THE MECHANISM OF CELL-DIVISION 11. OXYGEN CONSUMPTION DURING CLEAVAGE.

BY J. GRAY, M.A. King’s College, Cambridge.

(From the Marine Station, Millport, N.B., and the Zoological Laboratory, Cambridge.)

(Reckved 10 August, 1924.) (With Three Text-figures.)

IN a previous paper (Gray@)) it has been shown that the cleavage of echinoderm eggs depends upon the position and on the size of the asters which accompany the mitotic division of the nucleus. The cell cleaves at the moment the asters have reached their maximum size, and when the extremities of the astral rays lie very near the periphej of the cell. For this and other reasons, it follows that cleavage is the necessary conclusion to the process of aster formation. This goes on continuously during the whole mitotic cycle, and does not involve a sudden change in the surface energy of‘the cell at its equator, nor any contractile function on the part of the astral rays. Cleavage is essentially due to a rearrange- ment of the different phases round the two growing asters. I t is obvious that this conclusion is at variance with the views put forward by McClendon(11), Robertson ( l ~ ) , Spek (18) and Heidenhain (4). Although the schemes suggested by the first three authors differ materially from that of Heidenhain, yet all postu- late some sudden activity on the part of the cell, either by the liberation of a substance at the equator of the cell or by actual contraction of the astral rays. In support of these suggestions there is no direct experimental evidence. If the fully developed rays are capable of suddenly developing a tension sufficient to cleave the cell, the nearest comparison to them would be the contractile fibres of a muscle. If, on the other hand, cleavage be due to the liberation of some substance capable of producing a very marked change in the surface energy on part of the cell-surface, then some mechanism must exist for the formation of this sub- stance, and for its ultimate removal from the cell. In either case, but in the first case in particular, one might expect that the sudden activity of the cleaving mechanism would be marked by a change in the observable metabolism of the cell. Although, hitherto, no data have been available concerning the oxygen consumption during cleavage, an attempt was made in 1904 by Lyonw to measure the carbon- dioxide output of the cleaving eggs of Arb&, and more recently (1922) Vltscm) has measured by an indirect method that from the eggs of Paracentrotus livihs.

“ in nearly all experiments there was an increase in CO, production during the first ten or fifteen minute interval following fertilisation. The increase was slight and sometimes could not be detected. Following this came an interval in which the CO, production

Lyorl(9) found that

226 J . GRAY was small, visibly less indeed in two or three experiments than that of the unfertilisccl eggs and sperm. This is the mid-period of cleavage, approximating perhaps the time of nuclear growth and the early stages of karyokinesis. The interval during which the eggs were dividing into the first two blastomeres was one of active CO, production. After this period came an interval of lessened production. In one or two cases a second rise occurred at about the time of the second cleavage.”

In a later paper(10) dealing with the susceptibility of the egg to various reagents, he refers to these experiments as follows:

“ It may be stated that the apparent conclusion was that CO, production in the egg is not uniform throughout the whole series of morphological changes of cell division, but rather reaches a maximum at the time when the cytoplasm is actively dividing. Further- more it seemed that at the time when O2 is most necessary and presumably is being used in largest amount (as indicated by susceptibility to lack of 0, and to KCN) CO, is produced in least amount. If the conclusion above expressed should justify itself it would indicate that oxygen is chiefly used in the egg for synthesis rather than for combustion, and that the larger part of the CO, comes from splitting processes. One would also infer that the energy for cell-division comes from fermentative rather than oxidative processes.”

Lyon makes it abundantly clear, however, that he does not regard his experiments as of sufficient accuracy to permit of reliable theore.tica1 treatment.

“ These statements may need revision in the light of later and more accurate investigations.”

As far as I know, these have not been forthcoming. By observing the change in the p , of the fluid in contact with the eggs of

Z‘uracentrotus V l & s ( ~ found that well marked cyclical changes occur, and he attributes this, at least in part, to a periodic liberation of COO. Unless these observations are really due to CO, output, they are of course useless as a basis for determining energy changes in the cell. I n the present paper it will be assumed that Vks’ observations are not vitiated by the evolution or absorption of any other ions apart from those of COY, since positive evidence to the contrary is not apparent. Such difficulties are, of course, avoided by estimating the oxygen consumption of the cells.

