TIME-ASSOCIATED VARIATIONS O F MITOTIC ACTIVITY I N LIVERS O F YOUNG RL4TS1 B. JACKSON Department of Experimental Pathology, Esperirnental Therapeutics Research, Lederle Laboratories Division, American Cyanarnid Company, Pearl River, New Pork ONE FIQURE It is generally recognized that the number of dividing cells in a particular tissue or organ is not the same at different hours of the day. There exists an extensive literature which describes time-associated variations of mitotic rate in plants and laboratory animals as well as in humans. This literature has been reviewed recently by Halberg ( '53). The recognition of time-associated variations of mitotic rate seems to indicate an approach for analyzing the control of cell multiplication. Cell multiplication is one of the factors involved in the increase in matter or substance. In animals, cell enlargement, another factor of material increase has not been investigated in relation to changing rates of mitosis. This factor should be included in studies of time-associated variations of mitotic rate. The studies of time-associated variations of mitotic activity in animals which have been reported in the literature have been carried out either with relatively few animals sacrificed a t many times during 24 hours o r with many animals sacrificed at two times during this period. The marked variability of individual mitotic rates as seen in many of these experiments as well as unexplainable out-of-trend peaks suggested to us that large groups of animals (6 to 8) should be sacrificed A portion of a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at New Tork University. 365 THE ANATOMICAL RECORD, VOL. JULY 1959 134, NO. 3 366 B. JACKSON at rather close intervals to get adequate representation of the extent and succession of these variations. I n the course of pharmacological studies in this laboratory we became particularly interested in the mitotic activity of the liver in young rats. Time-associated variations of mitotic activity have been observed in the livers of young mice by Wilson (’48), and their presence in the livers of normal rats was assumed by McKellar ( ’49). Jaffe ( ’54) described these variations in regenerating livers of rats. The results of a preliminary experiment (Jackson and Willey, ’56) indicated that variations of mitotic rate, related to the time of day, were present in livers of young male rats. The purpose of the present investigation was to examine the extent and succession of time-associated variations of mitotic rate in the livers of young rats, and to investigate hepatic cell sizes and numbers of binucleate cells in relation to variations of mitotic rate. MATERIAL AND METHODS Male, albino, Wistar rats, about 24 days old were used. The rats were received and quartered in the animal room about 48 hours before the experiment. The animal room was air-conditioned so that relatively constant temperature and humidity were maintained. Hourly recordings of temperature showed that the mean temperature over the experimental period was 22°C (range of 18” to 24°C). During the daytime the animal room was lighted by artificial light as well as by sunlight. The lights were turned on at 6 : 00 A.M. and remained on until 4 : 00 P.M. (E.S.T.). The term “day” was applied to the hours between sunrise and sunset. In this experiment day included the hours from 6 :00 A.M. to 8 :00 P.M., and night included the remainder of the 24 hours. All times expressed are Eastern Standard Time. Supplies of food (ground-up Wayne Lab Blox) and tap water were available to the rats at all times. During the morning of the day preceding the experiment, full bowls of MITOTIC VARIATIONS IN RAT LIVER 367 the ration were given to the rats. During the afternoon of this same day and the next day, fresh bottles of water were placed in the cages. These supplies were not exhausted at the times of sacrifice. The rats were caged in groups of 4. The cages were numbered and the rats selected for sacrifice at a particular time were chosen using a randomization procedure (random orderings of numbers 1to 26). Groups of 8 rats were sacrificed at two-hour intervals commencing at 6 :00 A.M. on June 26, 1956 to and including 6 : 00 A.M. on June 27, 1956. The rats were killed by chloroform inhalation. The 8 rats were placed in the killing jar at the same time. About 10 minutes were required to remove the livers and