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The diurnal cycle in epidermal mitotic duration and its relation to chalone and adrenalin
Experimental
Cell
Research
43, 343-350
343
(1966)
THE DIURNAL CYCLE IN EPIDERMAL MITOTIC DURATION AND ITS RELATION TO CHALONE AND ADRENALIN W. S. BULLOUGH Birkbeck
College,
and E. B. LAURENCE
University of London,
London,
England
ReceivedJanuary 27, 1966 RECENTLY Bullough and Laurence [6, 7] have published evidence to indicate t.hat the duration of epidermal mitosis is not uniform and that it evidently varies especially in relation to the concentration of chalone and of adrenalin within the cells. In the presence of an effective concentration of these substances there is a reduced mitotic rate and a prolonged mitotic duration. These conclusions have emerged from a study of epidermal mitosis in vitro, and in the present paper a similar study is made of epidermal mitosis in vivo. MATERIAL
AND METHODS
The mice used were all adult males (4-8 months old) of the Kreyberg WLL and Strong CBA strains and only slight and negligible differences in mitotic rate and mitotic duration were found between them. All the mice followed the same diurnal rhythm: they were always awake about 09.30-12.30 hr (during which time they were fed and cleaned) and asleep about 12.30-18.30 hr (when the animal house was quiet). Only in one experiment was this routine deliberately disturbed. To study the effects of wounding, 3 radial cuts were made through each ear. In such wounds the broken sheets of epidermal cells do not re-establish their original continuity but instead they migrate acrossthe thickness of the ear sothat the dorsal sheet joins with the ventral sheet. Thus the total distance travelled equals the thickness of the ear and is the same in all cases. Only a-day-old wounds were studied; at this time mitotic activity is usually about its maximum. In some experiments each mouse was injected with 0.1 mg colcemid in 0.25 ml normal saline 4 hr before killing; in others the mice were killed at hourly intervals without colcemid. All mice were killed by neck-pulling and the pieces of ear were fixed in Bouin’s fluid, cut 7 p thick, and stained in haematoxylin and eosin. In normal ears the mitoses were counted in 1 cm lengths of sectioned epidermis, and in these experiments N equals the number of mice used. In wounded ears the counts were made in 1 or 0.33 mm lengths of epidermis adjacent to the wound edge, and in these experiments N equals the number of wounds (there was greater variation between wounds than between mice). The mitotic rate was estimated from the number of mitoses arrested by colcemid in 4 hr on the usual assumption that colcemid does not affect the numbers of cells Experimental
Cell Research
43
W. S. Bullough
and E. B. Laurence
entering mitosis. The mitotic duration was calculated by relating the average number (M,) of metaphases arrested by colcemid in 4 hr to the total number (EM,) of all phasesof mitosis seen(without colcemid) after the lst, 2nd, 3rd and 4th hr according to the formula: duration of mitosis (in hr) = CM,/M,. The sourcesof error were constant in all experiments and the results are sufficiently accurate to indicate large-scale changesin mitotic duration.
RESULTS
It is already well known that a higher epidermal mitotic rate is characteristic of sleep [2] but the duration of mitosis during the waking and sleeping hours has not previously been determined. The results are shown in Table I together with other figures obtained from mice which were forced to remain awake in a slowly rotating box during what would normally be their afternoon sleep period. These results for mice awake and asleep also represent the control figures with which the results in Tables II, III and IV can be TABLE