It therefore seemed to be desirable to investigate the kspiration of cleaving cells by the direct measurement of the oxygen consumed.

The material used for the following experiments was the eggs of Echinw escu- l e n t ~ . In order that an accurate estimation of the rate of oxygen consumption may be made at definite stages during the whole mitotic cycle, it is necessary that the conditions of the experiment should be such as will allow the eggs to develop normally and at the same rate, so that practically all the cells cleave at the same time. Again, in order that several determinations can be made during the comparatively short time occupied by the cleavage process, it is advisable to prolong this period by carrying out the whole experiment at a fairly low temperature. It was found that at I I’ C. the time required for the first cleavage was about half-an-hour ; that is to say, it takes half-an-hour from the time the eggs first begin to show traces of a cleavage furrow, until all the cells have completely divided into two blastomeres. The time required for subsequent cleavages is shorter.

The Mechanism of Cell-division 227

A typical experiment was performed as follows :-The eggs from a ripe female were fertilised, and washed repeatedly in clean sea-water. They were then divided into two approximately equal portions, and each portion was concentrated into 6 C.C. of sea-water and placed in the respiration chamber of a Barcroft respirometer. ‘l’he manometers were kept continuously shaken and the bottles kept in a bath of water at 11’ C. The form of respiration bottle used was of the Erlenmeyer type and had a volume of 28c.c. After about 20 minutes the taps were turned so as to place the bottles in communication with the manometers. In the case of one apparatus, the excursion of the meniscus was read every five minutes; the other apparatus was kept as a control whereby to follow the process of development. By removing eggs from this control apparatus, the stage reached by the eggs can be followed, and from time to time the stage of development can be compared with that reached in the apparatus used for determining the oxygen consumption. At the beginning of this work it was thought that with a large number of eggs developing in a small bulk of water, the development would tend to be very irregular and slow. This was found not to be the case, and is no doubt due to the fact that the eggs were continuously shaken, and that the C02 was being com- pletely absorbed by the caustic potash in the apparatus. Further, at fairly low temperatures it is easy to supply sufficient oxygen by gentle agitation. In all the experiments it was found that the eggs in both pieces of apparatus used for an experiment developed at the same rate, and that this was only slightly slower than isolated eggs immersed in a large bulk of sea-water.

It is of importance to know with what degree of accuracy to regard the observed figures. Two sources of error occur. One is due to the error in reading the meniscus; this has been found by practise to be of the order of i - I mm. ‘rhe other source of error lies in irregularities of the manometer when working under low pressures. It is difficult, even with thoroughly clean manometer tubes, to prevent occasional “lags” in the meniscus, but if a low reading is rapidly followed by one above the average, one may be tolerably certain that the cause lies in the meniscus. This error is spasmodic, and has an approximate value of rt -2 mm. Individual readings can, therefore, only be regarded as correct to within these errors. It has been found that the maximum probable error when measuring the respiration of tissues with a steady rate of respiration seldom exceeds i. ‘35 mm.

Table I gives the data derived from one experiment, and Fig. I shows graphically the total oxygen consumption in the same experiment plotted against time. Fig. 2 shows the actually observed amount of oxygen consumed per 5 mins. during the pe;iod occupied by three successive cleavages. Fig. 3 shows similar data from another experiment.

An examination of Fig. I shows that the amount of oxygen consumed is not a linear function of the time. Aftcr the second division has occurred there is distinct evidence of an acceleration in oxygen consumption although this acceleration is independent of the process of cell-division. By taking the slope of the curve in Fig. I at successive periods and plotting the points so derived on to Fig. 2 the thick

10 2

0 30

40

T

ime

in m

inut

es fr

om th

e be

ginn

ing of th

e ex

peri

men

t Fig. I

. G

raph

sho

win

g th

e total

oxyg

en a

bsor

bed

by t

he fe

rtili

sed

eggs

of

EcJ

inus

ucu

lmtu

s du

ring

thre

e su

cces

sive

div

ieio

ns.

The

bla

ck l

ines

on

the

time

axis

ind

icat

e th

e periods

occu

pied

by

the

proc

ew o

f cl

eava

ge.

