Age and Mitotic Activity in the Male Mouse, Mus Musculus L

In earlier analyses of mitotic activity in the male mouse (Bullough, 1948a, b) it was shown that the routine of waking and sleeping determines the form of the diurnal mitosis cycle, and that changes from the normal in this routine result immediately in changes from the normal in the mitosis cycle. The opinion was then expressed that factors such as the age and sex of the animals, and the habits of the laboratory staff, might be expected to influence the daily round of exercise and rest, and so to affect the form of the mitosis cycle.

Opportunity has now been found to discover the effect of age on the daily routine and mitotic activity of the mouse, and the following paper is a review of conditions in the male.

As in previous work, the observations were all made on mice of two pure line strains, Kreyberg’s white label albinos and Strong’s CBA agoutis. In the younger mice no differences were found between the strains, but with increasing age marked differences developed in both habits and mitotic activity. It is therefore unfortunate that so many of the mice of the older age groups were of one strain, the Strong’s CBA agouti. This was due to the fact that these males rarely fight, and are therefore far more easily kept for long periods than are the more irritable Kreyberg’s mice.

The results took about 12 months to collect, and thus represent mice examined at all seasons of the year. However, apart from unavoidable variations in the length of day, conditions were kept as uniform as possible. The room temperature was maintained at 20° C., and the mice received a regular diet of rat cake, dog biscuit, oats or flaked maize, and chopped carrots. Invariably they were given their food between 09.00 and 10.00 hr., and always it was given in excess so that at no time were they without something to eat.

The times of day recorded in the various experiments sometimes represent Greenwich mean time, sometimes British summer time, and occasionally double British summer time. In practice it was found unnecessary to record which of these systems was in operation when an experiment was performed, since the animals quickly adapted themselves to a change of the clock. It thus became abundantly clear that the daily habits of the animals were adjusted to the time of feeding, and they took only a few days to become accustomed to an hour’s change either way.

This has already been described in detail by Bullough (1948 a). Small pieces of ear were removed at intervals by means of a conchotome, and were fixed in Bouin’s alcoholic fluid. After sectioning at a thickness of 7μ, the mitoses were counted in section lengths of 1 cm. From each earclip ten such counts were made, and from these an average figure was obtained. As each experimental group usually consisted of clips from ten mice, ten average figures were available from which to derive the mean and standard error. The latter was calculated by the method for small samples recommended by Simpson & Roe (1939).

As a check on the results obtained by the earclip technique, mice were killed and examined after their mitoses had been arrested by means of colchicine. To each adult animal, weighing about 25 g., o-i mg. of colchicine dissolved in 0-25 c.c. of water was injected subcutaneously, but juvenile animals received proportionately less according to their weight. After 12 hr. the mice were chloroformed, dissected widely open, and fixed whole in Bouin’s alcoholic fluid.

Half of each group of animals was injected at 09.00 hr. and killed at 21.00 hr., while half was injected at 21.00 hr. and killed at 09.00 hr. Thus it was planned that a complete period of 24 hr. should be covered by each experiment. However, after the experiments had been completed and the stock of mice used up, it became evident that colchicine not only arrests mitosis in the metaphase, but that it also depresses the number of resting cells which enter the prophase. A separate investigation of this point was then made (Bullough, 1949b), and as a result it had to be concluded that the colchicine experiments as recorded here do not reveal as much as was hoped of the degrees of mitotic activity typical of the different age groups.

For comparison with the differences observed in the mitotic activity of the different age groups, the spontaneous bodily activity of the mice was also studied. For this purpose, five mice at a time were kept in a box containing two compartments connected by a small hole. In the hole was a hinged door which was pushed aside each time an animal passed, the movement being communicated to a spring arm and recorded on a revolving smoked drum.

Each group of mice remained in the apparatus for 20 days at a time, so that for each hour of the day and night twenty figures were obtained representing the spontaneous activity of the five mice. From these twenty sets of figures, averages and standard errors were calculated.

A study was first made of the mitosis cycles of normal male mice during each of the first 20 months of life. At the age of 20 months mice can be considered old, although there are great variations in this respect between different strains. Thus 20-month-old Strong’s CBA mice are usually in good condition and, if carefully tended, they may live for another year or more, while 20-month-old Kreyberg’s white label mice are usually thin and feeble and, in the best of conditions, they have only a short expectation of life. Difference in what has been termed the physiological age may also be induced by the conditions in which the mice live during their first 20 months, and by the diseases which they may have. In the experiments recorded here all the mice were subjected to the same conditions, and all were free from disease.

It appeared that the most satisfactory way to study the mitotic activity in each month of life would be to follow the cycle through a complete period of 24 hr., and then to repeat the experiment at a later date as a check on the first results. However, the effort required, and especially the numbers of mice needed, proved too great for this to be done. Consequently only those variations occurring between 08.00 and 20.00 hr. were analysed. This 12 hr. interval covered the feeding period between 09.00 and 10.00 hr. when the mice were always disturbed, the early afternoon sleep period during which the animal room was always quiet with all age groups resting, and the evening period of activity in which, by 20.00 hr., all animals were observed to be fully awake once more. Thus the interval 08.00-20.00 hr. could be expected to begin and end with periods of low mitotic activity, and to contain somewhere within it a period of high mitotic activity. During this time seven earclips were taken at 2 hr. intervals, and, since all of these could easily be obtained from one ear, it was possible to conserve the other ear for use in a subsequent month.

