MITOTIC ACTIVITY I N T H E THYROID GLAND O F FEMALE RATS1 THOMAS E. HUNT Department of Anatomy, Uniaersity of Alabama INTRODUCTION The rate of mitotic cell division in certain organs has been shown to vary with such factors as the phase of the estrous cycle, with age, and with the time of day. This paper is concerned with the extent to which these factors and especially the estrous cycle affect the rate of division in the thyroid gland of the female rat. By using the rat it will be possible to correlate the mitotic activity of the thyroid with that of other structures which have already been studied (Hunt, '40, '42, '43). Such a correlation is desirable since it would be helpful to know whether a factor acts specifically on one organ or on a group of related organs or whether it acts indirectly or not at all. During the estrous cycle the mitotic activity varies markedly in a number of organs. In the rat these include the ovaries (Lane and Davis, '39), genital epithelia (Allen et al., '43), the hypophysis (Hunt, '42), and adrenal gland (Hunt, '40). Some organs, however, such as the intestine (Hunt, '42) are not affected and of those that are, not all have have an increased activity at the same time. The genital epithelia and the hypophysis have more mitoses at late estrus and the adrenal more at diestrus. It thus appears that during the cycle the increased mitotic activity in the various structures is due to more than one factor that act in sequence. I n order to evaluate the effect of the estrous cycle on mitotic activity it is necessary to know the extent to which other factors such as age and time of day affect the activity. In the hypophysis the increased activity at late estrus becomes markedly reduced as animals become older (Hunt, '43) so that an animal of 300 days of age does not have many more mitoses at late estrus than one of 100 days at diestrus. The time of day at which an animal is killed is likewise an important factor to be controlled, since in practically all organs where mitoses have been studied there is an increased activity at one or more periods of the day. 'Aided by grants from the Jane Coffin Childs Memorial Fund f o r Medical Research and the University of Alabama Research Fund. 133 134 THOMAS E. H U N T Furthermore Blumenfeld (’42) has found that the periods of increased activity are not the same for all structures of the same animal and he concludes “that the factors which determine when and how often mitosis occurs act on or reside within the organ as a unit”. ACKNOWLEDGMENTS I wish to thank Dr. C. M. Goss for supplying the rats from his colony and for his helpful criticisms in the preparation of the paper. MATERIALS AND METHODS The 131 thyroid glands examined for mitotic activity were from the same rats that supplied the hypophyses used in previous reports (Hunt, ’42, ’43). Tissue was fixed in Allen’s B-15 fluid and cleared in dioxan after passing through alcohol from 10 to 70% in 10% steps. Sections were cut at 3 micra and every tenth section was mounted and stained according to Masson’s procedure. Four or five sections from each gland were examined for mitotic figures. This represents an area approximating 12.5 sq. mm. About half of the animals received colchicine 9 hours before being killed. By halting division at the metaphase during this period the mitotic count was approximately tripled and proved especially valuable in the counts from the glands of older animals and in those killed in diestrus where the mitotic figures were few and scattered. Animals receiving colchicine were, with fourteen exceptions, killed between 4 and 5 P.M. Those not receiving it were killed between 11and 12 A.M. The stages of the estrous cycle were determined by vaginal smears. RESULTS A definite correlation was found between the mitotic activity in the thyroid gland and the sexual cycle. I n groups not receiving colchicine and of comparable age, killed during each of the five phases of the estrous cycle (groups 1-5. table l), there was a significantly higher mitotic activity in the group killed in late estrus. Minimum activity was found during proestrus and diestrus and an intermediate activity occurred in early estrus and metestrus. The variation at different phases was even more striking in the animals that received colchichine. Comparison of groups 6c, 7c, 9c, 12c, and 13c (table l ) ,whi’ch were of comparable ages, shows that animals killed in late estrus have by far the highest mitotic activity. The invariable low counts of animals killed in proestrus and diestrus in this 135 MITOTIC ACTIVITY I N THE THYROID series is considered very significant since the use of colchicine rules