I. Effects of sleep and of exercise on mitotic rate and mitotic duration in mouse ear epidermis.
Average numbers of mitoses in unit lengths of 1 cm of ear epidermis sectioned 7 ,u thick. N, number of mice. Jletaphases arrested by colcemid in 4 hr State of mice
Experimental
All phases of mitosis hourly intervals without colcemid
Duration of mitosis =%l~f, hr
N,
Ml
2v P
M2
Asleep (early afternoon)
25
10.4 kO.42
25
6.7 kO.23 7.3 50.42 6.7 + 0.31 6.2 2 0.29
2.6
Awake (evening)
20
2.8 jo.19
20
2.7kO.19 1.5kO.16 2.2 +0.25 3.5 +0.24
3.5
Forced exercise (early afternoon)
20
1.8 kO.28
20
2.5 1.7 1.8 1.6
4.2
Cell Research
43
+ 0.26 kO.25 kO.31 +0.40
Epidermal
mitotic
duration
345
in vivo
compared. The conclusions are: (1) that there is a diurnal rhythm in mitotic duration, with each mitosis being completed more quickly when the mitotic rate is high during sleep; (2) that with forced exercise the mitotic rate is unusually low and the mitotic duration unusually long. TABLE
II.
Average
Effects of adrenalin and of adrenalectomy on mitotic rate and mitotic duration in mouse ear epidermis. numbers
of mitoses
in unit
lengths of 1 cm of ear epidermis N, number of mice.
Metaphases arrested by colcemid in 4 hr
All phases of mitosis hourly intervals without colcemid
.N,
Ml
N a
Injected 10 yg adrenalin (mice asleep)
15
1.5 +o.ao
20
Adrenalectomised for 2 days (mice asleep)
10
19.8 kl.90
10
11.3 11.1 9.3 10.0
Adrenalectomised for 2 days (mice awake)
10
18.1 + 1.71
10
10.8 + 1.10 9.8 f0.74 10.1+_0.56 10.3 10.58
State
of mice
sectioned
MS 2.0 LO.17 1.420.23 2.0 +0.28 1.7kO.18 +1.04 f1.30 +0.77 +0.52
7 p thick.
Duration of mitosis CM,IMl hr
4.7
2.1
2.3
Any period of wakefulness, whether normal or caused by forced exercise, is a period of increased stress (see [5]). It is therefore logical to consider the effects of the adrenal stress hormones and especially of adrenalin since this has a particularly powerful antimitotic action in normal epidermis. The results obtained after adrenalin injection and after adrenalectomy are in Table II. These show: (1) that adrenalin, in the concentration used, decreased the mitotic rate and increased the mitotic duration in sleeping mice to values beyond those normally seen in mice when awake; (2) that adrenalectomy increased the mitotic rate and decreased the mitotic duration to values beyond those normally seen in mice when asleep; (3) that adrenalectomy Experimenfd
Cell Research
43
346
W. S. Bullough
and E. B. Laurence
eliminated the normal diurnal cycles in both mitotic rate and mitotic duration. From these results, and especially from earlier work (see [5]), the suggestion can be made that all the variations in mitotic rate and mitotic duration III. Effects of excess of epidermal chalone and of lack of epidermal chalone (alongside Z-day-old wounds) on mitotic rate and mitotic duration in mouse ear epidermis. TABLE
Average numbers of mitoses in unit lengths of 1 cm in normal ears and of 1 mm in wounded ears of epidermis sectioned 7 p thick. For normal ears: N, number of mice. For wounded ears: N, number of wounds. Metaphases arrested by colcemid in 4 hr
All phases of mitosis hourly intervals without colcemid
Duration of mitosis =%/MI hr
State of mice
Nl
Ml
NZ
Injected 50 units mouse chalone (mice asleep)
17
1.6 +0.28
10
2.0 +0.35 1.0+0.19 2.3 + 0.16 1.1 kO.33
4.0
Wounded 2 days previously (mice awake)
35
10.6 iO.46
25 36 37 39
4.0 3.4 4.4 4.2
1.5
MS
+0.28 +0.19 kO.18 f 0.21
recorded in Tables I and II may be related to variations in the adrenalin content of the cells. However, it has previously been shown that adrenalin does not act directly on mitosis but that it augments the antimitotic action of the epidermal chalone [2]. This chalone evidently forms a critically important link in the chain of reactions on which mitotic homeostasis depends, and it is therefore important to discover what happens to mitotic duration when the chalone concentration is either increased or decreased beyond the normal level. Table III records the contrasting results of a general chalone excess, achieved by injection into sleeping mice of an extract of mouse epidermis obtained as an alcohol precipitate by the technique of Bullough, Hewett and Laurence [3], and of a local chalone lack, achieved by the infliction of a wound 2 days previously. It has already been established that the highest mitotic rate of which epidermis is evidently capable can be Experimental
Cell Research
43
Epidermal
mitotic
obtained by epidermal damage and it is most probably due to the loss of the If this conclusion is accepted, then the tion is inversely related to mitotic rate tion. TABLE
IV. Mitotic
duration
347
in vivo
has been concluded that this reaction epidermal chalone from the cells [4]. results show that chalone concentraand directly related to mitotic dura-
rate and mitotic duration in mouse ear epidermis adjacent to 2-day-old wounds.