Whenever possible the results for each month were confirmed by a second experiment performed at a different time with different mice. Because of the limited supply this was not always possible, but it was usually found that confirmation of the results for any one month was provided by the results for the months which preceded and followed it.

It quickly became obvious that striking changes in the mitotic activity of the ear epidermis do occur with advancing age, and it was possible to distinguish four types of cycle which differed from one another in amplitude and often also in timing. These four ages of the mouse can be named as follows:

The differences in mitotic activity between these various ages are clear cut, and an analysis of them is given below.

In this period of life the animals are still actively growing, and they range in weight from under 10 g. to just over 20 g. Sections of the testes showed that at 1 month spermatozoa were forming but had not yet been released into the epididymis. By 2 months the epididymis was full of spermatozoa, and the animals were presumably in breeding condition. When given the opportunity, mice of these strains have been known to breed at an age of 5 or 6 weeks.

The characteristics of the epidermal mitotic activity of these young animals are indicated in the following Table 1.

Important points to notice in this table are that the peak of mitotic activity was at 14.00 hr., and that the highest average number of mitoses observed per unit section length of 1 cm. was only about 5. Further, it will be noticed that the total of the average numbers of mitoses in each group of observations was about 22, and that there were no differences between the Strong’s CBA males and the Kreyberg’s white label males.

This age apparently begins quite abruptly when a mouse ceases to grow actively on reaching a weight of about 25 g., the testes then being fully active. This usually seems to happen some time about the beginning of the third month of life, and the first example, given in Table 2 below, is of a group of mice of that age. The mature age continues until the mouse is about a year old, and during that time little or no variation in the mitosis rate was found. The characteristics of the mitotic activity of this age can be seen from the Tables 2 to 4.

A survey of these tables indicates that between the ages of 3 and 12 months the time of maximum mitotic activity commonly remains, as in the immature animals, at about 14.00 hr. However, in these mature animals the maximum number of mitoses per unit section length of 1 cm. has risen from the immature figure of 5 to a new figure of about 8. Similarly, the totals of the average numbers of mitoses observed has risen from the immature figure of about 22 to a new figure of about 30. Thus the mature age is characterized by an apparent rise in the mitosis, rate. The question of whether this increase is real or illusory is dealt with in a later section.

A further point of interest also emerges from these tables. It can be seen that while the Kreyberg’s white label mice continued with great regularity to develop maximum mitotic activity at 14.00 hr., the Strong’s CBA mice did not. While these latter mice maintained their mitosis rate apparently unchanged, they tended after the age of about 10 months to develop maximum mitotic activity at an earlier hour. As will be seen in the following section, this tendency towards an earlier peak of mitotic activity is continued during middle age in mice of this strain.

The next change in the mitosis rate was apparently abrupt, and was observed in 13-month-old Strong’s CBA mice. About this time the animals seemed to be entering into what, by human analogy, can be called middle age. They usually appeared quieter and lazier, but the only positive sign of metabolic change was their increasing weight. The mature males had a steady average weight of about 25 g., but at 12 or 13 months almost all the animals began to lay down deposits of fat. This process was particularly marked in Strong’s CBA mice, some of which reached weights of over 50 g. at an age of about 16 months. There was no other external sign of advancing age, and the fur remained glossy and thick. Internally, apart from the fat deposits, there was also no apparent change. The testes remained indistinguishable from those of younger mice, and, with one exception, all the middle-aged males examined showed active spermatogenesis with the epididymes full of spermatozoa. The single exception, a Strong’s CBA mouse aged 16 months, had two shrivelled testes and lacked any spermatozoa. It was undoubtedly abnormal.

However, if it was difficult to point to any other feature which could be used as a positive sign that the middle-aged condition had been reached, the change in the mitosis rate at this time appeared to be certain and complete. This is brought out in the Tables 5 and 6 for Strong’s CBA mice from which the characteristics of this middle-age period can be readily perceived.

For an understanding of the peculiarities of mitotic activity in the middle-age period, the figures of the 13- and 14-month-old mice are the most valuable. They show a sudden apparent increase in the mitosis rate, so that the maximum number of mitoses per unit section length of 1 cm. rose from an earlier figure of about 8 to the new figure of about 14. Similarly, the total of the average numbers of mitoses observed during the 12 hr. period rose from the earlier figure of about 30 to the new figure of about 47. The question of whether this increase is real or not is dealt with in a later section.