out the possibility of killing animals at a low ebb of mitotic activity that might rise a few hours later. The intermediate activity in the early estrous phase (group 7c) is not apparent in the animals that received colchichine, because they were killed in the early part of the phase whereas in the comparable group without colchicine some were killed in the late part. TABLE 1 Mitot% activity in the thyroid gland of groups of female rats with and without colchicine. BmUP ~ * NUMBEB OF ANIMALS ~~ 1 2 3 4 5 6c 70 8c 9c 1oc 11e 12c 13c ~~ 7 11 19 14 14 8 5 7 11 11 11 7 6 AQE, AVERAGE. AND RANBE. DAYS STAQE OF CYCLE W H E N EILLrn MITOSESPER SQ. MM. AVERAGE & 5. E. Proestrus Early estrus Late estrus Metestrus Diestrus Proestrus Early estrus Late estrus Late estrus Late estrus Late estrus Metestrus Diestrus 0.35 -C 0.01 1.41 2 0.40 6.55 -C 1.07 1.37 -C 0.28 0.49 -C 0.12 1.09 -C 0.31 1.35 & 0.36 27.50 +- 9.87 12.60 -I: 3.31 9.10 2 1.75 4.25 ? 1.09 4.8 t 0.80 1.76 k 0.26 -~ 103 ( 61-204) 87 ( 53-106) 85 ( 52-126) 94 ( 66-114) 87 ( 66-114) 126 ( 67-172) 121 ( 89-142) 85 ( 60-111) 132 (117-139) 150 (140-169) 206 (171-242) 135 (127-146) 113 ( 79-142) There was considerable variation in the mitotic counts of the glands of animals killed in late estrus. I n those not receiving colchicine this ranged from 1.5 to 21.7 mitoses per square millimeter with five of the nineteen animals having unusually low counts. The variability can be explained in part by the variation in the time at which a maximum cell division is attained. Most of the high counts were from animals killed 25 to 30 hours after cornified cells were first found in the vaginal smears, but a few high counts were from animals killed 5 to 10 hours earlier. Since the maximum activity is probably maintained for a few hours only, it may very well have been missed in the animals with the low counts. Thus, although there is undoubtedly individual variation, the average mitotic count for animals in late estrus would be higher than 6.6 mitoses per square millimeter if all the animals had been killed at a time of maximum mitotic activity. The variability in mitotic activity associated with the age of the animal is shown by grouping according to age the forty animals receiving Groups 6c-13c received colchicine 9 hours before death. 136 THOMAS E. HUNT colchicine and killed in late estrus (groups 8c-llc, table 1).For the four age groups the average mitoses per square millimeter was as follows : A V E U Q E AQB, DAYS 85 132 150 206 MITOSES PER SQUARE MILLIMETER 27.5 12.6 9.1 4.25 I n a few individual cases older animals showed more mitoses than younger ones, but the difference between the 55- and 206-day age groups is highly significant statistically. There was some evidence of a diurnal mitotic rhythm in the thyroid. Of the twenty-two animals in groups 9c and 1Oc (table l), all of which had received colchicine 9 hours before death, seven were killed at 11P.M., seven at 8 A.M., and eight at 5 P.M. The average mitoses per square millimeter for these was 19.6, 8.5, and 9.2, respectively. Although the difference between those killed at 11P.M. and 8 A.M. is apparently considerable it is barely significant statistically (i.e., it might have been due to chance in about once in twenty times). However, whether or not this may be considered as evidence of a diurnal rhythm the fact remains that the estrous cycle is effective in causing an increased activity regardless of the time of day. It is interesting to note that in the hypophysis from these same animals the highest mitotic activity also occurred in animals killed at 11P.M. (49 mitoses per sq. mm.) and the lowest in those killed a t 8 A.M. (12.9 mitoses per sq. mm.). Chouke and Blumenthal ( '42) found there are generally more mitoses in the left lobe of the guinea pig thyroid than in the right lobe. This does not seem to be true for the Eat. I n thirty-one glands, selected because more numerous mitoses were present and the orientation was known, there were seventeen in which one side had more than 55% of the mitoses (five had more than 60%). Of the seventeen, nine had more on the left and eight, more on the right. The average mitoses per square millimeter in all the right lobes was 13.2 and in the left 13. Such minor differences can be accounted for by the errors in counting and are insignificant statistically. The position of the dividing cells may have some bearing on the problem of the origin of new follicles in the thyroid. The majority, or 65% of 1215 dividing cells recorded for this purpose touched the lumen of the follicle and presumably replaced follicular cells that died. Most of the others were separated from