Exp

erim

ent A

.

The Mechanism of Cd-divkbn 229

line AB (Fig. 2) is obtained as the theoretical change in rate during the whole period of the experiment. This curve conforms to the conditions that the logarithm of the rate of respiration is proportional to the time. From this it is possible to

B

50 150 250 350 Time in minutes

Fig. 2. Graph showing the rate of oxywn absorption during the process of cleavage. AB shows the rate equivalent to the smooth curve in'Fig. I . Experiment A.

5 5 1 1 I I I I I I I I I I I I I I I I I I I I I I I I

50

g 45 .- 'i 40 2 35

$ 20

30

5 25

0 % 15

8 10

2 0 40 60 80 100 120 140 160 180 200 220 240 260

Fig. 3. Graph showing the rate of oxygen absorption during the process of cleavage. ment B.

Experi-

calculate the theoretical value of the total oxygen consumption at any moment. These figures are shown in Table I and agree very closely with the observed readings.

A consideration of Figs. 2 and 3 shows that the fluctuations in the rate of oxygen consumption bear no relationship to the periods of cleavage. If an allowance

230 J. GRAY

Table I Details of Experiment A.

Eggs fertilised 10.45 a m . After thorough washing, eggs transferred to apparatus at 12.45 a.m. (zyKote nucleus stage). T = I 1 . 0 ~ .

Time in minutes

from beginning of exp.

5

15

25 30 35 40 45 50 55 60 6 5 70 75 80 85 90 (J 5

100

105 I 1 0 115 I20 12s 130 '35 140 145 I so

10

20

I 155 I 60

Total 0, consumed

Obs.

2.7 4.6 6.2 8.6

10'2 12'1

14'3 17'1 I 8.6

21.7 24.6 26.7 28.5 30'4 33'1

20'2

34'6 37' 1,

46.7 48.4

30.b 41'5 4 4 2

5 1 ' 0

52'7 54'4 56.5 58.9 61.2 62.8 65'9 68.7 71'0 72'9 74'9 77'4 80.0 82.3 84'Y 87.2 89.9 92'5 944) y6.8

100'0 102'1

104.4 106.4 10cj.3 I 12.4 I 14'7 1 1 5 4 I 18.8 121'2

Calc.

2' I

4'2 6.3 8.4 10.5 I 2.6 14'7 16.8 18.9

23.2 25'4 27'5 29.6 31'7 33'8 36.0 38.2 40'4 42.6 44-8 47'0 49'2 51.4

21'1

53.6 55.8 58.0 60.3 62.6 64.9 67.2 69.5 71.8 74' I 76.4 78.7 81.0 83.4 85.8 88.2 90.6 93'0 95'4 97.8

102.7 105.2

107.7

I I 2.7 115'3 117.9 I 20.5 123'1

100'2

110.2

Error

+ 0.6 + 0.4 - 0' I + 0 2 - 0'3 - 0.5 - 0.4 + 0.3 - 0.6 - 0.9 - 1.5 - 0.8 - 0.8

- 1'3 - 0.9 - 1'4

- 0.6

- 0.6 - 0.3 - 0.8 - 0.4 - 0.9 - 1'4 - 1'5 - 0.4 -- 1'4

- 1.3 - 0.8 - 1'7

- 1'5 - 1'3

- 1.1

- 1'1

- 1.1

- 2'1

- 1'2

- 1'0 - 1 '1

- 1'0 - 0.0

- 0.7 - 0 '5 - 0.8 - 1'0 - 0'2 - 0 6 ~ 0.8 - 1'3 - 0.9 - 0.3 - 0.6 - 2'1 - 1'7 - 1.9

Lmount of 0, consumed in 5 minutes

~

Obs. - Calc.