The other obvious change in the mitosis cycle shown by these tables is the steady shift of the period of maximum mitotic activity from 12.00 to 10.00 hr. and then from 10.00 to 08.00 hr. Further, it was observed that by the age of 16 months almost all the mitoses counted at 08.00 hr. were in the telophase. This was also true in the 17- to 20-month age groups, and it is therefore obvious that in all these cases the period of maximum mitotic activity had passed before the observations were commenced at 08.00 hr. This explains the lower total numbers of mitoses observed in these older groups.

It is also interesting to note that when the main peak of mitotic activity moved forward to 08.00 hr. or earlier, a small secondary burst of activity developed sometime between 12.00 and 16.00 hr. This was perhaps clearer in the sections themselves than it is in the tables because, while up to 12.00 hr. the mitoses were mainly in the ana- and telophases, in the early afternoon pro- and metaphases predominated. These in turn gave place to telophases by 16.00 and 18.00 hr.

These results for the Strong’s CBA mice have been described separately since they differ somewhat from those obtained with the Kreyberg’s white label mice. These latter results are given in Table 7.

In the first place it may be stressed that these figures for the Kreyberg’s mice confirm the fact that an apparent increase in the mitosis rate accompanies the transition to middle age. Compared with the maximum figures of about 8 mitoses obtained for mature mice, the new maximum lies between about 12 and 18. Similarly, the total of the average numbers of mitoses observed during the 12 hr. period of the experiment has risen in middle age from the earlier figure of about 30 to a new figure of between 46 and 60.

However, it will be seen that the Kreyberg’s white label mice differed markedly from the Strong’s CBA mice in that the period of maximum mitotic activity remained at about 14.00 hr. as in younger animals. The timing of this period of maximum mitotic activity was certainly more irregular than in the younger mice, but there is no evidence of any progressive forward shift.

The general conclusions can therefore be reached that the period of middle age, which begins at about 13 months, is characterized by an apparent rise in mitotic activity, and that, according to the strain of mouse used, this change may or may not be accompanied by changes in the timing of the mitosis cycle.

Relatively few data are available for the senile age, which is characterized by feebleness and great loss of weight. In the Kreyberg’s white label mice these changes were apparent before the age of 20 months, but in the Strong’s CBA mice they were not. The Kreyberg’s mice of 19 and 20 months, on which Table 8 is based, were emaciated with weights of less than 20 g. Their backs were permanently arched, and their fur was thin and poor. They were extremely feeble, and they spent almost all their time lying quietly in a corner.

In these old animals the mitosis rate apparently fell to a slightly lower level than that seen in the immature males (Table 1). The time of maximum mitotic activity remained at about 14.00 hr., but at that time the average number of mitoses per unit section length of 1 cm. was only about 5 as compared with almost 13 in the 18-month-old Kreyberg’s mice. A similar fall is seen in the total of the average numbers of mitoses observed during the 12 hr. period of the experiment. In these senile mice the figure was only about 18 or 19 as compared with about 50 for the 18-month-old animals.

Following this preliminary survey, the observations were checked and extended by a study of each type of cycle in greater detail. The ages so examined were 2, 6 and 17 months, these being typical respectively of the immature, mature, and middle ages. Each experiment covered a full period of 24 hr., and the earclips were taken at 2 hr. intervals from 08.00 hr. on one day to 08.00 hr. on the next. It is unfortunate that no senile Kreyberg’s mice were available for this experiment. Because of this the senile age group was omitted altogether, and the observations were restricted to Strong’s CBA mice.

The results obtained with the 2-month-old males are recorded in Table 9 and shown graphically in Fig. 2. They afford still further confirmation of the results previously obtained with immature males (Table 1). They also indicate that, in the conditions of the experiment, an immature male experiences periods of maximum mitotic activity at 04.00, 08.00 and 14.00 hr., and periods of minimum mitotic activity at 06.00, 10.00 and 22.00 hr.

It can also be seen that in none of the periods of maximum activity did the average numbers of mitoses present per unit section length of 1 cm. rise above 5*8, while the lowest recorded figure at 10.00 hr. was 1.4. The total of the average numbers of mitoses observed between 08.00 hr. on the first day and 06.00 hr. on the second day was approximately 40.

The second group of males, ten Strong’s CBA mice aged 6 months, gave the figures recorded in Table 9 and in the graph in Fig. 2.

These results are in confirmation of those recorded above for mature males (Tables 2–4). Compared with the immature animals, there were only two periods of maximum mitotic activity at 06.00 and 14.00 hr., and two periods of minimum activity at 10.00 and 20.00 hr., and hour by hour the mitosis rate was on a higher level. The greatest average number of mitoses present per unit section length of 1 cm. was 9·1 and the lowest was 2·2. These figures can be compared with 5·8 and 1·4 respectively in the immature animals. As a final point of contrast, it should be noted that the total of the average numbers of mitoses observed between 08.00 hr. on the first day and 06.00 hr. on the second was about 63, which represents a rise of approximately 55% over the figure for the immature animals.

