-00005 to 0-001 amperes the basophil constituents of the nucleus and of the cytoplasm showed a marked tendency to moA’e to the

anode, as was also the case of the c\-toplasm. With currents of O-Ol amperes the pressure of the migrating chromatin was sufficient to distort the nucleus into a pear-shaped structure.

/

Fig. 62. Effect of a direct current on mitosis in the root tip of onion bulb. (From McClendon.) a, Control: note uniform distribution of chromatic elements in nucleus and c\i:opIasm; b, 0*0001 amperes: note basophil material carried to anode; c, 0*01 amperes: note anodic distortion of nucleus; d, 0*0015 amperes: the spireme of the prophase has moved; e, 0*0002 amperes: no movement;/, 0*0006 amperes: the whole of the spindle has moved bodily ; g, 0*003 amperes : the whole of the spindle has moved.

Pentimalii (1909) stated that during the progress of mitosis the chromosomes became more and more sensitive to an external electric field, but this was not confirmed by McClendon, who showed that some at least of Pentimalii’ s results were due to displacement of chromosomes not by the electric field but by the microtome knife. McClendon states that as the process of mitosis advances the chromatin becomes less and less sensitive to the current; whereas 0-0015 amperes are sufficient to more the prophase spireme, at least twice that current is required to move the spindle. Further, during the anaphase the whole spindle with the contained chromosomes move as one unit, and there is no displacement of chromosomes relative to the spindle.

It is obvious that the general configuration of astral rays and ‘spindle fibres’ in a normal cell is no evidence of the nature of the force rvliieh is their underlying cause. Models of amphiastral figures can be made not only from magnetic or electrical fields but also from those of thermal, osmotic, and other types of energy. Btitschii’s (1876) figure, here reproduced (fig. 636), shows that particles of gelatin vill orientate themselves suitably when they are subjected to the

Fig. 63. a, Artificial amphiaster formed round two centres of coagulation. The matrix is a solution of globulin in alkali, and the coagulation is effected by platinic chloride. (Prom Fischer.) b, Artificial amphiaster formed round two air bubbles enclosed in a warm matrix of gelatin. Fixed in formol and chromic acid and stained by acid fuchsin. (From Biitsciili.)

stresses caused by two bubbles of air contracting ^wthiii a gelatin matrix. Similarly, the figures (figs. 64 and 65) obtained by Lediic (1914) are equally good mitotic models obtained in osmotic fields. Until it can be shown that the mitotic field is sensitive to forces of one particular type it is quite arbitrary which theory to adopt; presumably one gives preference to whichever theory provides the most picturesque model.

When an attempt is made to analyse the nature of the force operating in the spindle by applying an external field of a particular type, some support is forthcoming for the conceptions of an osmotic field of diffusion, for at least we know that the stability of the spindle and the behaviour of the chromosomes are upset by disturbances in osmotic pressure (see fig. 69).

As already mentioned there is, in all echinoderm eggs, an unmistakable accumulation of fluid at the poles of the spindle during

Fig. 04. Diffusion field between two drops of hypertonic salt solution %nt!i a hypotonic drop between them. The lines of diffusion are marked by particles of Indian Ink. (From Leduc.)

Fig. 65. A triastral field of diffusion between three contiguous drops. (From Leduc.)

the whole period of mitosis; Biitschli (1876) suggested that the anaphasic movement of the chromosomes might be due to similaT protoplasmic currents set up in the spindle itself; these currents he assumed to be caused by localised changes in surface tension.

Elaboration of these conceptions have been made from time to time by Ehumbler (1896-1908) and others, but it is difficult to picture such phenomena when we remember that the spindle is an elastic solid. Perhaps one of the most interesting of the hydrodynamic theories of mitosis is that put forward by Lamb (1908). This author suggested an application of Bjerknes’ (1900) observations on the field of force surrounding adjacent bodies which are oscillating or pulsating in a fluid medium. If, within a fluid, two bodies are pulsating synchronously and in opposite phase they repel one another: or if they are pulsating synchronously in the same phase they attract one another. Between the aphasic pulsating or oscillating bodies there is a field of force which is morphologically identical in form with that between unlike magnetic poles (see fig. 66). Lamb

Fig. 66. Diagram of the field of force between two bodies pulsating out of phase with each other.