the lumen by at least one non-divicling cell and appeared to be the beginninq of a bud of a preexisting follicle. Very few divisions were seen in what might have been inter- MITOTIC ACTIVITY IN THE THYROID 137 follicular cells, so that it seems unlikely that new follicles would arise from them. DISCUSSION Since the greatest mitotic activity of the thyroid and hypophysis occurs in both glands during late estrus, it might be assumed that a single factor is responsible for the activity. I n the thyroid, however, the increase and decrease in number of mitoses occurs somewhat earlier than it does in the hypophysis. For example, an animal killed in early estrus may show increased mitotic activity in the thyroid but not in the hypophysis. This suggests that the mitosis stimulating factor is not necessarily the same for the two glands. Further support for such an opinion has been obtained from experiments now in progress in which injections of estrogens result in an increase in mitoses in the hypophysis but not in the thyroid. It thus seems probable that the rise in mitotic activity of the thyroid during the estrous cycle is not the direct result of changes in the estrogen level but rather an indirect effect. That the effect is indirect, working through the hypophysis, is suggested by the work of Blumenthal and Loeb ('42) who found that administration of an acid extract or implants of the hypophysis resulted in an increased mitotic activity in the thyroid of the guinea pig. During the estrous cycle in the guinea pig the time of highest mitotic activity in the thyroid (Chouke et al., '35, '42) occurs in what corresponds t o the early part of diestrus in the rat. Since mitoses in the rat were at a low level in diestrus, it is difficult to find an explanation that accounts for the rise in both animals. The discrepancy, however, may be more apparent than real and be due to the difference in the timing of the phases of the estrous cycles in the two forms. There is also a possibility that the mitotic activity is an expression of physiological actions that occurred in the preceding cycle. Thus the factor that causes the increased activity might be the same but requires different lengths of time to become effective. SUMMARY S N D CONCLUSIONS The mitotic activity of the thyroid gland of the female rat varies with the different phases of the estrous cycle. It is increased somewhat in early estrus, attains a maximum in late estrus, declines in metestrus, and is at a minimum in diestrus and proestrus. The maximum activity does not quite correspond in time with that in the hypophysis but is somewhat earlier. This and other evidence lead to the conclusion that the increased mitotic activity in the thyroid is not due t o a direct effect of an ovarian hormone but rather to an indirect effect. 138 THOMAS E. HUNT As the animals advance in age to 200 days there is a decline in mitotic activity so that the vqriation during the estrous cycle is hardly perceptible unless colchicine is given. There is some evidence of a diurnal rhythm, since more mitoses were found in colchicine injected animals killed at 11P.M. than in such animals killed at 8 A.M. or 5 P.M. LITERATURE CITED J. G. WILSONAND D. A. HESSION 1943 Differential growth in the ALLEN,E., T. B. THOMAS, ovaries and genital tract near the time of ovulation in rats treated with colchicine. Am. J. Anat., vol. 72, pp. 291-337. BLUMENFELD, C. M. 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., vol. 33, pp. 770-776. BLUMENTHAL, R. T., AND L. LOEB 1942 Parallelism i n the response of thyroid and parathyroid to various hormones and hormone-like substances. Endocrinology, vol. 30, pp. 502-510. CHOUKE,K. S., H. FRIEDMAN AND L. LOEB 1935 Proliferative activity of the thyroid gland of the female guinea pig during the sexual cycle. Anat. Rec., vol. 63, pp. 131-137. CHOUKE,K. S., AND H. T. BLUMENTHAL 1942 Further investigations on the proliferative activity of the thyroid gland of the guinea pig during the sexual cycle. Endocrinology, vol. 30, pp. 511-515. HUNT,T. E. 1940 Mitotic activity of the adrenal gland of rats in different phases of the sexual cycle. Anat. Rec., vol. 78, (Suppl.), p. 152. 1942 Mitotic activity in the anterior hypophysis of female rats. Anat. Rec., vol. 82, pp. 263-276. 1943 Mitotic activity in the anterior hypophysis of female rats of different age groups and a t different periods of the day. Endocrinology, vol. 32, pp. 334-339. LANE,C. E., AND F. R. DAVIS 1939 The ovary of the adult rat. I. Changes in growth of the follicle and in volume and mitotic activity of the granulosa and theca during the estrous cycle. Anat. Rec., vol. 73, pp. 4 2 9 4 4 2 .