Table 9 and Fig. 2 also include an analysis of the mitosis counts from the group of ten Strong’s CBA males aged 17 months. Once again confirmation is provided for the results given earlier (Tables 5 and 6), and it is very clear that the diurnal mitosis cycle of these older males differs from that of the younger males both in its timing and in its amplitude. The main periods of maximum mitotic activity were at 08.00 and 24.00 hr., and there was a lesser burst of activity at 14.00 hr. on which comment has already been made. The maximum average number of mitoses present per unit section length of 1 cm. reached the high level of 15·3, while the minimum number fell no lower than 2·6. The total of the average numbers of mitoses observed between 08.00 hr. on the first day and 06.00 hr. on the second was about 80, which is an increase of about 30% over the figure for the mature males and of about 100% over the figure for the immature males.

From all the experiments recorded above it can safely be concluded that the diurnal cycles of epidermal mitotic activity in Strong’s CBA males differ in timing in the immature, mature and middle ages, while in Kreyberg’s white label males the timing of the cycles of mature, middle-aged, and senile animals is essentially the same.

However, the most important point still remains obscure. This is the question of whether in fact the ear epidermis of the immature male has a lower mitosis rate than that of the mature male, and whether this in turn has a lower mitosis rate than that of the middle-aged male. While all the evidence suggests that this is so, the conclusion is so unexpected that other explanations must be sought. The most obvious alternative possibility is that the results obtained may be due to age changes in the speed at which each division is completed. If in a mature animal the speed of completion of a mitosis is less than in an immature animal, then it might be expected that the former would show more mitoses at any given moment than would the latter. Again, if the speed of completion of a division is still further reduced in middle age, then the numbers of mitoses visible at any given moment might be expected to rise once more.

However, in the case of the senile animal it can hardly be supposed that a sufficient increase in the speed of division could occur to account for the fall in the numbers of mitoses observed, and it appears reasonable to draw the immediate conclusion that this age is in fact characterized by a real reduction in the mitosis rate.

An attempt to answer this whole question was made with the use of colchicine, which is considered to arrest all mitoses at about the metaphase. The theory underlying these experiments was that by means of a single injection all the mitoses beginning during the subsequent 12 hr. period could be arrested, so that, after sectioning and counting in the manner already described, a fairly accurate estimate could be obtained of the numbers of mitoses which normally occur during this period. In this way any complication due to the speed of completion of the divisions could be eliminated, and, with two experiments, the full period of 24 hr. could be covered and an estimate made of the total number of mitoses which occur daily.

In practice, however, difficulties were encountered which are best discussed after a consideration of the results. These difficulties made it necessary to consider the Kreyberg’s mice separately from the Strong’s mice, as is done in Tables 10 and 11. The mice concerned were injected with colchicine at 09.00 hr. and killed at 21.00 hr. to cover the 12 hr. of day, or injected at 21.00 hr. and killed at 09.00 hr. to cover the 12 hr. of night. Each adult mouse received 0·1 mg. of colchicine dissolved in 0·25 c.c. of water which was injected subcutaneously. Each immature mouse, being approximately half the weight of an adult, received only 0·05 mg. of colchicine in 0·125 c.c. of water.

From Table 10 it would appear that the total numbers of mitoses occurring in a period of 24 hr. in each cm. length of sections of ear epidermis cut 7 μ thick was in immature males about 9, in mature males about 16, and in middle-aged males about 28. While this affords strong confirmation that real increases in the mitosis rate do occur with increasing age, all these figures are considerably smaller than would be expected from the other tables given above. Thus a doubt immediately arises as to the accuracy of the method, and this doubt is increased by a consideration of the results from the Strong’s CBA mice given in Table 11.

From this table it is evident that the results for the immature males, with a daily mitosis total of about 8, and for the mature males, with a daily mitosis total of about 18, are similar to those obtained with the Kreyberg’s mice. The difference is seen in the middle-aged males, which in this table show little or no increase over the mitosis rate typical of the mature animals. The daily mitosis totals of the middle-aged Strong’s males lie between about 17 and 21, and so do not approach the middle-aged Kreyberg’s males’ figure of 28.

From all these colchicine results a strong suspicion arises that the drug not only arrests mitosis in the metaphase, but that it also slows down the rate at which the resting cells enter the prophase. If this is so, then the results obtained cannot be regarded as an indication of normal conditions, and in order to clear up this point it was necessary to carry out an investigation into the action of colchicine using the earclip technique (Bullough, 1949 b). This investigation showed that only for a period of about 5 hr. after the injection of o-i mg. of colchicine does the epidermal mitosis rate remain normal. After 5 hr. a depressing effect rapidly develops, and after 6 hr. mitosis stops altogether. Thus, in the colchicine experiments recorded in Tables 10 and 11, the mitoses observed were those which developed during only the first 5 hr. of the 12 hr. period.

With this in mind, the results can be interpreted as follows. In all the three age groups of Kreyberg’s mice injected at 09.00 hr., the colchicine must have arrested in or about the metaphase those mitoses which developed during the period up to 14.00 hr., the usual time of maximum mitotic activity associated with the afternoon sleep period. Thus these three sets of figures are strictly comparable, and it can be concluded that the numbers of mitoses which are involved in the rise to maximum activity in immature, mature, and middle-aged animals are in the approximate proportion of 1:2:3.

