A RHYTHMICAL PERIODICITY IN THE MITOTICDIVISION OF ANIMAL CELLS

BY ALICE CARLETON, M.A. (OxoN), M.B., N.U.I.Department ofHuman Anatomy, University of Oxford

A PERIODIC 24-hourly rhythm has often been observed in the karyokinesis ofplant cells. Different forms of plant life show a maximum number of mitosesoccurring chiefly at night, and a minimum in the daytime. The relative differ-ence between maximum and minimum varies from plant to plant, as does alsothe hour at which the extremities of the curve are attained.

Karsten(9) has shown that in Spirogyra, a maximum nupiber of mitosesoccurs about midnight. Kellicott(1o), working with Allium, found a doublerhythm, a primary maximum at 11 p.m. and a primary minimum at 7 a.m.,with a secondary maximum and minimum at 1 and 3 p.m. respectively.Stalfelt(13) observed in the roots of Pisum sedativum under constant outwardconditions a daily rhythm with a maximum about 9-11 in the morning and aminimum about 9-11 at night.

This rhythm is, however, not invariably found. Karsten observed aperiodical karyokinesis in the young buds of Zea Mais, reaching a maximumduring the night, but in the root tips of Vicia Faba no such rhythm was to befound. He concluded that root growth is non-periodic ("entbehrt der Perio-dizitat"). Berinsohn(l), however, working with Allium Cepa, found that theroot tips showed more cell division in the dark than in the light'. On the otherhand, Friesner (6), examining a variety of seedlings and roots from germinatingbulbs, found a number of oscillations, as a rule three, in the 24-hour period,and came to the conclusion that the occurrence of maxima and minima in themitotic curve was dependent not on the time of day by the clock but on thetime of initiation of metabolic activity.

A great deal of work has been done on the details of karyokinesis in theanimal world, and on the various possible causes which induce or inhibit it.But I have been able to find very little reference to the possibility of a periodicrhythm such as has been observed in plant cells. Mrs Droogleever Fortuyn-van Leyden(3) has, however, investigated this point. She used for her firstobservations six newly born kittens from two different litters. These werekilled at intervals of 4 hours on the second day of life. She does not mentionthe conditions of temperature or of light, though the light was, by inference,the ordinary light of night and day. In the mesentery, mitoses began to appearat 2.30 p.m., increasing in number till 2.30 a.m. In the crypts of Lieberkiihn,the maximum was at 6.30-10.30 p.m., the minimum at 10.30 a.m. In a secondseries of observations, again on kittens, she records her findings in the thymus,lymph gland and spleen. A maximum number of mitoses was found from10.30 p.m. to 2,30 a.m., and a minimum at 10,30 a.m. The bone marrow of

252 Alice Carleton

these animals showed a maximum at the same time, i.e. 10.30 p.m.-2.30 a.m.,but the minimum occurred at 2.30 p.m. Six mice were also examined. Theirparentage was not known, and lighting and temperature conditions are againnot mentioned. The crypts of Lieberkuhn in the mouse had a maximumnumber of mitoses at 11 a.m. and a minimum at 3 a.m.

Mrs Fortuyn-van Leyden, therefore, found a periodic 24-hourly rhythmin the karyokinesis of cats and mice, though the hour of the maxima andminima varied for the two animals, and to a lesser extent for different tissuesin the same animal. The number of observations was small, and in criticismof the results one might say that the animals were of different parentage,thus increasing the chance of individual variations, and no effort apparentlywas made to stabilise temperature conditions.

This paper records, firstly, observations on eight litters of mice for thepurpose of confirming Fortuyn-van Leyden's results. The animals in eachseries belong to one litter only. The lighting conditions for the doe before birthand for the litter were of the natural diurnal sequence. They were all killedin the same way-by chloroform. Immediately after death, a square piece ofskin was cut from the lower part of the right side of the abdomen, and fixedat once in Carnoy's fluid. I would like to emphasise that removal of the tissueand fixation were both done with the minimum of delay, in view of the sug-gestion that cells undergoing mitosis might complete their division if fixationwere delayed. Four hours after fixation in Carnoy, the tissues were changedinto 96 per cent. alcohol. Though the method used was the same in each case-this fixative having been found by preliminary trial and error to be the mostsuitable-the results varied considerably, and in one, fixation was so poor thatthe colon, which had been removed at the same time, was used instead.