immediately
Average numbers of mitoses in unit lengths of 0.33 mm of ear epidermis sectioned 7 p thick. N, number of wounds. Metaphases arrested by colcemid in 4 hr State of mice Wounded 2 days previously (mice awake)
All phases of mitosis hourly intervals without colcemid
Nl
Ml
N,
M2
Duration of mitosis =f,/M, hr
35
6.6 20.29
25 36 37 39
2.2 kO.17
1.3
1.9 20.13 2.3 +0.13 2.1 kO.11
The range of mitotic duration recorded above is 1.5-4.7 hr, which compares with 1.2-5.8 hr for similar epidermis in vitro [6]. It is therefore interesting to discover whether in vivo a duration of less than 1.5 hr is possible. It is known that within the 1 mm wide zone of raised mitotic activity alongside skin wounds (Table III) there is a mitotic gradient with the highest point adjacent to the wound edge [4]. The figures for the inner and most active zone, 0.33 mm wide, were therefore separately analysed and the results are in Table IV. In this narrow zone the mitotic rate was extremely high but the mitotic duration was reduced to only a little less than 1.3 hr. Thus from both the in vivo and in vitro results it seems that mitotic duration in ear epidermis is not commonly reduced to less than about 1.25 hr.
DISCUSSION
The evidence suggests that in mouse ear epidermis the variations in both mitotic rate and mitotic duration are controlled by that homeostatic mechanism in which the epidermal chalone and adrenalin both play a part [a]; Experimental
Cell Research
43
348
W. S. Bullough
and E. B. Laurence
as the concentration of either or both these substances falls the mitotic rate increases and, within limits, the mitotic duration decreases (see Fig. 1, and compare [B] for the similar inverse relation in u&o). This confirms the suggestion of Evensen and Iversen [12] that “the old dogma of a constant mitotic duration for each cell type in a given species may be open for re-
Duration
Fig. I.-Relation
between mitotic
of mitosis
rate and mitotic
in hours
duration
in mouse ear epidermis
in vivo.
evaluation”, and the most that can now be said is that for each cell type in a given healthy animal of a given sex and age there is probably a relatively constant average mitotic duration. In recent years a new dogma has been growing that in adult mammalian tissues the period of DNA synthesis has an approximately constant duration of about 7 hr [ 13,14,15]. However, the figures on which this conclusion is based are mostly derived from tissues with relatively high mitotic rates and already it is known that in tissues with lower mitotic rates DNA synthesis may occupy as long as 13.5 hr (mouse forestomach [17]) and even 30 hr (mouse ear epidermis [9, 161). More important, Epifanova [ll] has recently shown that an oestrogen-induced increase in the mitotic rate of uterine epithelium is accompanied by a sharply decreased duration of the phase of DNA synthesis. It is therefore possible that the duration of the phase of DNA synthesis, like the duration of mitosis, may vary inversely with the mitotic rate. If this is true the figure of about 7 hr may represent an approach to the minimum time required for DNA synthesis. Experimental
Cell
Research
43
Epidermal
mitotic
duration
349
in vivo
It now seems probable that, within limits, an increased mitotic rate is accompanied by the reduced duration of the inter-mitotic period (or Cl), of the phase of DNA synthesis, of antephase (or G2), and of mitosis itself. Bullough and Laurence [S] have recently reviewed evidence to suggest that all these changes may be dependent on the timing and intensity of that
mRNA -
I I II I I II Fig. 2.-Diagram illustrating the two preparation of a cell for mitosis.