2' I 2' I 2' I 2' I 2' I

7' 1 2'1 2' I 2' I 2' I 2' I 2' I 2.1 2' I 2' I 2' I 2'2 2'2 2'2 2'2 2'2 2'2 2'2 2'2 2'2 2'2 2.2

2'3 2'3 2.3 2'3 2'3 2'3 2.3 2.3 2'3 2'3 2.4 2'4 2'4 2.4 2'4 2.4 2'4 2.4 2.5 2'5 2.5 2.5 2'5 2.6 2.6 2.6 2.6 -

Error

+ 0.6

- 0.5 + 0 . 3 - 0 '5

+ 0.7 - 0.6 - 0.5 - 0.6

- 0 '2

- 0'2 -t 0' I

+ 0.8 0

- 0'2 - 0.3

+ 0.6 - 0.7 + 0.3 + 0.5 - 0.5 -I- 0.5 + 0.3 - 0.5 + 0.4 - 0.5 - 0.5 - 0'1 +O'I +O'I - 0.7 + 0.8 + 0.5

- 0.4 - 0 . 3

+ 0 .3

0

+ 0 ' 2

- 0'1 + 0'2 - 0'1

+ 0'2

- 0'2

+ 0 ' 3

- 0.3

+ 0.8 - 0.4

- 0.5 + 0.4 -1 0' I + 0.4

~ 1'5 + 0.4

- 0'2

-- 0'2

First cleavage completi

Second cleavage beginning

Second cleavage complete

The Mechanism of Cell-division 23 1

Time in minutes

begmning of exp.

275 280 285 290

ffom

--

2r 3 0 305 310 315 320 325 330 335 340 345 350 355 360 365 370

Table I (contd.)

Total Op consumed

Obs.

123.8 I 26.6 I 29.6 132.5 135.0 137'7 140.4 143'4 I 46.6 148.6 151.5 154'0 157'7 161.3

168.2 171.1 173'5

I 80.7

164.5

176.7

Calc.

125.7 I 28.4 131.1 133.8 136.5 139'2 142.0 144.8 147.6 150'5 153'4 I 56.3 159'3 162.3 165.3 168.4 171.5 174'7 177'9 181.1

Error

- 1'9 - 1.8 - 1.5 - 1.3 - 1'5 - 1'5 - 1.6 - 1.4 - 1.9 - 1.9 - 2.3

- 0.8

- 0.4

- 1'0

- 2'0 - 1'0

- 0'2

- 1'2 - 1'2 - 0.4

h o u n t of O1 consumed in 5 mInutes State of cleavage

Obs.

2.6 2.8 3'0 2'9 2.5 2'7 2.7 3 '0 3'2

2'9 2'5 3 '7

3'2 3'7 2'9 2.4 3'2 4'0

___

2'0

3'6

Calc.

2.6 2'7 2.7 2.7 2'7 2.7 2.8 2.8 2.8 2'9 2.9 2'9 3'0 3'0 3'0 3.1 3'1 3'2 3'2 3'2

- Error1

0 +O'I + 0.3 + 0 2 - 0 2

0 -0.1 + 0'2 + 0.4 - 0.9 - 0.4 + 0.7 + 0.6 + 0'2 + 0.6 - 0'2 - 0.8 0

+ 0 8

0

Third cleavage beginning

Third cleavage complete

The oxygen is shown as millimetres pressure of 0, and can be converted into cubic millimetres of volume by multiplying by 2.5.

be made for the probable error of each determination, the large majority of the observed fluctuations are eliminated, whilst those which remain are irregular in their occurrence and are of very short duration. It may therefore be inferred that if the process of cleavage involves any change in the oxygen consumption of the cell, this change is so small and of such short duration that the total amount of oxygen involved can only be a very small fraction of the total oxygen being used by the cell.

Again, the following figures, Table 11, show that the oxygen consumption during the half hour periods prior to, during, and after the first two divisions is very nearly the same.

Table I1

M m pressure 0, used per half hour

No. of Immediately During Immediately I exp. I p~~~~~ 1 division 1 division after