The percentage number is based in every case on a count of 5000 cells,made on five different groups of 1000. The epithelial cells of the epidermis andhair follicles were counted. Cells which are beginning or completing karyo-kinesis present difficulties to the observer. All the counts were made bymyself, so that the same standard, as far as possible, was applied in each case.In cases 1, 2 and 3, no endeavour was made to stabilise the temperature of thesurroundings.

Case 1. A litter of white mice. The first of the litter was killed about8 hours after birth. The skin was examined. The percentage of mitoses was asfollows1:

12 o'clock ... ... 0 416 ,, .. ... 0.6

0 ,, ... ... 2-04 ,, ... ... 0-28 ,, ... ... 0.6

1 The continental mode of counting the hours of the day as 1 to 24, instead of two series of1 to 12, has been used to avoid confusion.

Rhythmical Periodicity in Mitotic Division of Animal Cells 253

Case 2. A litter of white mice. The first was killed about 24 hours afterbirth. The skin was examined. The percentage of mitoses was as follows:

12 o'clock ... ... 0.816 ,, ... ... ,, 1-320 ,, ... ... 2-30 ,, ... ... 2X04 ,, ... ... 1P58 ,, ... . . .

Case 3. A litter of white mice. The first was killed about 36 hours afterbirth. The skin was examined. The percentage of mitoses was as follows:

12 o'clock ... ... 0-816 ,, ... ... 0720 ,, ... ... 1.90 ,, ... ... 1P34 ,, ... ... 1P28 ,, ... ... 0-6

As it struck me after these results that the differences in growth ratemight be due to the varying temperature of the surroundings, the animalsfrom now on were kept as far as possible at a constant temperature, the varia-tion never being more than 80 F.

Case 4. A litter of white mice. The first was killed about 48 hours afterbirth. The skin was examined. The temperature of the room was 700 F. atnight and 750 F. by day. The percentage of mitoses was as follows:

12 o'clock ... ... 0-516 ,, ... ... 04

20 ,, ... ... 0'8

0 ,, ... ... 1*04 ,, ... ... 0.98 ,, ... ... 0-5

Case 5. A litter of white mice. The first was killed about 24 hours afterbirth. The skin was examined. Temperature conditions as in case 4. Thepercentage of mitoses was as follows:

12 o'clock ... ... 0-416 ,, ... ... 0*620 ,, ... ... 1*30 ,, ... ... 1-24 ,, ... ... 1-18 ,, ... ... 0-6

17Anatomy LXVIIJ

254 Alice Carleton

Case 6. A litter of white mice. The first was killed 6 days after birth. Theskin was examined. Temperature conditions as in case 4. The percentage ofmitoses was as follows:

12 o'clock ... ... 0-616 ,,, ... ... 0-620 ,, ... ... 1

0 ,, .. .. 1-24 ,, ... ... 1-08 ,, ... ... 0-6

Case 7. A litter of white mice. The first was killed 7 days after birth. Theskin was examined. Temperature: 580 F. by night, 630 F. by day. The per-centage of mitoses was as follows:

12 o'clock ... ... 0 416 ... ... 0-620 ,, ... ... l1.0 ,, ... ... 1-J4 ,, ... ... 0*98 ,, .. .. 1.0

Dr L. D. H. Buxton now suggested to me the possibility that the curvewas not, as I had taken for granted, a 24-hourly one, but conceivably stretchedover a longer period. I waited for a litter of eight, and counted eight periodsat 4-hourly intervals.