periods
of synthesis
during
early
and late prosphase
in the
mRNA synthesis on which the whole mitotic process depends. In conditions promoting mitosis such synthesis begins earlier, thus reducing the intermitotic period, and proceeds more rapidly. There is evidence that this mRNA synthesis is at a maximum during a short period (called early prosphase [ 11) just before the onset of DNA synthesis and again during antephase (see Fig. 2); that the speed at which DNA synthesis proceeds may be directly related to the concentration of essential enzymes formed in early prosphase; and that the speed at which mitosis proceeds may be directly related to the concentration of essential enzymes formed in antephase. The evidence further shows [S, lo] that early prosphase and antephase are approximately the two periods when chalone and adrenalin exert their maximum inhibitory actions (see Fig. 2), and suggests that the mitotic homeostatic mechanism of which these substances form a part most probably acts by limiting the synthesis of that special mRNA on which mitosis depends.
Experimental
Cell Research
43
350
W. S. Bullough
and E. B. Laurence
SUMMARY
1. It is shown that in mouse ear epidermis the duration of mitosis follows a diurnal rhythm, ranging between 2.6 hr when the animals are asleep and 3.5 hr when they are awake. 2. With injections of adrenalin into sleeping mice the mitotic rate is decreased and the mitotic duration increased beyond the levels normally found in mice when awake. After adrenalectomy the mitotic rate is increased and the mitotic duration decreased beyond the levels normally found in mice when asleep. Adrenalectomy also destroys the diurnal rhythm in both mitotic rate and mitotic duration. 3. The injection of epidermal chalone also decreases the mitotic rate and increases the mitotic duration, while the reduction in chalone concentration after wounding has the converse effects. 4. The nature of the inverse relation between mitotic rate and mitotic duration is shown in Fig. 1. It is concluded that both rate and duration are determined by the homeostatic mechanism of which adrenalin and chalone form parts. REFERENCES 1.
BULLOUGH,
2. ~ 3. 4.
W.
Cancer
BULLOUGH, BULLOUGH,
5. ~ 6. ~ 7. --
Proc. Expfl Nafure
S., Nature
199, 859 (1963). (1965). W. S., HEWETT, C. L. and LAURENCE, E. B., Expfl Celf Res. 36, 192 (1964). W. S. and LAURENCE, E. B., Proc. ray. Sot. B 151. I 517 (1960). ~ I 1 roy. Sot. B 154, 540 (i961). Cell Res. 35, 629 (1964).
Res. 25, 1683
210,
715 (1966).
8. __
in W. MONTAGNA (ed.), Advances in biology of skin. Carcinogenesis. Pergamon New York, 1966. 9. ~ Unpublished. 10. CAMERON, I. L. and CLEFFMANN, G., J. Cell Biol. 21, 169 (1964). 11. EPIFANOVA, 0. I., Exptl Cell Res. 42, 562 (1966). 12. EVENSEN, A. and IVERSEN, 0. H., Nature 196, 383 (1962). 13. KOBURG, E. and MAURER, W., Biochim. Biophys. Acfa 61, 229 (1962). 14. KRAYSCH, D., BECK, V. and OEHLERT, W., Beifr. Path. Anat. 128, 416 (1963). 15. MAURER, W., PILGRIM, C., WEGENER, K., HOLLWEG, S. and LENNARTZ, J., in FELLINGER, and H~FER, R. (eds.), Radioactive Isotope in Klinik und Forschung, vol. 6. Urban Schwarzenberg, Miinchen, 1965. 16. SHERMAN, F. G., QUASTLER, H. and WIMBER, D. R., Expfl Cell Res. 25, 114 (1961). 17. WOLFSBERG, M. F., Expfl Cell Res. 35, 119 (1964).