I 1- I A A A B B C D

13.0

16.4 14-1 13'3 6.5

14.8

21'0

12.3 15'0 15'9 13.6 13'4 6.1 19.0

14.0 13'7 18.3 13.0 13.7 7'1 21'0

No. of division

1st 2nd

2nd

3rd 1st

I St 1st

232 J. GRAY

In view of the above results it is necessary to investigate the fact, established by Lyon(~) and by Matthews(l2) that echinoderm eggs exhibit a marked periodicity in their susceptibility to cyanide poisoning. From the evidence given by these authors there can be little or no doubt that prior to fertilisation the egg is relatively resistant to cyanide. For the first 20 minutes after fertilisation, on the other hand, the eggs of Arbanu are relatively sensitive; after this period the resistance rises and no further susceptibility is found until immediately before and during each of the cell-cleavages. Loeb(7) found that in the absence of oxygen cleavage did not occur, and Matthews foundethat reagents such as cold, quinine, and anaesthetics which are known to reduce oxidations, also prevent cell-division and cause a disappearance of the astral rays. On this evidence Matthews(l2) concluded that the whole process of cell-division is intimately associated with the oxidative processes in the egg, and that the periodicity of the former is due to a periodicity in the capacity of the cell to carry out certain oxidations involving the use of atmospheric oxygen, this periodicity in oxidative power being due to the periodic liberation from the nucleus of an oxidase whenever the nuclear membrane breaks down. In opposition to this conclusion is the fact established by Warburg(21) that the level of oxygen consumption of the eggs can be maintained almost un- changed when the periodic changes of the nucleus are entirely inhibited.

The paradox presented by these facts is, in my opinion, only apparent, In the first place the periodic susceptibility of echinoderm eggs to cyanide is exactly paralleled by that of the eggs to a variety of reagents having little or no obvious direct effect on the respiration. A similar periodicity to that shown by Lyon for cyanides has been shown to exist by Spaulding(17) for ether, heat and hypertonic sea-water; by R. S. Lillie (6) for hypotonic sea-water; and by Baldwin (1) for alcohols. Now it has been shown in a previous paper (Gray@)) that for the first 15-20 mins. after fertilisation thc surface of the fertilised egg is undergoing an active change in the differentiation of a definitive ectoplasm or cortical layer. This layer is fully formed after about 30 mins., and remains equally distributed over the egg surface until just before the appearance of the cleavage furrow. During the process of cleavage there is a marked change in the- distribution of this surface layer. I t accumulates at the equator and becomes very thin at the poles. lfter cleavage the polar ectoplasm thickens again but never reaches its original thickness. The two periods of susceptibility to reagents share, therefore, the characteristic that the surface layer is abnormally thin either over all the egg surface or over its poles. Just(5) and Lilliew) have both observed that the region of the cleaving egg most susceptible to reagents lies at the surface of each pole. There is, therefore, some ground for believing that the properties of the surface layer are such as to inhibit the effect of reagents in general, including KCN*. This is confirmed by the fact that the final effect of KCN on the blastomeres of Ctmolabrus is to cause

A peculiarly interesting example of the instability of the polar surfaces of dividing cells is described by Strangeways(19). In certain cells the surface layer at the poles appears to rupture temporarily as soon as the cell begins to cleave. According to Strangeways these cells have no visible asters. It would be interesting to know whether microdissection would reveal two enlarging areas of high viscosity comparable to the asters of echinoderm eggs, since the astral rays of the latter are probably not the direct cause of the higher viscosity of the rcgion they occupy.

The Mechanism of Cell-divkion 233 them to fuse together-and since the cortical layer prevents this in the normal egg (&e Gray@)), it is fairly clear that the initial effect of KCN is to render soluble this surface layer. Until the surface layer is fully formed and whilst it is temporarily reduced at the poles of the cell the egg of the sea-urchin is more susceptible to reagents than at intermediate times. This is again confirmed by the fact that with successive divisions the thickness of the ectoplasm is reduced, and at the same time the sensitivity to KCN increases (Lyonce)).

Certain authors, including Matthews (121, have developed attractive hypotheses of the mechanics of cell-division from the evidence of the effect of KCN. Whilst it is true that this reagent depresses the oxidations in echinoderm eggs, it does not abolish it. Loeb and Wasteney'sc'la) figures show that a -oooo3 yo solution of KCN reduced the respiration to about one-third of its noxinal value; but the distinctive property of KCN is that although very dilute solutions have a detectable effect on the oxidations, successive increases in the concentration of KCN have less and less effect, and it is impossible to reduce the respiration of tissues below about 20 % of the normal. This maximum effect is reached by 50 x 10-6 M , KCN, Graycz). If we compare this concentration with those used by Lyon to demon- strate the susceptibility of sea-urchin eggs, viz. G-200, it becomes obvious that the KCN used in this latter connection is operating in some way other than iis direct effect on the oxidation. This fact is' of interest when attempts are made to correlate susceptibility to KCN and oxygen consumption.