place the liver samples in fixative. Samples of liver tissue consisted of a slice from base to apex from the right part of the median lobe. The location of the sample was chosen as suggested by Eranko ( ’55).2 After the tissue samples were placed in fixative, several slices were made through the remainder of the liver and the cut surfaces examined. No evidence of parasites or other pathologic processes were seen in any of the livers. Tissues were fixed in Carony’s fixative (6 parts absolute ethanol, three parts chloroform, one part acetic acid). Paraffin sections were cut at 10 p and stained with Weigert’s iron hemat oxylin. Counts of hepatic cells in mitosis were carried out at a magnification of 970 x. The number of mitoses in 250 fields was determined for each liver. Binucleate cells in mitosis, of which there were extremely few, were included in the total number of mitoses. All stages of mitosis were seen and counted. Precautions were taken while moving the slide during counts to avoid the same field. Mitotic activity was expressed as the number of mitoses per 1,000 cells, a figure n“Even if it has been demonstrated that nuclei are similar in all parts of the liver it is wise to take a piece from the same lobe and moreover from the same part of the same lobe in each animal, because different lobes may be differently affected in the experiment.” 0. Eriinkii ( ’55). 368 B. JACKSON based on the number of mitoses in 250 fields and the mean number of cells per field. Mononucleate cells and binucleate cells were distinguished in counting but both were included in counts of the total number of cells per field. Counts in 25 fields in each liver were used to obtain an estimate of the number of cells per field as well as the percentage of binucleate cells. The values from cell counts and mitotic counts for the different times are summarized in table 1 by their arithmetical means. Standard errors of the means were calculated from sz=s/dG where s is the standard deviation of a single value (rat) and YZ is the number of values in the particular mean. The statistical significance of time-associated differences over the 24 hours was tested by the analysis of variance. Differences between individual means were examined by ttests. OBSERVATIONS Mitotic actiuity. The results of the mitotic counts are shown in table 1 and figure I. During the 24-hour period TABLE I Mitotic activities, cell densities and percentages of hinucleate cells in livers of young male rats sacrificed at two-hour intervals TLME O F DAY I.R.T. 6 A.M. 8 10 12 M. 2 P.M. 4 6 8 10 12 2 A.M. 4 6 MEAN MITOTIC * +- ACTIYITY ST. ERROR 5.07 2 1.27 13.10 f 3.10 5.07 -+ 1.14 10.28 -I1.45 7.76 -C 1.59 4.95 2 0.72 3.56 & 0.85 2.68 2 0.61 2.16 & 0.46 2.33 2 0.27 2.16 -+ 0.43 2.06 -t 0.48 3.19 -+ 0.55 MEAN NO. O F CELLS PER FIELD 2 ST. ERROE 54.6 -C 1.96 56.2 & 1.29 60.1 5z 3.66 61.9 f 2.91 60.9 f 2.38 65.4 f 2.29 62.0 -t- 1.64 70.8 f3.83 65.2 f 2.02 61.5 f 2.89 58.7 & 1.84 59.4 2 2.16 55.3 f 1.81 'Each mean value in table based on 8 rats at each time. Mitotic activity is number of mitoses per 1,000 cells. MEAN PERCENTAGE O P BINUCLEATE CELLS & ST. ERROR 20.3 1.58 22.4 f 1.80 23.1 f 1.68 20.0 & 0.95 25.6 f0.86 22.7 f1.59 26.9 -C 1.12 24.2 1.08 23.9 f 2.17 23.7 & 0.81 24.6 & 0.98 22.4 f 1.54 20.9 f 1.87 I r l l l r l l l l OF BINUCLEATE CELLS I u I u I u N I u w . e . U I : z PERCENTAGE (DEE - c t c t t t t t t c t 0 P P UI ' 0 Q m -J '0 o) D - REL.CELLSIZE(l/noof cells per field) o r , ~ P m c o 5 , i 3 6 MITOTIC ACTIVITY (mitosesA000 Cells) 370 B. JACKSON studied, the mitotic rate varied as much as 5 to 6 times the minimal rate seen. Mitotic activity was found to be higher during the day than during the night. Highest mitotic activity was seen at 8: 00 A.M. Following this peak, a drop occurred at 10: 00 A.M. At 12: 00 M. the mitotic activity was again high but showed a gradual downward trend throughout the rest of the day. During the night the mitotic activity was uniformly low but was slightly increased at 6: 00 A.M. the next day. Cell sixe. Counts of cells per field were found to exhibit time-associated differences. Fewest cells per field were seen in livers of rats sacrificed at 6 : 00 A.M., and most cells per field were seen at 8 : 00 P.M. Obviously, in a cell mosaic, variations of cell density (number of cells per field) reflect variations of cell size. The reciprocals of the