suggested that at one pole of the mitotic spindle there is a body (the eentrosome) which pulsates or oscillates synchronously in opposite phase to its mate at the other pole. This hypothesis would account for the fact that the two centrosomes move apart as mitosis proceeds and yet are united by a field of force similar to that between the unlike poles of a magnet. Since bodies heavier than the surrounding medium are attracted by the poles of such a hydrod}mamic field whereas lighter bodies are repelled. Lamb suggested that a change in the specific gravity of the chromosomes might account for the fact that they are at first (metaphase) located as far as possible from the centrosomes, whereas afterwards they are attracted to the poles. Lamb put forward his suggestion as an ad hoc h}^othesis of only ‘hypothetical value ’ and no direct evidence in its favour has, as yet become available. An extension of the suggestion has, however, been made by Cannon (1923) by appMng the h^^othesis to ‘an hypotlieticai, isolated, ideal cell ' and then extending these deductions to the results of actual experiment. That such a procedure is fascinating there can be no doubt, but it is at the same time a negation of the experimental method. There is no evidence that the chromosomes are suspended in a fluid medimn; all the experimental facts support the view that the spindle is a structure possessing considerable rigidity. If the chromosomes lie vithin a gelated matrix of this t}T)e it is quite improbable that they would move in the way outlined in Bjerknes’ experiments. The second objection to Lamb’s hypothesis lies in the fact that there is no evidence which suggests that at each end of the spindle is a pulsating or oscillating body. Until these two objections have been met, it hardly seems profitable to attempt to apply the theory itself.

Summary of mitotic movement

It is obvious that no theory of mitotic movement has yet established itself as a working hypothesis for the obvious reason that none of the proposed theories indicates a conceivable line of experimental analysis. From a purely mechanical point of view it would be of interest to know the kind and magnitude of force which can move a particle of nucleic acid through a gelated matrix, bearing in mind that the speed of movement may be extremely slow (e.g. 1 mm. per day). Until something of this sort is attempted, it seems futile to put forward theories for which there is no real foundation in fact. That there is a force which operates between the chromosomes and the poles of the spindle seems almost certain and until it is identified with any of the known physical forces there is no harm done by giving it a name. In this respect there is something to be said for Hartog’s (1905, 1914) mitokinetism.

From an experimental point of view it seems useful to stress the following facts :

(i) The only structure which is necessary for the anaphasic movement of chromosomes is a bipolar or multipolar spindle. Neither asters nor definitive centrosomes are universally present, (ii) The longitudinal division of the chromosomes is independent of the presence of a spindle, (iii) In a few cases chromosomes are capable of autonomous movement, but daughter chromosomes do not move apart except when enclosed in a spindle. The distance which these chromosomes move is proportional to the length of the spindle. I iv) Since fibrils are found in the spindle when the latter is coagulated, it is reasonable to suppose that there is normally a state of tension or other localised distribution of energy along the axis of the spindle, (v) The orientation of the chromosomes at the equatorial metaphase suggests that the spindle itself generates the forces which are responsible for the orientation of the chromosomes.

The whole phenomenon of mitotic movement is baffling in the extreme. We are dealing mththe movement of particles of very small size, in an elastic medium at a very low velocity, and all attempts to detect an attendant output of energy by the cell have failed.

Modification of mitosis by external reagents

Like other properties of the cell, mitotic activity can be readily and reversibly inhibited by such reagents as cold, acids, or anaesthetics. The effect of variations in temperature on the successive phases was investigated by Jolly (1904) and more recently- by Ephrussi (1926), who concluded that each phase has its own temperature coefScient— that of the prophase being high (2-5-1-66)

Fig. 67. Diagram illustrating the temperature coefficients of the various phases of mitosis. (Prom Ephrussi.)

whereas that of the anaphase movement is low" (1*0-1*22).

The application of cold, quinine, anaesthetics, or KCN (Mathews, 1907), during the period of mitosis leads to a disappearance of the asters and spindle, although the chromosomes themselves do not appear to be affected. It is perhaps curious to find that an exposure to X-rays does not affect mitosis (Strangeways and Hopwood, 1926). whereas the rays can prevent a cell undergoing further mitoses when it is irradiated during the interkinetic period. The effect of acid on mitosis has not as yet been investigated in detail, but Smith and Clow^es (1924) have shown that, when the CO 2 tension of sea water is increased, the velocity of cell division in Arbacia and in Asterim is decreased, and that, at a definite tension of the gas, mitosis ceases altogether.