Experimenfal
Cell
Research
43
Press,
K. and
Cell
Research
43, 343-350
343
(1966)
THE DIURNAL CYCLE IN EPIDERMAL MITOTIC DURATION AND ITS RELATION TO CHALONE AND ADRENALIN W. S. BULLOUGH Birkbeck
College,
and E. B. LAURENCE
University of London,
London,
England
ReceivedJanuary 27, 1966 RECENTLY Bullough and Laurence [6, 7] have published evidence to indicate t.hat the duration of epidermal mitosis is not uniform and that it evidently varies especially in relation to the concentration of chalone and of adrenalin within the cells. In the presence of an effective concentration of these substances there is a reduced mitotic rate and a prolonged mitotic duration. These conclusions have emerged from a study of epidermal mitosis in vitro, and in the present paper a similar study is made of epidermal mitosis in vivo. MATERIAL
AND METHODS
The mice used were all adult males (4-8 months old) of the Kreyberg WLL and Strong CBA strains and only slight and negligible differences in mitotic rate and mitotic duration were found between them. All the mice followed the same diurnal rhythm: they were always awake about 09.30-12.30 hr (during which time they were fed and cleaned) and asleep about 12.30-18.30 hr (when the animal house was quiet). Only in one experiment was this routine deliberately disturbed. To study the effects of wounding, 3 radial cuts were made through each ear. In such wounds the broken sheets of epidermal cells do not re-establish their original continuity but instead they migrate acrossthe thickness of the ear sothat the dorsal sheet joins with the ventral sheet. Thus the total distance travelled equals the thickness of the ear and is the same in all cases. Only a-day-old wounds were studied; at this time mitotic activity is usually about its maximum. In some experiments each mouse was injected with 0.1 mg colcemid in 0.25 ml normal saline 4 hr before killing; in others the mice were killed at hourly intervals without colcemid. All mice were killed by neck-pulling and the pieces of ear were fixed in Bouin’s fluid, cut 7 p thick, and stained in haematoxylin and eosin. In normal ears the mitoses were counted in 1 cm lengths of sectioned epidermis, and in these experiments N equals the number of mice used. In wounded ears the counts were made in 1 or 0.33 mm lengths of epidermis adjacent to the wound edge, and in these experiments N equals the number of wounds (there was greater variation between wounds than between mice). The mitotic rate was estimated from the number of mitoses arrested by colcemid in 4 hr on the usual assumption that colcemid does not affect the numbers of cells Experimental
Cell Research
43
W. S. Bullough
and E. B. Laurence
entering mitosis. The mitotic duration was calculated by relating the average number (M,) of metaphases arrested by colcemid in 4 hr to the total number (EM,) of all phasesof mitosis seen(without colcemid) after the lst, 2nd, 3rd and 4th hr according to the formula: duration of mitosis (in hr) = CM,/M,. The sourcesof error were constant in all experiments and the results are sufficiently accurate to indicate large-scale changesin mitotic duration.
RESULTS
It is already well known that a higher epidermal mitotic rate is characteristic of sleep [2] but the duration of mitosis during the waking and sleeping hours has not previously been determined. The results are shown in Table I together with other figures obtained from mice which were forced to remain awake in a slowly rotating box during what would normally be their afternoon sleep period. These results for mice awake and asleep also represent the control figures with which the results in Tables II, III and IV can be TABLE