cell densities were used to represent relative cell Variations of cell size are shown in figure 1, ad the cell densities are contained in table 1. Maximal cell size was found at 6: 00 A.M., and minimal cell size at 8: 00 P.M. There was a gradual decline in cell size between these two times with a subsequent increase during the night. The increase in cell size during the night was consistent except for an interval of no change between 2 : 00 and 4 :00 A.M. Biwucleate cells. The percentages of binucleate hepatic cells were calculated from the cell counts. These were found also to show time-associated variations. The percentages of binucleate cells increased during the day, remained fairly constant for part of the night and dropped off abruptly during the early morning (4 : 00 A.M.). The greatest percentage of binucleate cells was found at 6: 00 P.M. aMeasurements were made of the size of mononucleate and binucleate cells in sections of livers from rats sacrificed at 6 : 00 A.M. (first group) and 8: 00 P.Y. The major and minor diameters of 10 mononucleate and 10 binucleate cells were measured for each rat of these two groups. Cell areas were calculated. The cell areas of both types of cells at 6: 0 0 A . ~ .were greater than at 8: 0OP.M. A plot of cell densities and cell areas showed good correspondence between these two measures of cell size. 371 MITOTIC VARIATIONS IN RAT LIVER Statistical fidirvgs. The results of statistical ananlyses of mitotic activities, cell densities and percentages of binucleate cells are summarized in tables 2 and 3. The analyses of variance showed significant differences among times with respect to all three measurements. Differences between pairs of means were examined by t-tests. TABLE 2 Summary of analyses of variance of data of table 1 (exclusive of second 6: 00 A.M. group) PEWENTAllBS SOUROE OF DEGREES OB VABIATION FREEDOM MITOTIC ACTIVITIES m.s.1 CELL DENSITIES F.2 m.8. OP BINVCLEATE CELLS F. F. m.8. Among times 11 3.53 8.61' Within times (between rats) 84 0.41 Total 95 m.s. = mean square. ratio. Statistically signscant at 'Statistically signifleant at a F. = variance 148.72 2.83' 14.47 2.30' 6.28 52.23 < 0.05 probability level. < 0.01 probability level. TABLE 3 Summary of t-tests for significance of differences between means Mitotic activities MEANS TESTED T-VALUES PFKIBABILITY 6 : 00 A.M. (first) vs. 6 : 00 A.M. (second) 0.85 NS 8 : 00 A.M. vs. 8 : 00 P.M. 4.79 < 0.01 6: 00 A.M. (first) vs. 6: 00 A.M. (second) 0.03 NS 7.07 < 0.01 6: 00 A.M. (first) VS. 6: 00 A.M. (second) 0.22 NS 12 : 00 M. vs. 6 : 00 P.M. 3.82 < 0.01 Cell densities 6: 00 A.M. (first) VS. 8 : 00 P.M Binucleate cells 1 NS = not significant. 372 B. JACKSON Student's t-test was used to determine the significance between means of cell densities and percentages of binucleate cells, but not mitotic activities. The variances of the mitotic activities of the different groups were not homogeneous : thus the Behrens-Fisher test was employed (as described by Fisher and Yates, '43). No significant differences between cell densities, percentages of binucleate cells, or mitotic activities of the two 6 : OOA.M. groups were found. The differences between high and low values examined were all highly significant. DISCUSSION The tissues and organs of the rat in which time-associated variations of mitotic activity have been reported are shown in table 4. Mitotic activity was found to be higher in rats sacrificed during the day in all instances except in the adrenal glands (Miihlemann et al., '55; Marthaler, '56). I n the intestine, mitotic activity was found to be high at one time during the day and equally as high at one time during the night (Klein and Geisel, '47). The occurrence of higher mitotic activity in the liver during the day as observed in the present experiment is in agreement with the results of Wilson ( '48), and those of Jaffe ('54). Cell size decreased and the percentage of binucleate hepatic cells increased during the day. I n contrast, during the night when mitotic activity was minimal, there was an increase in cell size and a decrease in the percentage of binucleate cells, Considering first the relation of time-associated variations of mitotic rate to cell size it thus appears that decreasing cell size is attendant with a prolonged high level of mitotic activity. This suggests that the quantity of liver with which the animal