Fig. 6S. a, Accessory asters formed in fertilised egg of E. esculentus after exposure to h\'pertonic sea water; b, egg of E. esculentus exposed to h^^ertonic sea water after fertilisation. Note the normal asters and the very abnormal chromosomes, (From Gray, 1913.)

In the above cases, the inhibiting agent simply reduces the velocity of mitosis, and if its effect is not too intense mitosis is resumed at its normal pace when the inhibitor is removed. On the other hand, mitosis is extremety sensitive to many external changes which seriously affect the process in an irreversible manner. As a type of such action w^e may take exposure to an abnormally high osmotic pressure (Koiiopacki, 1911: Gray, 1913). If the eggs of echinoderms are exposed to concentrated sea water it is frequently found that one or more chromosomes fail to move to the poles during the anaphase. If the osmotic pressure reaches a critical figure the whole mitotic system may be affected, the asters disappear, and the whole of the chromosomes form an irregular mass drawn out between the poles of

• die (see figs. 68 and 69). It is interesting to note that many of hese experimentally induced irregularities occur naturally -when an = (T is fertilised by the sperm of a foreign species. When the eggs of the sea urchin Sphaerechinm granularis are fertilised by the sperm of Strongylocentrotus lividus the mitoses of the segmenting eggs are usually quite normal, although in a few cases abnormalities occur which recall the effects of hypertonic sea water on other types of ecrcr (fig. 69). In the reverse hybrid Strongylocentrotus $ x SpkaerecUniis S, many of the paternal chromosomes fail to pass to the

Fig 69 a and h, The effect of hypertonic sea water (50 c.c. sea w’ater -f S-8 c.c. 2UI NaCl) on the nucleus of a fertilised egg of Strongylocentrotus. (From Konopaeki.)

Abnormal zygote nucleus of hybrid egg, Sphaerechinus $ x Strongylocentrotus £. Xote tetraster and compare the form of the nucleus with that m 6.

poles of the first mitotic spindle (fig. 70) and are omitted from the daughter nuclei (Baltzer, 1910). Similarly the abnormal mitoses found in the cross fertilised eggs of Echinus esculentus x E, acutus (Doncaster and Gray, 1913), can be artificially induced in normally fertnised eggs of E. acutus by treatment with hypertonic sea water (Gray, 1913). There can be little doubt that abnormal mitoses can be the result of a variety of causes, and that the final effect on the nucleus is largely independent of the particular agent employed — ^heat, high or low osmotic pressure, electrolytic ions

Fig. 70. Cleavage spindles of hybrid Sirongylocentrotus $ x Sphaerechinus Note that some of the chromosomes ha%^e failed to pass to the poles of the spindle. (From Baltzer.)

Fig. 71. Polyastral figures in echinoderm eggs: a, induced by treatment with hypertonic sea water (Konopacki); h-d, by treatment with strychnine (Hertwig).

and some drugs all induce the same type of irregularity in the asters or chromosomes; it is therefore difficult to gain from such experiments any clear conception of the mitotic mechanism, although the results may have an important application to the study of malignant growths.

Mitotic stimulation

The reasons which cause a cell nucleus to undergo the process of division are difficult to analyse, since mitosis is usually only part of a long and complicated series of cell changes which go on hand in hand with growth (see Chapter XI). Growing cells often exliibit a regular rhythm of mitosis and newly divided cells usually grow; hence, peculiar interest is attached to those cases where mitosis can be made to occur in tissues whose rate of groivth is reduced to a minimum. Dustin (1921, 1922) found that if 1~3 c.c. of a foreign blood serum was injected into the peritoneal cavity of a mouse, a remarkable outburst of mitosis occurred in the cells of various organs after a latent period of 2-3 days. The organs involved were the thymus gland, skin, lymphatic ganglia, and intestinal epithelium. The induced mitoses, having followed their normal course, were not followed by others although other injections might be given. Dustin correlates the onset of mitosis with the liberation of a substance which is released from other cells in the process of degeneration and in this respect his views are supported by Gutherz (1925), who believes that degenerating nuclei produce substances (necrohormones) which are the cause of the premature and incomplete maturation divisions observed in the ooc 3 rtes of sexually immature mammals and in the formation of oligopyrene spermatozoa in molluscs.