I. Effects of sleep and of exercise on mitotic rate and mitotic duration in mouse ear epidermis.
Average numbers of mitoses in unit lengths of 1 cm of ear epidermis sectioned 7 ,u thick. N, number of mice. Jletaphases arrested by colcemid in 4 hr State of mice
Experimental
All phases of mitosis hourly intervals without colcemid
Duration of mitosis =%l~f, hr
N,
Ml
2v P
M2
Asleep (early afternoon)
25
10.4 kO.42
25
6.7 kO.23 7.3 50.42 6.7 + 0.31 6.2 2 0.29
2.6
Awake (evening)
20
2.8 jo.19
20
2.7kO.19 1.5kO.16 2.2 +0.25 3.5 +0.24
3.5
Forced exercise (early afternoon)
20
1.8 kO.28
20
2.5 1.7 1.8 1.6
4.2
Cell Research
43
+ 0.26 kO.25 kO.31 +0.40
Epidermal
mitotic
duration
345
in vivo
compared. The conclusions are: (1) that there is a diurnal rhythm in mitotic duration, with each mitosis being completed more quickly when the mitotic rate is high during sleep; (2) that with forced exercise the mitotic rate is unusually low and the mitotic duration unusually long. TABLE
II.
Average
Effects of adrenalin and of adrenalectomy on mitotic rate and mitotic duration in mouse ear epidermis. numbers
of mitoses
in unit
lengths of 1 cm of ear epidermis N, number of mice.
Metaphases arrested by colcemid in 4 hr
All phases of mitosis hourly intervals without colcemid
.N,
Ml
N a
Injected 10 yg adrenalin (mice asleep)
15
1.5 +o.ao
20
Adrenalectomised for 2 days (mice asleep)
10
19.8 kl.90
10
11.3 11.1 9.3 10.0
Adrenalectomised for 2 days (mice awake)
10
18.1 + 1.71
10
10.8 + 1.10 9.8 f0.74 10.1+_0.56 10.3 10.58
State
of mice
sectioned
MS 2.0 LO.17 1.420.23 2.0 +0.28 1.7kO.18 +1.04 f1.30 +0.77 +0.52
7 p thick.
Duration of mitosis CM,IMl hr
4.7
2.1
2.3
Any period of wakefulness, whether normal or caused by forced exercise, is a period of increased stress (see [5]). It is therefore logical to consider the effects of the adrenal stress hormones and especially of adrenalin since this has a particularly powerful antimitotic action in normal epidermis. The results obtained after adrenalin injection and after adrenalectomy are in Table II. These show: (1) that adrenalin, in the concentration used, decreased the mitotic rate and increased the mitotic duration in sleeping mice to values beyond those normally seen in mice when awake; (2) that adrenalectomy increased the mitotic rate and decreased the mitotic duration to values beyond those normally seen in mice when asleep; (3) that adrenalectomy Experimenfd
Cell Research
43
346
W. S. Bullough
and E. B. Laurence
eliminated the normal diurnal cycles in both mitotic rate and mitotic duration. From these results, and especially from earlier work (see [5]), the suggestion can be made that all the variations in mitotic rate and mitotic duration III. Effects of excess of epidermal chalone and of lack of epidermal chalone (alongside Z-day-old wounds) on mitotic rate and mitotic duration in mouse ear epidermis. TABLE
Average numbers of mitoses in unit lengths of 1 cm in normal ears and of 1 mm in wounded ears of epidermis sectioned 7 p thick. For normal ears: N, number of mice. For wounded ears: N, number of wounds. Metaphases arrested by colcemid in 4 hr
All phases of mitosis hourly intervals without colcemid
Duration of mitosis =%/MI hr
State of mice
Nl
Ml
NZ
Injected 50 units mouse chalone (mice asleep)
17
1.6 +0.28
10
2.0 +0.35 1.0+0.19 2.3 + 0.16 1.1 kO.33
4.0
Wounded 2 days previously (mice awake)
35
10.6 iO.46
25 36 37 39
4.0 3.4 4.4 4.2
1.5
MS
+0.28 +0.19 kO.18 f 0.21
recorded in Tables I and II may be related to variations in the adrenalin content of the cells. However, it has previously been shown that adrenalin does not act directly on mitosis but that it augments the antimitotic action of the epidermal chalone [2]. This chalone evidently forms a critically important link in the chain of reactions on which mitotic homeostasis depends, and it is therefore important to discover what happens to mitotic duration when the chalone concentration is either increased or decreased beyond the normal level. Table III records the contrasting results of a general chalone excess, achieved by injection into sleeping mice of an extract of mouse epidermis obtained as an alcohol precipitate by the technique of Bullough, Hewett and Laurence [3], and of a local chalone lack, achieved by the infliction of a wound 2 days previously. It has already been established that the highest mitotic rate of which epidermis is evidently capable can be Experimental
Cell Research
43
Epidermal
mitotic
obtained by epidermal damage and it is most probably due to the loss of the If this conclusion is accepted, then the tion is inversely related to mitotic rate tion. TABLE
IV. Mitotic
duration
347
in vivo
has been concluded that this reaction epidermal chalone from the cells [4]. results show that chalone concentraand directly related to mitotic dura-
rate and mitotic duration in mouse ear epidermis adjacent to 2-day-old wounds.