In the same way, the figures for the. immature and mature Strong’s CBA males injected at 09.00 hr. are also comparable, and are in the same approximate proportion of 1:2. It is the figures for the middle-aged Strong’s males which cannot be compared, since it is evident from Tables 5, 6 and 9 that in this strain the period 09.00–14.00 hr. is not a time of greatly increasing mitotic activity. Instead, the maximum activity is passed before 08.00 hr., and the increased activity which has been noted at about 14.00 hr. is slight by comparison.

Of the figures obtained for the period 21.00–09.00 hr., little can be said except that in general they support the evidence provided by the figures for the period 09.00–21.00 hr. It is important to bear in mind that the mitosis cycle is less regular during the night, probably because of the lack of a feeding time by which it can be stabilized.

While admitting the generally unsatisfactory nature of these colchicine experiments, it is legitimate to conclude that they offer the strongest evidence that a real increase in the mitosis rate of the ear epidermis occurs between the immature and mature ages, and again between the mature and middle ages. As stated earlier, it is also reasonable to conclude that the senile age is characterized by a reduction in mitotic activity to a level slightly below that of the immature mice. It is not yet possible to state accurately the sizes of these increases and decreases, but, judging from all the figures available, they are perhaps in the approximate proportions of immature, 1; mature, 2; middle age, 3; senile, 1.

An attempt was also made to account for the observed changes in the timing of the diurnal mitosis cycles in the different age groups. Since these changes in timing were most pronounced in the Strong’s CBA strain, all the experiments were performed with these animals.

In view of earlier results (Bullough, 1948 a, b), which related the diurnal changes in the mitosis rate to the periods of waking and sleeping, it was immediately suspected that the age changes in the diurnal mitosis cycles were merely reflexions of age changes in the animal’s daily habits. A study was therefore made of activity and rest in immature, mature, and middle-aged mice. All the experiments were conducted in the same way. In each case five males were put into a box with two compartments connected by a small hole, and, by means of a recording device, it was possible to discover the number of times which the five animals passed through the hole in each hour of the day and night. In each experiment the five animals remained in the box for 20 consecutive days, so that twenty sets of figures were obtained from which the averages and standard errors were calculated.

The five males used were 4 weeks old at the beginning of the experiment, and 7 weeks old at the end, and the results which they gave are expressed in Table 12 and in Fig. 3. In the figure the results are represented for convenience by a line graph, instead of more correctly by a block graph, and superimposed is the graph of the diurnal mitosis cycle of immature Strong’s CBA males (Table 9). It is evident that these young animals were never still for long, so that even in the quietest hour of the day there was an average of about eleven passages through the hole. However, it is clear from the graphs that the quieter periods at about 04.00, 08.00 and 14.00 hr. were also the times of maximum mitotic activity, while the periods of greatest bodily activity at about 06.00, 10.00 and 21.00 hr. were the times of minimum mitotic activity. Of these periods of greatest bodily activity, that at about 10.00 hr. coincided with the daily feeding time, while those at 06.00 and 21.00 hr. approximately coincided with dawn and dusk respectively.

For later comparisons it should also be noted that the total of the average numbers of times which the five animals passed through the hole each day was approximately 565.

The Strong’s males examined in this experiment were 5 months old, and the results are given in Table 12 and in Fig. 4.

Like the immature animals, these males become extremely active at about 10.00 hr., when they were in the habit of being fed, and were inactive at 14.00 and 15.00 hr., when they observed an afternoon rest period. They became active again in the evening, and they maintained a moderate degree of activity throughout the night. However, unlike the immature males, they did not show any increased activity about dawn, but instead they observed several hours of rest until feeding time.

These points are illustrated in Fig. 4 to which, for comparison, a graph of mitotic activity has been added (Table 9). Once again it is obvious that the times of high bodily activity are also the times of low mitotic activity, while the times of low bodily activity are the times of high mitotic activity.

It should also be noticed that the mature males indulge in less spontaneous bodily activity than do the immature males. Their rest periods are longer and more pronounced, and the total of the average numbers of times which they passed through the hole each day was only about 460 as compared with about 565 for the younger animals.

The third experiment was performed with 17-month-old males, and the results are also shown in Table 12. They are illustrated graphically in Fig. 5.

The differences between this cycle of spontaneous activity and those of the younger animals are considerable. In the first place it can be seen that the middleaged mice were not greatly disturbed when the food was put into their box at about 10.00 hr., so that the early morning rest period became almost continuous with the afternoon rest period. The animals were closely watched at their feeding time, and it was found that they ate very little and quickly returned to rest. Thus their daily routine was a simple one with almost continuous rest during the 12 hr. period 06.00–18.00 hr., and almost continuous activity during the 12 hr. period 18.00–06.00 hr.