Somewhat parallel to Dustin’s observations are those of Isawaki (1925), who found that the injection of l/60th c.c. of a bacOlus culture into the caterpillar of Galleria melorella induced active mitoses in certain types of leucocytes. In one case the number of mitoses rose from three per thousand cells to 136 (fig. 72); and the intensity of the effect produced by the injection varied markedly with the temperature of incubation. A somewhat different t^’^pe of mitotic stimulant is that isolated by Chambers and Scott (1925), who found that, during autolysis, malignant tumour cells produce a substance which increases the rate of growth of tumour tissues in vitro, and this stimulant they believe to be derived from the nuclei of the autolysed cells. Perhaps the most active supporter of specific mitotic stimulants is Haberlandt (1922, 1923), wlio has elaborated the theory of mitotic hormones in plants. Haberlandt cut thin sections of potato tuber and after five or six days found numerous mitoses whenever a section had included a portion of the vascular bundles. Sections without vascular bundles showed no mitoses. If, on the other hand, such sections \vere closely attached by a film of agar to a vascularised section, both fragments of tuber showed mitoses. Similarly mitosis could be induced by contact with freshly triturated vascular tissues. From these facts Haberlandt concludes that there must be associated with the vascular bundles a substance capable of inducing nuclear division.

Fig. 72. Graph showing induction of mitosis in blood cells of Galleria by injection of bacilli. The abscissa shows the time in days after injection. (Prom Isawaki, 1923. j

Somewhat analogous to Haberlandt’s wound hormones are the ‘desmones’ described by Fischer (1925 6). This author states that isolated animal cells when grown in vitro fail to divide because they lack an essential constituent which can only be supplied by way of the intercellular bridges which he claims unite all the cells in a normal culture. These conclusions have, however, been denied by Wright (1925), who believes that isolated cells in a tissue-culture are capable of division, although they usually divide synchronously.

ir • It interprets this simultaneous type of mitosis as the result of iiiiiform environment on a homogeneous population of cells. It ^ be doubted, however, how far this explanation is complete. An elated bacterium in a satisfactory medium soon gives rise to a lation which is heterogeneous in respect to the moment of deava^ye whereas when organic continuity between nuclei is clearly

Fia. 73. Periblast of embryo of Belone. Note synchronisation of mitosis. ’ (From Gurwitsch.)

Fi?. 74. Section of testis of Salamander showing synchronisation of mitosis.

(From Gurwitsch.)

established there is very clear evidence of synchronisation of cleavage. In syncytia all the nuclei divide together (fig. 73), and simultaneous mitosis is very common in the spermatoc}i:es mthiii a testicular tubule (fig. 74). How far these phenomena are due to the fact that all the nuclei concerned are or may be m protoplasmic connection with each other is doubtful; in this respect the observations of Polowzo\v (1924) are of interest. This author found that if fertilised eggs of sea urchins are exposed to dilute concentrations of alcohol, the mitotic figures are abnormally small and that nuclear division proceeds without cell cleavage: in such cases all the nuclei within any single cell divide simultaneously (fig 75) If an egg is allowed to develop normally in sea water the firJt two blastonreres usually cleave simultaneously, whereas if one blastomere is separated from its mate nuclear division often falls out of step Between the early blastomeres of many echinoderm e.^as fine protoplasmic bridges undoubtedly exist (Andrews, 1899; Grav. 1925), and it is possible that the presence of these bridges enables adjacent cells to undergo simultaneous nuclear division. It

Fig. 73 SvncWial mitosis in sea urchin eggs after treatment with alcohol. Note that alf the 'nuclei' in one blastomere are dividing simultaneously: in the other cell no nuclei are dividing. (From Polowzow, 1924.)

would be of interest to know whether all the cells of a developing larva divide together as long as they are in organic connection tilth each other and whether, when such cells as 4 d in a trochophore larva remain quiescent, organic connection with their neighboun

has been lost.