immediately
Average numbers of mitoses in unit lengths of 0.33 mm of ear epidermis sectioned 7 p thick. N, number of wounds. Metaphases arrested by colcemid in 4 hr State of mice Wounded 2 days previously (mice awake)
All phases of mitosis hourly intervals without colcemid
Nl
Ml
N,
M2
Duration of mitosis =f,/M, hr
35
6.6 20.29
25 36 37 39
2.2 kO.17
1.3
1.9 20.13 2.3 +0.13 2.1 kO.11
The range of mitotic duration recorded above is 1.5-4.7 hr, which compares with 1.2-5.8 hr for similar epidermis in vitro [6]. It is therefore interesting to discover whether in vivo a duration of less than 1.5 hr is possible. It is known that within the 1 mm wide zone of raised mitotic activity alongside skin wounds (Table III) there is a mitotic gradient with the highest point adjacent to the wound edge [4]. The figures for the inner and most active zone, 0.33 mm wide, were therefore separately analysed and the results are in Table IV. In this narrow zone the mitotic rate was extremely high but the mitotic duration was reduced to only a little less than 1.3 hr. Thus from both the in vivo and in vitro results it seems that mitotic duration in ear epidermis is not commonly reduced to less than about 1.25 hr.
DISCUSSION
The evidence suggests that in mouse ear epidermis the variations in both mitotic rate and mitotic duration are controlled by that homeostatic mechanism in which the epidermal chalone and adrenalin both play a part [a]; Experimental
Cell Research
43
348
W. S. Bullough
and E. B. Laurence
as the concentration of either or both these substances falls the mitotic rate increases and, within limits, the mitotic duration decreases (see Fig. 1, and compare [B] for the similar inverse relation in u&o). This confirms the suggestion of Evensen and Iversen [12] that “the old dogma of a constant mitotic duration for each cell type in a given species may be open for re-
Duration
Fig. I.-Relation
between mitotic
of mitosis
rate and mitotic
in hours
duration
in mouse ear epidermis
in vivo.
evaluation”, and the most that can now be said is that for each cell type in a given healthy animal of a given sex and age there is probably a relatively constant average mitotic duration. In recent years a new dogma has been growing that in adult mammalian tissues the period of DNA synthesis has an approximately constant duration of about 7 hr [ 13,14,15]. However, the figures on which this conclusion is based are mostly derived from tissues with relatively high mitotic rates and already it is known that in tissues with lower mitotic rates DNA synthesis may occupy as long as 13.5 hr (mouse forestomach [17]) and even 30 hr (mouse ear epidermis [9, 161). More important, Epifanova [ll] has recently shown that an oestrogen-induced increase in the mitotic rate of uterine epithelium is accompanied by a sharply decreased duration of the phase of DNA synthesis. It is therefore possible that the duration of the phase of DNA synthesis, like the duration of mitosis, may vary inversely with the mitotic rate. If this is true the figure of about 7 hr may represent an approach to the minimum time required for DNA synthesis. Experimental
Cell
Research
43
Epidermal
mitotic
duration
349
in vivo
It now seems probable that, within limits, an increased mitotic rate is accompanied by the reduced duration of the inter-mitotic period (or Cl), of the phase of DNA synthesis, of antephase (or G2), and of mitosis itself. Bullough and Laurence [S] have recently reviewed evidence to suggest that all these changes may be dependent on the timing and intensity of that
mRNA -
I I II I I II Fig. 2.-Diagram illustrating the two preparation of a cell for mitosis.