Case 8. A litter of white mice. The first was killed 3 days after birth. Thetemperature variation was 70-72° F. The skin was used. The percentage ofmitoses was as follows:

12 o'clock ... ... 14116 ,, ... ... 1-220 ,, ... ... 1-40 ,, .. .. 1-44 ,, ... ... 0*98 ,, ... ... 0-8

12 ,, ... ... 1.116 ,, ... ... 1P3

The 24-hourly rhythm continued to hold good.If the average of the figures is taken for the eight cases the following is

the result:12 o'clock ... ... 0-616 ,, ... ... 0-75

0 ,, ... ... 1-44 ,, ... ... 0-958 ,, ... ... 0-7

Represented graphically, the curves for the eight cases run as in figs. 1 and 2.

Rhythmical Periodicity in Mitotic Division of Animal Cells 255

The result of these observations on mice is to confirm those of Mrs Droo-gleever Fortuyn-van Leyden. A daily periodic rhythm is observed, with amaximum between 8 p.m. and midnight, and a minimum at midday.

In 1876 Strassburger (14) found that if Spirogyra was left in an unheatedroom at night, and the temperature sank below 50 C., cell division was delayeduntil next morning, when on the room being warmed, it took place in fulldaylight. No inhibition of cell division seems to have occurred in the present

Hour of day12 16 20 0 4 8 12

2-21.8 Hour of day

1 0 12 16 20 0 4 8 12 16

2 case I 1\| -

2 Case 52.2 1418 _ F

14

1. F 1.1. 22CaCasea)

case fromate slgh lowrin oftencunltmertrt hch

As in each of these eight cases, them ragebeofngaesd to th ae itr

1.8-1.4

.62- Case 3

variations of temperature, it seemed possible that the periodic rhythm wascaused by the differences in illumination.

Fortuyn-van Leyden(3) observed that if growing onions were kept in thedark in a box, the lowest and highest points of the periodic curve occurred at7 p.m. and 11 a.m. respectively, whereas under ordinary conditions thesepoints were at 11 p.m. and 7 a.m.

17-2

256 Alice Carleton

Karsten (9) in his work already mentioned on the root tips and growingbuds of various plants found that:

(1) Continuous exposure to electric light eliminated the period and in-hibited growth.

(2) Irradiation with an electric lamp at night, with darkness by day, gavetwo maxima instead of one, with a time difference of 12 hours. He suggestedthe presence of two warring factors, firstly, an inheritance factor from parentsaccustomed to the natural diurnal variation, and secondly, a direct adaptationto the changed conditions.

To test the effect of varying light conditions, several litters were now keptin a dark room and exposed at intervals to the light of a 75 watt electric lamphanging over the cage at a distance of 38 in. When not exposed, the cageswere protected from light by loose wooden covers. Four-hourly counts weremade, as before, over a period of 24 hours, but whenever a larger litter thansix presented itself, further counts were made.

The following three cases were exposed to continuous illumination.

Case 9. A litter of white mice. Exposure to light began the day afterbirth, and was continued for 7 days, when the litter was killed. The tem-perature variation was minimal, 70-71° F. The skin was examined. The per-centage of mitoses was as follows:

12 o'clock ... ... 0 916 ,, ... ... 1-3

0 ,, ... ... 0784 ,, ... ... 0.98 ,, ... ... 0-6

12 ,, ... . .. 0.9

Case 10. A litter of white mice. Exposure to light began the day afterbirth, and was continued for 7 days, when the litter was killed. The tem-perature variation was 62-70° F. The skin was examined. The percentage ofmitoses was as follows:

12 o'clock ... ... 0o716 ,, .. .. 0920 ,, ... ... 1V5O , ... ... 1-04 ,, ... . .. I -

8 ,, ... ... 1-112 ,, ... ... 1-216 ,, ... ... 09

Case 11. A litter of white mice. Exposure to light began the day afterbirth, and was continued for 8 days, when the litter was killed. The tem-

Rhythmical Periodicity in Mitotic Division of Animal Cells 257

perature variation was 62-70° F. The skin was examined. The percentage ofmitoses was as follows: 12 o'clock ... ... 08