Lyon showed that for the first 15 mins. after fertilisation the eggs of Arban'a are abnormally sensitive to a lack of atmospheric oxygen, and, as mentioned above, at a later stage an absence of oxygen causes individual blastomeres to fuse together. It therefore looks as though the surface layer of the cell is only stable in the presence of oxygen. I t is hoped that further work will throw light on this iiiteresting phenomenon.

The nature of the rhythm exhibited by all these reagents is, however, of a different nature to that described by V l b for the evolution of CO,. In this case the period of maximum CO, evolution comes apparently immediately after each cleavage, and the rate falls off gradually during the next mitotic cycle. The graph given by VlCs does not, unfortunately, give sufficiently accurate data to determine the actual rate of CO, production at different periods of time. The figure pub- lished by him is, however, comparable to Fig. I of this paper. It is at once obvious that the marked periodicity observed by VlCs is not accompanied by a corre- sponding periodicity of oxygen consumption. The variations in the rate of CO, production at different periods are of an altogether different order to the very slight and Transient variations in oxygen consumption.

It may perhaps be permissible to point out one or two peculiarites in 'Vlts' curve. It would appear that just prior to the third cleavage there is an actual absorption of CO,, unless this point is due to experimental error. Again, after the third cleavage, the eggs do not appear to have given off any CO, for more than one hour. Before attaching implicit faith to these facts one would like to know more precisely the conditions under which the experiment was carried out. In experiments

M M

23 4 J. GRAY dealing with small variations in the respiration of cellsit isessential that the conditions should be accurately defined. For this reason, the results of V l b cannot strictly be compared to those here described. In Vlks’ experiments the CO, was allowed to accumulate, and the eggs were not agitated*. In the present work the CO, was con- tinuously removed and the solution saturated with oxygen by agitation. On general grounds there can be little doubt that the oxygen consumption is a more reliable guide to the metabolic activity of the eggs than is the evolution of CO,, particularly when the latter is determined by an indirect method. At the same time there is no n priori reason why the CO, output should bear a definite relationship to the 0, consumption except over prolonged periods. If one considers the nev ly fertilised egg, it is reasonably clear from the work of Shearer(16) that the rate of 0, con- sumption fairly rapidly attains its new level. The amount of CO, which will escape during this period will possibly depend upon three factors: ( I ) the alkali reserve of the egg, (2) the alkalinity of the surrounding medium, and (3) the surface area of the egg. Eventually one would expect an equilibrium in CO, output to be reached, so that the latter will bear a definite relation to the 0, consumption. When cleavage occurs this equilibrium may be disturbed by the fact that the surface of the egg has increased, and some of the CO, held in the egg may be free to escape. That there is a lag between the 0, consumption and the CO, output has been shown to accompany sudde‘n increases in the activity of other tissues (Gray(2)). Whether such causes play any part in the case of echinoderm eggs is impossible to say from the data at present available.

I t is clear, however, that since the periodicity in evolution of CO, is not accom- panied by a periodicity in 0, uptake, the former cannot be regarded as of significance in respect to energy changes in the egg, unless they be due to some obscure form of anaerobic activity for which there is no evidence. I t will have been noticed perhaps that the periodicity in CO, production reported by Vlks does not correspond to that described by Lyon. In the former case the period of maximum CO, evolution is immediately after the division, in the latter case this period is one of reduced CO, evolution.

It is just possible that the process of aster formation involves the use of CO,, or that some associated process prevents the loss of CO, from the egg. Such con- ditions would, of course, lead to a periodic liberation of CO, whenever the asters faded away, and would be in harmony with some of the data given by Vlks.

I t has been claimed that the nucleus plays an active r6le in the oxidative mechanism of the cell (Matthews (E), Osterhout (13)). The present experiments show that if this is the case, the presence of a nuclear membrane or definitive nucleus is unnecessary since the rate of oxidation is independent of the phase of nuclear activity.