Balfour was inclined to ascribe the variation in cleavage rhythni noticeable during the segmentation of eggs to a variation in the amoun of volk present, e.g. the yolk-laden blastomeres of the lower hennsphere divide slower than those at the animal pole. There can, however, be little doubt that this explanation is insufficient to account for the marked variation in cleavage rh\i;hm noticeable during invertebrate development. As pointed out by Wfison (1925, p. 997) the two upper sister eeUs m each quadrant of the 16-celled stage of annelids and molluscs differ marked y from each other in their rhydihm. One divides many times in quick succession, the other divides only t%vice, although there is no visihl. difference m yolk content between them. The clewaae rhvthml ^ i

iiore closely related to the functional requirements of the e^r! to simple mechanical causes (F. R. Lillie). nibr\ o than

By far the most original conception of mitotic stimulation is however, that of G^ivitsch (1923 seq.). This author, together with hi, pupils, claims that certain types of living tissues emit a SDecific type of ray (mitogenic rays) which induces the onset of mito^sis in

ndghbouring cells. Gurwitsch’s original experiment consisted in exposing one side of a root-tip of an onion bulb to contact with the tip ot another actively growing root (fig. 76). After a short period of inductance, the number of mitoses found on the exposed side of the induced root was significantly greater than that on the opposite side, lhat this result is not due to the diffusion of materials from one root

fiimi w contact is unnecessary

I )• From this experiment it is inferred that induced mitosis is

caused by rays of very short wave length, since the ' mitogenic ’ ravs can readily pass through glass and quartz, but are absorbed bv gelatin from Avhich it looks as though their wave length Avas of tliforder of 2000 A. Xot only actively groA\dng root-tips are able to induce mitosis in other roots, for inductance occurs when the 'inducting’ root is replaced by yeast (Baron, 1926), tadpoles (Giiiwitsch, A. and L., 1925), or oxygenated blood (Sorin, 1926). In nearly all his experiments GurA\itsch has used the root -tip of plant bulbs as 'detectors’ of the mitogenic rays, although Baron (1926 ^ found that yeast cells can be induced to di^dde more rapidly bv exposure to groAA'iiig plant roots. In one experiment the number of budding yeast cells rose from 8 to 22 per 100 cells Avhen exposed to root -tip emanations, whereas in the root -tip the number of mitoses rose by 22-26 per cent, on exposure to actively sprouting yeast.. So far no animal cells have been used as detectors.

Table XXVII

Number of mitoses in sections of root -tip of Viciafaba

! Spireme

Monaster

Diaster

Telophase

Normal

37

30

19

38

After h hour in 0*125 % ether

160

61

40 1

25

It is not altogether easy to assess the significance of Guiwitsch's experiments, but if the results are substantiated b}^ further work they must represent an entirely new line of biological research. It is unfortunate, perhaps, that it has been too often assumed that an arbitrary difference of numbers of mitoses between the control section and the induced section of the root-tip is truly significant, since a statistical justification of this point is fundamental to all the experimental conclusions. That the root-tips of plants are peculiarly sensitive to mitotic stimulation is confirmed by Mainx (1924), Avho found that after exposure to low dilutions of ether the number of mitoses was greatly increased.

From time to time the determining cause of mitosis in animal cells has been sought by variation in environmental factors. In this connection it seems fairly clear that mitosis tends to occur after a period of starvation. For example, many amphibia are capable of Avithstanding prolonged starvation during which the cytoplasmic portion of their cells is markedly reduced in volume. As soon as food is pro\'ided, rapid mitosis frequently occurs (Kornfeld, 1922); -vhether in plants the rhythmical formation of food-stuffs during daylight is the factor responsible for the prevalence of mitoses during the night (Karsten, 1915, 1918) is not certain. On the other hand, active mitosis has been described in the tissues of starving animals Ipigeons (Morpurgo, 1888); eats (Hofmeister, 1890); salamanders illorgulis, 1911)]. There can be no doubt that, at times, the phenomena of mitosis and of grovi;h are independent of each other, for in dhiding eggs the rate of increase in the amount of respiring protoplasm changes quite independently of the mitotic rhythm (Gray, 1927). Even in Protozoa, where the cells maintain the same a%'erage size over a number of successive cleavages, the absolute size of the cell depends on the nutrient level of the surrounding medium and mitosis does not necessarily occur when the cell has reached a critical size.

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