Another point of difference from the younger males was that at no time did these animals develop such a high rate of activity. The highest average number of passages through the hole in 1 hr. was only 23·9, as compared with 49·6 for the mature males and 46·4 for the immature males, while the lowest number of passages was 2·4, as compared with 5·0 and 11·1 respectively. This drop in the spontaneous activity of the middle-aged mice is most clearly shown by the total of the average numbers of passages through the hole per day. This figure was only about 280, as compared with 460 for the mature males and 565 for the immature males.

In Fig. 5 the comparison is made between the bodily activity (Table 12) and the mitotic activity (Table 9) of middle-aged Strong’s CBA males. Once again the usual inverse relationship is demonstrated. The beginning of the early morning rest period coincides with a rise in the mitosis rate, and the beginning of the evening activity coincides with a fall. In the night there is a slackening of bodily activity about midnight which is accompanied by a second rise in the mitosis rate, and an increase in activity about 04.00 hr. which is accompanied by a second fall. In addition, there are minor fluctuations in bodily and mitotic activity between 10.00 and 18.00 hr. which also show an inverse relationship.

However, these times of minor fluctuations by night and by day are particularly interesting since they provide an apparent contradiction. It can be seen that the rest period between 10.00 and 17.00 hr. is the longest and most clearly defined in the whole day, and yet it is accompanied by only relatively slight mitotic activity. By contrast, the reduction in bodily activity between 22.00 and 02.00 hr. is negligible, but the increase in the mitosis rate which accompanies it is great. This curious state of affairs has considerable theoretical importance, and it is dealt with in some detail in the next section.

With this apparent anomaly set aside, the general conclusions emerging from these results can be summarized as follows. In all age groups high bodily activity is associated with a low rate of mitosis, while rest or sleep is associated with a high rate of mitosis. Thus the differences observed in the timing of the diurnal mitosis cycles of immature, mature and middle-aged Strong’s CBA males are related to, and apparently dependent on, differences in the timing of the diurnal cycles of spontaneous activity. Finally, it might appear to be significant that the immature animals which have the highest rate of bodily activity have also the lowest rate of mitotic activity; that the mature animals which have a lower rate of bodily activity have a higher rate of mitotic activity; and that the middleaged animals which have the lowest rate of bodily activity have the highest rate of mitotic activity.

As described above, the cycle of mitotic activity in the ear epidermis of middleaged Strong’s CBA males offers an apparent modification of the general rule. While the animals observed 12 continuous hours of almost uninterrupted rest, this did not result in 12 continuous hours of high mitotic activity. The mitosis rate rose to a very high level at the beginning of this rest period, but by 10.00 hr., when the mice were fed, it fell to a relatively low level. After the insignificant disturbance due to feeding, the mitosis rate rose only slightly at 14.00 hr., and then fell steadily until the end of the rest period. With the beginning of the evening period of wakefulness it fell still further.

In a previous publication (Bullough, 1949 a) the tentative conclusion was reached that the critical factor which allows the development of a high rate of mitosis is probably the high glycogen content of the tissue concerned, and further that such a high glycogen content is normally developed with the onset of sleep because of the deposition of blood sugar which takes place at that time. However, it may be surmised that the greatest deposition takes place only at the beginning of sleep while the blood sugar level is actually being lowered, so that the process is not a continuous one. Consequently it may well be that if sleep is unduly prolonged, so that thé glycogen content of the tissue becomes depleted, the mitosis rate must fall. In a younger mouse the sleep period does not normally last for more than a few hours at a time, and it appears that sufficient energy is stored in the epidermis to maintain a high level of mitotic activity until the animal wakes. In a middle-aged mouse the sleep period is apparently too long for this to happen, and the slight disturbance at about 10·00 hr. is seemingly followed by only slight further deposition of sugar so that the mitosis rate shows only a slight recovery at 14.00 hr. After 14.00 hr. the fall in the mitosis rate is continuous until after the next burst of activity and of feeding. Then the relatively slight period of rest about midnight is accompanied by the development of a very high rate of mitotic activity.

If this theory is correct, it should be possible to cause an almost immediate rise in the mitosis rate of a sleeping middle-aged male by supplying extra carbohydrate by injection, since it could be expected that this carbohydrate would be taken up by the blood stream and deposited in the tissues. The following series of experiments was performed to test this. Sleeping Strong’s CBA males, all middle-aged, were injected subcutaneously with starch solution using the technique developed by Bullough (1949 a). This was done at 11.00 hr. when it was anticipated that the mitosis rate would be low, and each mouse received 20 mg. of starch dissolved in 0·4 c.c. of normal saline. Earclips were taken from these mice, and from saline-injected controls, at intervals from 08.00 to 20.00 hr., and it was observed that neither the injections nor the removal of the earclips caused any significant disturbance of the rest period. The results of four separate experiments are shown in Tables 13 and 14.

All the results were essentially similar. A single injection of 20 mg. of starch induced an immediate rise in the epidermal mitosis rate, so that between 1 and 3 hr. later a rate of cell division similar to that normally seen at about 08.00 hr. was induced. This is in agreement with the theory that an exceptionally long sleep period results in a depletion of the carbohydrate content of the ear epidermis with a consequent drop in the rate of cell division.