begins the day is being cut up into smaller units. During the night, when the mitotic rate is low, cell size increased to the value seen previous to the onset of active division. Apparently, an increase in the number of cells (cell multiplication) alternates with an increase of cell size. Although both processes contribute to the over-all increase of Halberg, et al., '54 Oral tissues a. Retromolar mucosa b. Conn. tissue periodontal membrane e. Epith. interdental a. Blumenfeld, '39 b. Halberg et al., '54 Epidermis a. Abdominal b. Earlobe 'Six 24-hour periods in all. 8 15 and 16 8 12; a t 2-hour intervals Blumenfeld, '42 Submaxillary a. 12 ;at 2-hour intervals b. 2 ; about 1 2 hours apart 8 12 ; at 2-hour intervals Kidney Blumenfeld, '38 Klein and Geisel, '47 Intestine 3 or 4 15 and 16 16 16 1 2 ; at 2-hour intervals ~ 2 ; about 12 hours apart Miihlemann et al., '55 Adrenal 2 ; about 12 hours apart 2 ; about 12 hours apart Miihlemann et al., '55 8 2 ;about 12 hours apart Thyroid No. of rats at each time No. of times sampled per 24-hr.period Jaffe, '54 AUTHOR Liver (regenerating) TISSUB SAMPLING TIME OP HIGHER MITOTIC AOTIVITY P.M. high ~~ except a. Day (8-10 A.M.) b. Day (6: 35-8: 48 A.M.) Uniformly 2-4 A . N Day ( 2 - 4 P.M.) Day (12 M.) Night (4 A.w.) c. No difference b. Day (6: 35-8: 48A.u.) a. Day (6: 35-8: 48 A.M.) Night (9: 30-10 Day (7-7 : 30 A.M.) Day (6-10 A.N) Besearch on time-associated variations of mitotic activity in tissues and organs of the rat ______ during 374 B. JACKSON liver substance (matter), it must be realized that these cytological changes may incorporate effects of function and maintenance activities of the liver and the organism as well. Decreased numbers of binucleate cells accompanying active cell division have been observed by McKellar ( ’49). I n the present study the general trend of the mitotic activity and percentages of binucleate cells seems to favor the possibility of a reciprocal relation between them. The over-all gain in the number of binucleate cells during the day was balanced by a decline at night. If the binucleate cells which accumulate during the day are cells in which cytokinesis has failed then it appears that they somehow manage to complete division during the 24 hours. Perhaps it is significant that a drop in binucleate cell number occurred during the night just prior to the time when increased mitotic activity was found and following an interval of little change in cell size. The latter observation lends additional support to the idea that cell multiplication, in this case formation of mononucleate cells from binucleate cells, alternates with cell enlargement. Many ideas have been proffered to explain time-associated variations of mitotic activity. In order to explain any biological phenomenon which is associated with time, it is necessary to correlate the phenomenon as well as the time function with other biological phenomena inside or outside the system studied (“integration into a physiological sequence by a physiological mechanism,’’ Halberg, ’53). I n rats, food consumption (Higgins et al., ’33), water intake (Young and Richey, ’52), body weight (Jaffe, ’54), liver weight (Higgins et al., ’32), water fat, protein and carbohydrate content of the liver (Higgins et al., ’32), and blood sugar (Pitts, ’43) have been found to exhibit time-associated variations. These offer attractive correlates with which the mitotic activity of hepatic cells might be correlated. In fact, certain phases of carbohydrate metabolism, e.g., glycogen content of cells (Bullough, ’52), have found favor with Jaffe ( ’54)in explaining time-associated variations of mitotic activity in regenerating livers of rats. MITOTIC VARIATIONS IN RAT LIVER 375 It is unlikely that time associated variations of mitotic activity can be explained in a simple manner by any single correlation with a metabolic property or process such as those listed above. Moreover, it is probable that the integration of variations of mitotic rate into a physiological sequence can better be established by considering first the events occurring at the same level of organization, i.e., changes in hepatic cell size and number of binucleate cells as was done in the present study. Further work will be directed toward an attempt to dissociate these events by experimental means. SUMMARY 1. The extent and succession of time-associated variations of