16 ,, .. . 0 720 ,, ... ... 0-80 ,9 ... ... 0*84 ,, ... ... 0*8

8 ,, ... ... aG912 ,, .. ... 0.9

These results agree with those of Karsten, already referred to, in that theperiodicity is eliminated by continuous exposure to light; but the inhibitionof growth which he noted in plants is only observed'in case 11. There is asudden irregular rise at 16 o'clock in case 9, and 20 o'clock in case 10, but nosteady wave. If an average is taken of the figures in these three cases, thefollowing results are found:

12 o'clock ... ... 0-816 ,, ... ... 1.020 ,, ... ... 100 ,, ... ... 0.94 ,, ... ... 0'98 ,, ... ... 0.9

12 ,, ... ... 1016 ,, ... ... 1D0

In fact, the periodicity has been completely eliminated.Hour of day Hour of day

12 16 20 0 4 8 12 16 12 16 20 0 4 8 12 16

1-4 - , , 1.4 _-.n0 1:6 I

.6- .~~~~~~~~~~~~~6*2 - Case 9 *2 Case 14

1.4 1~~~~~~~~~~~~~~~~~~.41,0 _-m-,0 /

-6 .~~~~~~~~~~~~~6*2 -Case IO -2 -Case 15

1414

1-0 _ 1.0.6 -

2 Average ofCases 9, 10,112 -Case 11.4

.6* \6

*2 - / - .2 Average of Cases 14 to 18 l

1-4 Fg.13 Fi. 4

-I

Fig. 3. Fig. 4.

258 Alice Carleton

The next two cases were kept in continuous darkness.

Case 12. A litter of black and white rats. Weaned 23 days after birth, andthen kept in darkness for 14 days. They were fed on mixed corn, brown bread,lettuce and liver. The temperature variation was minimal, 70-71° F. The skinwas examined. The percentage of mitoses was as follows:

12 o'clock ... ... 0-516 ,, ... . .. 0.320 ,, ... ... 070 ,, ... ... 1*54 ,, .. .. 1.38 ,, ... ... 0-2

Case 13. A litter of white mice. Kept in continuous darkness for 7 daysafter birth, and then killed. The temperature variation was 62-70° F. The skinwas examined. The percentage of mitoses was as follows:

12 o'clock ... ... 0-716 ,, ... ... 0*520 ,,9 ... 1.10 ,, ... ... 1-54 ,, ... ... 1.08 ,, ... ... 0*8

12 ,, ... ... 0-716 ,, ... ... 0.9

Continuous darkness appears hardly to affect the normal diurnal periodi-city. There is, however, a "lag."' The minimum is at 16 o'clock instead of12 o'clock, and the maximum at midnight instead of 20 o'clock to mid-night.

The following five cases were exposed to light from 11 or 12 o'clock noonto 11 or 12 o'clock at night.

Case 14. A litter of white mice. Exposed to light from 3 days after birth,and killed 10 days later. The temperature variation was 70-71° F. The fixationof the skin was so bad that the colon was used for counting. The percentage ofmitoses was as follows:

12 o'clock ... ... 0-816 ,, ... ... l 020 ,, ... ... 1.1

0 ,, ... ... 0-34 ,, ... ... 0-88 ,, ... ... 0-8

Case 15. A litter of white mice. Exposed to light from 11 to 23 o'clock,from the day after birth, and killed 8 days later. The temperature variation

Rhythmical Periodicity in Mitotic Division of Animal Cells 259

was 70-71° F. The skin was examined. The percentage of mitoses was asfollows:

12 o'clock ... ... 0-616 ,, ... ... 0*720 ,, ... ... 1-20 ,, ... ... 1*24 ,, ... ... 1-08 ,, .. .. 1.5

Case 16. A litter of white mice. Exposed to light from 12 to 0 o'clockfrom the day of birth for 10 days, and then killed. The temperature variationwas 65-70° F. The skin was examined, and the percentage of mitoses was asfollows:

12 o'clock ... ... 1.216 ,, ... ... 10

20 ,, ... ... 0.90 ,,9 . ... 0.94 ,, ... ... 0o78 ,, ... ... 05

Case 17. A litter of white mice. Exposed to light from 12 to 0 o'clock for7 days after birth, and then killed. The temperature variation was 62-70° F.The skin was used, and the percentage of mitoses was as follows:

12 o'clock ... ... 0 716 ,, ... ... 0920 ,, ... ... 0-50 ,, ... ... 0.94 ,, ... ... 0-38 ,, ... ... 0-912 ,, .. ... 1.016 ,, ... ... l-1

Case 18. A litter of white mice under exactly the same conditions as thepreceding case. The skin was used, and the percentage of mitoses was as follows:

12 o'clock ... ... 0 716 ,, ... ... 0720 ,, ... ... 0-80 ,, ... ... 1.0

4 ,, ... ... 1.0

8 ,, ... ... 0-812 ,, .. .. 07

260 Alice Carleton

If we take the average results of these five cases, the figures are as follows:

12 o'clock16 ,,20 ,,

0 ,,

4 ,

8 ,,

.... ... 0-8

... ... 0-9

... ... 0-9

... ... 0-9

... ... 0-8

... ... 0-9

The effect of retarding the normal period of illumination to a later hour ofthe day seems to have been to destroy the periodicity completely.

Hour of day12 16 20 0 4 8

c)

bo0

5)

1-41-06-2

1-41-0*6-2

141-c

-6-2

1.41-0

-6.2

.-41.0.6-2

12 16

Fig. 5.

The following four cases were exposed to light from 11 or 12 at night to11 or 12 noon.

Case 19. A litter of white mice. Exposed to light from 23 to 11 o'clockfrom the day of birth for 5 days, and then killed. Temperature variation70-71o F. The skin was examined, and the percentage of mitoses was as follows:

12 o'clock16 ,,

20 ,,

0 9

4

8 ,

1-6... ... 1-0... ... 0-7... ... 1.0... ... 1-0... ... 1-1

Case 20. A litter of white mice. Exposed to light from 0 to 12 o'clock frombirth for 8 days. Temperature variation 62-70° F. The skin was examined,and the percentage of mitoses was as follows:

-Case 19

-Case 20

-Case 21

-Case 22

7 Average of Cases 19 to 22, . I_1 I

Rhythmical Periodicity in Mitotic Division of Animal Cells 261

12 o'clock ... ... 1*216 a, ... ... 1-220 ,, ... ... 1-40 ,, ... ... 0*84 ,, ... ... 098 ,, ... ... 1-0

12 ,, ... ... 1-2

Case 21. A litter of white mice. Exposed to light from 0 to 12 o'clockfrom the day of birth for 8 days. Temperature variation 62-70° F. The skinwas examined, and the percentage of mitoses was as follows:

12 o'clock ... ... 1-216 ,, .. .. 1-320 ,, ... ... 090 ,, ... ... 0*84 , ... ... 0*78 ,, ... ... 0*8

12 ,, ... ... 1.116 ,, ... ... 1.l

Case 22. A litter of white mice. Conditions as in preceding case. Per-centage of mitoses as follows:

12 o'clock ... ... 0-616 ,, ... ... 0*920 ,, ... ... 141

0 ,, ... ... 1.0

4 ,, ... ... 1.0

8 ,, ... ... 10

An average taken from the figures in the last four cases produces thefollowing results:

12 o'clock ... ... 1.116 ,, ... ... 1-1

20 ,, ... ... 1.00 ,, ... ... 094 ,, ... ... 0*98 ,, ... ... 10

Thus, illumination from midnight to midday causes considerable irregularityand eliminates the periodicity.

An apology appears to be due for the number of apparently purposelessvariations in the conditions of temperature, the periods of illumination, andduration of life. There was much difficulty in obtaining material. Litters ofmice are often less than six, and thus useless for the present purpose. Whenthey were of six or more, the mother frequently reduced the number of her

Alice Carleton

brood by eating one or two. The litters had therefore to be taken whereverthey were available, and thus the work was done in three different laboratories,in only one of which temperature conditions were stable. Again, when themice had to be changed every night from dark to light or vice versa, one wasdependent on the services of the night porter, who did his rounds in one caseat 11 o'clock, in another at midnight. And finally, with regard to duration oflife the mice had to be killed when one could be free of any other engagementsfor 24 hours or more.