Throughout this investigation it was found convenient to concentrate on conditions in the ear epidermis, but it is obviously of the greatest importance that an attempt should be made to discover whether the results obtained are also typical of other tissues. It was hoped that precise and detailed information on this point would become available from a study of the colchicine injected animals, but the difficulties encountered in the use of this drug have already been described. However, valuable, if limited, information was obtained from some of the tissues of the Kreyberg’s white label mice which had given the most reliable results for the ear epidermis.

The first tissue to be examined from these mice was the epidermis of the antero-dorsal region of the back, the region above the scapulae. This was done in order to determine whether the conditions already found in the ear epidermis could be considered as typical of the epidermis as a whole. The mitoses were counted in unit lengths of 1 cm. of sections cut 7μ thick, and the results, given in Table 15, are therefore directly comparable to those already obtained from the ear.

In this table there is one result which is strikingly different from anything which has been described before, namely that the highest mitotic activity occurred in the i-month-old mice. All the results obtained with the ear epidermis have shown that low mitotic activity is typical of the immature age group, and it follows that, in this particular at least, the ear epidermis cannot be taken as typical of the epidermis as a whole. However, the results for the middle-aged mice confirm those already obtained with the ear epidermis in showing an increase in mitotic activity over that recorded for the mature mice.

The second tissue examined was the stratified epithelium lining the oesophagus. Sections, 7 μ thick, were cut transversely in the region just anterior to the diaphragm, and the numbers of mitoses were counted in unit section lengths of 1 mm. The results are given in Table 16.

Again the immature mice gave a mitosis count which was considerably higher than that given by the mature mice, and again the middle-age period was characterized by a sharp rise in the mitosis rate.

The third tissue examined was that of the salivary gland. This was cut into sections 7μ thick, and the mitoses were counted in unit section areas of 0·5 mm.². The results are shown in Table 17.

Once again the results are similar with a relatively high mitosis rate in the immature and middle-age groups.

The final tissue examined was the epithelium lining the tubules of the epididymis. This was chosen as a representative of the accessory sexual organs, and the counts were made on.sections, cut 7 p, thick, of that region of the caput epididymis in which the epithelial cells have a particularly tall columnar form. For the present purpose the tissue was regarded as homogeneous, and the numbers of cell divisions were estimated in unit section areas of 0·5 mm.². They are recorded in Table 18.

In spite of the fact that these figures do not really represent the numbers of mitoses occurring in a period of 12 hr., two conclusions emerge the validity of which can hardly be doubted. The first is that the ear epidermis is abnormal in developing fewer mitoses in the immature stage than in any other stage except the senile. The results for the other tissues indicate unanimously that mitotic activity is greater, and sometimes far greater, in the immature than in the mature male.

The second conclusion is that these results do not contradict the evidence of the ear epidermis that a rise in mitotic activity is typical of middle age.

The main conclusion arising from the foregoing data is that, when judged from the point of view of mitotic activity, there are four distinct ages in the life of a male mouse. Of these it seems generally true to say that the first is characterized by a high rate of cell division, and, since the animals are actively growing at this time, this is what would be anticipated. The second age begins when the adult body size has been attained. Then there is an abrupt change to a lower rate of mitosis, which is maintained at a remarkably steady level until the age of about 12 or 13 months when a further change marks the onset of middle age. This third age is characterized by an increased mitosis rate, the precise degree of increase apparently varying from tissue to tissue. The final change occurs with the onset of senility, and, although fewer data are available concerning it, it is probably true, and certainly logical, to say that it is characterized by a mitosis depression which affects the whole body.

Apart from the evidence of the mitosis rate, the difficulty of defining these four ages is considerable. The state of the reproductive system is of no assistance, and no other internal criterion has been discovered except the size of the fat deposits. Externally it is often possible, and with practice usually possible, to distinguish the four ages by the size and general appearance of the animals. Thus immature mice have not yet reached their full stature, while middle-aged mice have exceeded it by the deposition of quantities of fat. Coincidentally, the immature animals are excitable and active, while the middle-aged animals are placid and quiet. The senile animals are feeble and shrunken with arched backs and poor fur, so that they are particularly easily distinguished.

This general vagueness of definition makes it difficult to suggest any obvious basis for the changes in the mitosis rate, and the curious fact that the change from age to age is apparently quite sudden adds to the difficulty. It is extraordinary how the transition from the immature to the mature plan of mitotic activity is accomplished in no more than a week or two, and the same phenomenon is evident in Kreyberg’s mice during the transition from middle age to senility. The change from maturity to middle age is perhaps equally abrupt, but this is not yet certain.