periods
of synthesis
during
early
and late prosphase
in the
mRNA synthesis on which the whole mitotic process depends. In conditions promoting mitosis such synthesis begins earlier, thus reducing the intermitotic period, and proceeds more rapidly. There is evidence that this mRNA synthesis is at a maximum during a short period (called early prosphase [ 11) just before the onset of DNA synthesis and again during antephase (see Fig. 2); that the speed at which DNA synthesis proceeds may be directly related to the concentration of essential enzymes formed in early prosphase; and that the speed at which mitosis proceeds may be directly related to the concentration of essential enzymes formed in antephase. The evidence further shows [S, lo] that early prosphase and antephase are approximately the two periods when chalone and adrenalin exert their maximum inhibitory actions (see Fig. 2), and suggests that the mitotic homeostatic mechanism of which these substances form a part most probably acts by limiting the synthesis of that special mRNA on which mitosis depends.
Experimental
Cell Research
43
350
W. S. Bullough
and E. B. Laurence
SUMMARY
1. It is shown that in mouse ear epidermis the duration of mitosis follows a diurnal rhythm, ranging between 2.6 hr when the animals are asleep and 3.5 hr when they are awake. 2. With injections of adrenalin into sleeping mice the mitotic rate is decreased and the mitotic duration increased beyond the levels normally found in mice when awake. After adrenalectomy the mitotic rate is increased and the mitotic duration decreased beyond the levels normally found in mice when asleep. Adrenalectomy also destroys the diurnal rhythm in both mitotic rate and mitotic duration. 3. The injection of epidermal chalone also decreases the mitotic rate and increases the mitotic duration, while the reduction in chalone concentration after wounding has the converse effects. 4. The nature of the inverse relation between mitotic rate and mitotic duration is shown in Fig. 1. It is concluded that both rate and duration are determined by the homeostatic mechanism of which adrenalin and chalone form parts. REFERENCES 1.
BULLOUGH,
2. ~ 3. 4.
W.
Cancer
BULLOUGH, BULLOUGH,
5. ~ 6. ~ 7. --
Proc. Expfl Nafure
S., Nature
199, 859 (1963). (1965). W. S., HEWETT, C. L. and LAURENCE, E. B., Expfl Celf Res. 36, 192 (1964). W. S. and LAURENCE, E. B., Proc. ray. Sot. B 151. I 517 (1960). ~ I 1 roy. Sot. B 154, 540 (i961). Cell Res. 35, 629 (1964).
Res. 25, 1683
210,
715 (1966).
8. __
in W. MONTAGNA (ed.), Advances in biology of skin. Carcinogenesis. Pergamon New York, 1966. 9. ~ Unpublished. 10. CAMERON, I. L. and CLEFFMANN, G., J. Cell Biol. 21, 169 (1964). 11. EPIFANOVA, 0. I., Exptl Cell Res. 42, 562 (1966). 12. EVENSEN, A. and IVERSEN, 0. H., Nature 196, 383 (1962). 13. KOBURG, E. and MAURER, W., Biochim. Biophys. Acfa 61, 229 (1962). 14. KRAYSCH, D., BECK, V. and OEHLERT, W., Beifr. Path. Anat. 128, 416 (1963). 15. MAURER, W., PILGRIM, C., WEGENER, K., HOLLWEG, S. and LENNARTZ, J., in FELLINGER, and H~FER, R. (eds.), Radioactive Isotope in Klinik und Forschung, vol. 6. Urban Schwarzenberg, Miinchen, 1965. 16. SHERMAN, F. G., QUASTLER, H. and WIMBER, D. R., Expfl Cell Res. 25, 114 (1961). 17. WOLFSBERG, M. F., Expfl Cell Res. 35, 119 (1964).
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