In a subsequent paper, the steady increase in the rate of oxygen consumption during the first few hours of development will be considered in more detail. I t is, however, already clear that this increase is independent of the phases through which the nuclei are passing. This fact has a direct bearing on an ingenious hypothesis put

Personal communication.

The Mechanism of Cell-division 235

forward by Robertson (15). This author suggests that the possibility for cell-division and growth is dependent upon the relative distribution of a catalyst between the nucleus and the cytoplasm of the cell. This autocatalyst is formed within the nucleus and can only escape into the cytoplasm when the nuclear membrane breaks down at each prophase. Although Robertson does not suggest that this autocatalyst is actually an oxidising agent, yet he does accept the view that the distribution of this substance can satisfactorily be foliowed by the rate of cellular respiration. The autocatalyst increases within the resting nucleus and is only shared with the cytoplasm when the nuclear membrane breaks down. If this be so, one would undoubtedly expect to find a periodic change in the oxygen consumption. This is not the case.

One other point remains to be considered. It is clear that the independence of cleavage and the rate of oxygen consumption indicates that the asters are not using up oxygen at a rate proportional to their volume, and that their r61e during actual cleavage does not demand an increased oxygen supply. Yet, Matthews(12) has shown that in the absence of oxygen the astral rays disappear. This does not show in any way that the asters use oxygen, it indicates that when the normal oxidative processes of the cell are upset the resulting conditions destroy the asters.

Contrary to Matthews(l2) and Osterhout (134 Warburg(21) concluded that the respiration of echinoderm eggs is a function of the cytoplasm, and is independent of nuclear synthesis. Warburg's conclusions have been criticised by Robertson (15) ; and it is true that the data given by 'Warburg from experiments with normal cells are insufficient to give a decisive proof of his conclusion. At the same time, his evidence from the effects of narcotics on respiration and on nuclear synthesis, taken in conjunction with the data provided in this paper, support the view that there is no direct association between the rate of oxygen consumption and the amount or activity of the nuclei.

SUMMARY. The process of cell division in echinoderm eggs has under normal conditions

no effect upon the rate of oxygen consumption. If the nucleus plays any direct rde in the oxidative processes in the cell it does so independently of any particular phase of nuclear activity. The development of the egg is associated with an acceleration in the rate of oxygen consumption.

REFERENCES. (I) BALDWIN, F. M. (1920). Biol. Bull. 38, 123. (2) GRAY, J . (1924). ROC. Roy. SOC. 98 B, 95. (3) (4) HEIDENHAIN, M. (1895). Arch. Entw. Mech. 1, 4. ( 5 ) JUST, E. E. (1922). Ame~Jourlr . Phyriol. 61,565. (6) LILLIE, R. S. (1916). Joum. Exp. Zod. 21,401. (7) Lo=, J. (1895). PpiisCr's Archh. 82, 249. (7 a) Lorn, J. and WASTENEW, H. (1911). Bioclunr. Zed. 38,345.

- (1924). Roc. Comb. Phil. SOC. (Biol. Sn'.) 1, 164.

16 PSPBS 1.

J. G R A Y (8) LYON, E. P. (1902). Amer.Journ. Phys. 7, 56. (9) - (1904). Science, 19, 350. - (1904). Amw.Journ. Phys. 11, 52. (10)

(11) MCCLENDON, J. F. (1913). Arch. Enm. Mech. 37, 233. (12) MATTHEWS, A. P. (1907). Amw. Journ. Phyr. 18,89. (13) OSTBRHOUT, W. J. V. (1907). ScimCe, 46, 367. (14) ROBERT~ON, T. B. (1909). Arch. Entw. Mech. 27, 29. (15) - (1924). The C M c u l Buris of Growth and Senescence. Philadelphia. (16) SHEARER, C. (19+2). Proc. Roy. Soc. 9313, 213. (17) SPAULDINC, E. G. (1904). Biol. Bttll. 6, 224. (IS) SPEK, J. (1918). Arch. Enhu. Mech. 44, 5. (19) STRANGEWAYS, T. S. (1922). Proc. Roy. Soc. 946, 137. (20) V h , F. (1922). Comptu Rendits, 643. (21) WARBURG, 0. (1914). Ergeb. d. Physiol. 14, 253.