Approximately coincident with these changes in the mitosis rate are the changes in spontaneous bodily activity. These can be regarded as furnishing a complete explanation for any alteration in the timing of the mitosis cycle, as, for instance, that between the immature and mature ages, and again that between the mature and middle ages in the Strong’s CBA mice. However, in spite of the general inverse relationship which exists between bodily activity and mitotic activity, it seems unlikely that changes in spontaneous bodily activity alone can account for the observed alterations in the mitosis rate. The transition from mature to middle age is characterized by a great reduction in spontaneous bodily activity and by an increase in the mitosis rate, but, while the one may assist in the development of the other, it appears probable that it is not solely responsible for it. It is interesting to notice here that during pregnancy in the rat the spontaneous bodily activity is also greatly reduced (Wang, 1925). Again the reduction in muscular activity might be expected to favour the development of a high rate of mitosis, but obviously this reduction cannot be held solely responsible for the raised mitosis rate. Further, it must be remembered that immature mice are very active and restless and yet have a high mitosis rate, while conversely senile mice spend almost the entire day lying at rest and yet only develop a low mitosis rate.

The conclusion seems inescapable that the age changes in the mitosis rate are due mainly to some factor other than that of exercise. In analysing this point it is obviously important to discover whether these changes, which are so abrupt in the ear epidermis, are equally abrupt and have the same timing in all other tissues. Such evidence as is available at the moment suggests that this may be so, and, if this is proved, then perhaps the critical factor or factors may lie not in the tissues themselves but in some discreet part of the body. In this case, a critical change in the composition of the blood might be suspected, and here it is of interest to recall the tendency for both the blood-sugar level and the renal threshold to rise with increasing age (see review by Cannon, 1942). From previous results on the effects of the blood-sugar level on mitotic activity (Bullough, 1949 a), it would be expected that any such rise would be accompanied by an increase in the mitosis rate, and this might perhaps furnish some explanation for the condition in middle age. Of course, as already mentioned, the reduction in the spontaneous bodily activity during middle age may also assist in the development of excessive reserves of sugar, and the deposition of fat at this time might be taken as evidence that such an excess does, in fact, develop.

An interesting side issue which may be mentioned here is the effect on mitotic activity of the prolonged rests of the middle-aged animals. That rest and sleep are favourable to mitotic activity is now well known (Bullough, 1948 a, b), but the present results show clearly that full stimulation is achieved only during the first few hours. Thereafter the mitosis rate falls unless more carbohydrate is added to the system for deposition into the tissues. This happens if the animal wakes, eats and sleeps again, or if carbohydrate is injected.

The fact that middle age is characterized by an increase in mitotic activity which is apparently general throughout the body is of particular interest. While male mice do not usually develop spontaneous tumours, it may be said of mice generally that the cancer age begins at about 12 months. If it should now transpire that an increase in the mitosis rate is normal during mammalian middle age, when spontaneous tumours are especially liable to develop, it may be a matter of considerable importance. Mottram (1944), and others working on experimental carcinogenesis, have distinguished between the blastogenie action of a carcinogen in producing cancer cells, and the developing action of non-carcinogenic factors which, by inducing hyperplasia, assist in the formation of a tumour. Thus Berenblum & Shubik (1947) have insisted that the initial action of a carcinogen is to induce a sudden and irreversible change whereby a few normal cells are converted into ‘latent tumour cells’ which then lie dormant. The development of these ‘latent tumour cells* is an altogether different process which can be assisted by any treatment causing hyperplasia, and, in the absence of such treatment, many of these cells would never receive the stimulus to develop. Thus, while the development of a raised mitosis rate in middle age would not of itself be expected to cause, or even assist in, the formation of cancerous cells, it might be expected to increase the chances of development of any latent cancer cells which were already present.

It would follow from this that if the high mitotic activity of middle age could be reduced, a reduction in the incidence of spontaneous tumours might also result. In this connexion it is now known that underfeeding has a powerful effect in reducing cancer incidence. The reviews of Tannenbaum (1947) and Boyland (1948) include evidence that a reduction in the diet of a mouse to two-thirds of what it would eat if it fed ad lib. markedly reduces the incidence of a variety of tumours, both spontaneous and induced, and also retards the time of appearance of those which do form. It has now been shown (Bullough, unpublished) that such starvation has the effect of causing an immediate and pronounced reduction in the mitosis rate of the ear epidermis of the male mouse, and thus it is evident that a restriction of diet acts in an opposite manner to that of a developing agent which induces hyperplasia.

It may therefore be suspected that any factor which restricts mitotic activity in middle age, and so induces what can be called hypoplasia, will also hinder the formation of tumours. At the moment the most potent restricting agents known are starvation and insulin, both of which act by lowering the blood-sugar level, and a similar effect can be induced by phloridzin, which acts by reducing the availability of whatever sugar is present in the body (Bullough, 1949 a). While work on these lines is still in progress, preliminary results have already indicated that mice kept phloridzinized during middle age are considerably less liable to develop spontaneous tumours than are the controls. In view of these results it appears highly significant that, in the experiments reported by Tannenbaum concerning the effect of restrictions of diet on carcinogenesis, it is the carbohydrate fraction of the food which is the most important. A reduction in the protein fraction has no effect on tumour development, while a reduction in the fat fraction produces irregular results.