SUMMARY

1. Under normal conditions of light, there appears to be a 24-hourlyrhythm in the mitotic division of animal cells, with a maximum from 8 o'clockin the evening to midnight, and a minimum about noon.

2. This periodicity is destroyed by-continuous exposure to light; but notby continuous darkness.

3. Exposure to artificial light for 12 out of the 24 hours causes irregularityin the curve of mitotic division, with a complete suppression of the rhythm(independent of the period of illumination).

4. The numbers of individuals on which the counts were taken are toosmall for certainty, but the figures are suggestive.

My thanks are due to Prof. Thomson, Department of Human Anatomy,Prof. Dreyer, F.R.S., Sir William Dunn, School of Pathology, Prof. Gunn,Department of Pharmacology, and Prof. Peters, Biochemical Laboratory, foraccording to me the facilities of their Departments; to Dr L. H. DudleyBuxton for his valuable criticism and suggestions; and to Miss Kempson,Messrs W. Chesterman, Thomas Marsland and Jesse Wheal for assistance inkeeping animals, section cutting and staining.

BIBLIOGRAPHY

(1) BERINSOHN, H. W. (1919). "The influence of light in the cell increase in the roots of AlliumCepa." Proc. Sec. of Sciences, Amsterdam, p. 457.

(2) BURROWS, MONTROSE T. (1927). " Mechanism of cell division." Amer. J. Anat. vol. XXXIX,p. 83.

-(3) DROOGLEEVER FORTUYN-VAN LEYDEN, Mrs (1916). " Some observations on periodic nucleardivision in the cat." Proc. Sec. of Sciences, Amsterdam, vol. xix, p. 38.

(4) (1926). "Day and night period in nuclear division." Proc. Sec. of Sciences, Amsterdam,vol. XXix, p. 979.

(5) EVANs, NEWTON (1926). "Mitotic figures in malignant tumours as affected by time beforefixation of tissues." Arch. of Path. and Labor. Med. vol. i, p. 894.

(6) FRIESNER, R. C. (1920). "Daily rhythm of cell division in certain roots." Amer. J. Botany,vol. vII, p. 380.

(7) GuRwrrSCH, A. (1924). "Die Natur des spezifischen Erregers der Zeilteilung." Arch. f.mikroskop. Anat. u. Entw. Mechanik, Bd. c, S. 11.

(8) -- (1910). "Untersuch. uiber den zeitlich. Faktor der Zellteilung." Arch. f. mikroskop.Anat. u. Entw. Mechanik, Bd. xxx, 1. S. 133.

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Rhythmical Periodicity in Mitotic Division of Animal Cells 263

(9) KARSTEN, G. (1915-18). "tOber Tagesperiode der Kern- u. Zellteilungen." Zeit. f. Bot.7. Jahrg. H. 1; 10. Jahrg. H. 1.

(10) KELLICOTT, W. (1904). "The daily periodicity of cell division and of elongation in the rootof Allium." Bull. Torrey Bot. Club, vol. xxxi.

(11) KORNFELD, W. (1922). "Uber den Zeilteil. Rhythmus u. seine Regelung." Arch. Entw.Mech. H. 50, S. 526.

(12) LEVI, G. (1916). "Il ritmo e la modalita della mitosi nelle cellule viventi coltivati in vitro."Arch. Ital. Anat. vol. xv, pt 2.

(13) STALFELT, M. G. (1921). "Stud. uber die Periodizitat d. Zellteilung u. sich daran anschlies-sende Erscheinungen." Kunst. Svenska Vetensk. Hand. vol. LXII, P. 1.

(14) STRASSIBURGER, E. (1876). tber Zellteilung und Zellbildung. 2. Aufl.(15) THURINGER, J. M. (1928). "Studies in cell division in the human epidermis." Anat. Record,

vol. XL, P. 1.(16) WILSON, E. B. (1925). The Cell in development and heredity. 3rd. ed. New York.