mitotic rate during 24 hours were determined in livers of young male rats. The relation of these variations to hepatic cells sizes and t o the numbers of binucleate cells was investigated also. 2. During the 24-hour period studied, the mitotic rate varied as much as 5 to 6 times the minimal value seen. High mitotic activity was found during the day while at night mitotic activity was uniformly low. Day included the hours from 6 : 00 A.M. to 8: 00 P.M. 3. During the day, when mitotic activity was high, cell sizes decreased. I n contrast, during the night, cell sizes increased to the value seen at the beginning of the previous day. The increase of cell sizes was continuous except for a period of no change between 2 : 00 A.M. and 4 : 00 A.M. 4. The percentages of binucleate cells increased during the day but remained constant during the night except for a marked drop during the early morning (4: 00 A.M.), just before the time of active cell division and just after the interval of no change in cell sizes. 5. The relation of cell sizes and percentages of binucleate cells to the time-associated variations of mitotic rate suggests that cell multiplication alternates with cell enlargement during 24 hours in the livers of young rats. 376 B. JACKSON ACKNOWLEDGMENTS I am indebted to Dr. C. H. Willey and Dr. M. J. Kopac, of New York University, and to Dr. F. I. Dessau, of the Lederle Laboratories Division of the American Cyanamid Company, for their interest and valuable suggestions. Thanks are due especially to Dr. Edmund Mayer for his assistance in preparing this manuscript, and to other members of the Lederle Laboratories Division of the American Cyanamid Company for their encouragement and advice. The financial aid of the Educational Assistance Plan of the American Cyanamid Company is gratefully acknowledged. LITERATURE CITED BLUMENFELD, C. M. 1938 Periodic and rhythmic mitotic activity in the kidney of the albino rat. Anat. Rec., 72: 43-43. ~- 1939 Periodic mitotic activity in the epidermis of the albino rat. Science, 90 : 446-447. 1942 Normal and abnormal mitotic activity; I. Comparison of periodic mitotic activity in epidermis, renal cortex and submaxillary salivary gland of the albino rat. Arch. Path., 33: 770-776. BULLOUGR, W. S. 1952 The energy relations of mitotic activity. Biol. Rev., 27: 133-169. ERLNxO, 0. 1955 Quantitative Methods i n Histology and Microscopic Histochemistry. Little, Brown and Co., Boston and Toronto. FISHER, R. A., AND F. YATES 1943 Statistical Tables for Biological Argicultural and Medical Research, 2nd ed. Oliver and Boyd, Ltd., Edinburgh. HALBERQ, F. 1953 Some physiological and clinical aspects of 24-hour periodicity. J. Lancet, 7 8 : 20-32. HALBERG, F., R. A. ZANDER,M. W. HOIJGHLUN A N D H. R. MUHLEMANN1954 Daily variations in tissue mitoses, blood eosinophils and rectal temperatures of rats. Am. J. Physiol., 177: 361-366. HIGGINS,G. M., J. BERKSONAND E. FLOCK1932 The diurnal cycle in the liver. I. Periodicity of the cycle, with analysis of chemical constituents involved. Ibid., 102: 673-682. 1933 The diurnal cycle in the liver of the white rat. 11. Food, a factor in its determination, Ibid., 105: 177-186. JACKSON, B., AND c. H. WILLEY 1956 Hourly variations of mitotic activity in the livers of young rats. Anat. Rec., 125: 583. JAFFE, J. 3. 1954 Diurnal mitotic periodicity in regenerating rat liver. Ibid., 180: 935-954. KLEIN, H., AND H. GEISEL 1947 Zum Nachweis eines 24-Stundenrhythmus der Mitosen bei Ratte und Maus. Klin. Wschr., 24-25: 662-663. MITOTIC VARIATIONS IN RAT LIVER 377 MCKELLAR, M. 1949 The postnatal growth and mitotic activity of the liver of the albino rat. Am. J. Anat., 85: 263-308. MARTHALER, T. M. 1956 Tag- und Naehtsehwankungen der mitotisehen Aktivitiit in der Nebennierenrinde der unbehandelten und colehizinbehandelten weissen Ratte. Buchdruckerei, Marthaler and Go., Heerbrugg, Zurich. MUHLEYANN, H. R., T. M. MARTHALER AND P. LOUSTALVT 1955 Daily variations i n mitotic activity of adrenal cortex, thyroid, and oral epithelium of the rat. Proc. SOC.Exp. Biol. Med., 90: 467-468. PITTS,G. C. 1943 A diurnal rhythm in blood sugar of the white rat. Am. J. Physiol., 139 : 109-116. WILSON,J. W. 1948 Diurnal rhythm of mitotic activity in the liver of the mouse. Anat. Ree., 101: 672. YOUNG,P. T., AND H. W. RICHEY 1952 Diurnal drinking patterns in the rat J. Comp. Physiol. Psychol., 45: 80-89.