Phase difference between 24-hour rhythms in cortical adrenal mitoses and blood eosinophils in the mouse.код для вставкиСкачать
PHASE DIFFERENCE BETWEEN 24-HOUR RHYTHMS I N CORTICAL ADRENAL MITOSES AND BLOOD EOSINOPHILS I N T H E MOUSE1 FRANZ HALBERG, MARTHELLA J. FRANTZ AND JOHN J. BITTNER Division of Cancer Biology, Department of Pathology, University of Minnesota Medical School and Cambridge State School and Hospital, Cambridge, Minnesota ONE FIGURE INTRODUCTION Various physiologic aspects of the daily adrenal cycle have been investigated (for reviews see Halberg, '53 ; DiRaimondo and Forsham, '56; Tatai and Osada, '56), but only a few reports have dealt with the corresponding morphologic aspects. Engstrom, et al. ('38) described a maximum at night for the RBC-content of the fasciculata in the adrenal cortex of rats and mice. Bander ('50), working with mice, noted a maximum for the lipid content of the fasciculata in the morning and a minimum at night. Muhlcmann, et al. ('55) established under standardized conditions the occurrence of significant day-night differences in mitoses of the adrenal cortex in male rats. I n earlier reports, Blumenthal ('40a, '40b, '50) has studied daily changes in cortical adrenal mitoses, primarily in relation to the feeding schedule. I n many other studies of mitosis in the adrenal cortex of various species, including mice and rats, the possibility of periodicity was ignored (cf. Bachmann, '54). The present study on mice was undertaken, first, in order to cxplorc a possible 24-hour periodicity in number of cortical 'Supported by the Elsa U. Pardee Foundation and by funds from the U.S. Public Health Service, the American Cancer Society, tho Graduate School at the University of Minnesota and the Department of Public Welfare, State of Minnesota. 349 350 F. HALBERG, M. J. FRANTZ AND J. J. BITTNER adrenal mitoses in an inbred stock of mice. Second, in order to compare the timing of the anticipated mitotic rhythm with that of the eosinophil rhythm in the same stock. MATERIALS AND METHODS The study was carried out on mice of the C strain (Bagg albino), maintained by brother to sister mating for over 20 generations in the Division of Cancer Biology at the University of Minnesota Medical School. Separate groups of these C mice, composed each of a male and a female subgroup, were used for counts of cortical adrenal mitoses and of tail blood eosinophils. The mice used f o r “mitoses” were about 5 weeks old, those available for “eosinophils” were 8 weeks old. F o r 7 days prior to the respective phase of study, the mice were kept singly caged in a room maintained at 78 2 1”F, illuminated from 6 A.M. to 6 P.M. and darkened from 6 P.M. to 6 A.M. Purina Laboratory Chow and tap water were available to the mice ad libitum from the time of weaning until the killing by cervical dislocation. For mitoses, as wcll as for eosinophils, separate mice were studied at noon and at midnight. The assembly-line procedures employed for cosinophil counts have been described (Hdberg et al., ’51). For mitotic counts, the adrenals were removed within one minute after killing. The glands were fixed in Bouin’s solution and embedded in paraffin. From each pair of glands at least 10 nearly-equatorial sections, about 5 p thick, were mounted, stained with Heidenhain’s iron-hematoxylin, and faintly counterstained with eosin. I n such sections from each pair of mouse adrenals, on the average 80 oil immersion fields were examined at a magnification of 900 x. The total number of mitoses, their respective stages and their location were recorded. While most mitoses were in the glomerulosa and outer fasciculata, some also were found in the rest of the cortex and occasionally in the medulla (of these still immature mice). Irrespective of their location, all cortical mitoses were pooled for the purpose of this report, whereas medullary mitoses were omitted. ADRENAL CORTICAL MITOSES 351 RESULTS AND DISCUSSION A noon “high” and a midnight “low” in eosinophils of tail blood is readily apparent in the right-hand section of figure 1. For the males as well as for the females studied herein, the noon-night difference in eosinophil count was significant at the one per cent level, in close agreement with the results of earlier work (Halberg and Visscher, ’50; Panxenhagen and Speirs, ’53; Louch et al., ’53, Brown and 0 I/ I Adrenal cortex 0 Tail ood b Y Night h -6 P 0) .- ’ mice Fig. 1 Day-night differences in cortical adrenal mitoses and in tail blood eosinophils of mice of the C strain (Ragg albino). Dougherty, ’56). But by contrast to earlier results (Halberg et al., ’56) a sex difference in eosinophil count was not found. It was previously suggested that a sex difference in eosinophil count may not necessarily describe the entire life span of the B, (C5,Black) stock, for which it was recorded, and, also, that it may not be extended to other stocks without further study. The C mice studied differed from the mice studied oarlier in that they were younger and from a different stock. 352 F. HALBERG, M. J. FRANTZ A N D J. J. BITTNER The left-hand section of figure 1 shows, next, that cortical adrenal mitoses were more frequent at midnight than at noon, in males as well as in females of the stock and age group studied. For each sex, the noon-night difference in cortical mitoses was significant at the 5% level. A possible sex difference, which is not apparent herein, cannot be ruled out with the limited available data. But it is the noon-night difference in mitotic activity, which constitutes a main point of this report. This difference, recorded herein for immature C mice, of both sexes, is roughly comparable to that recorded for young male rats by Muhlemann et al. (’55). The direction of change from a noon “low” to a night “high” in these mice is in close agreement with the corresponding data on rats, kept under similar lighting conditions. Since earlier work in this laboratory on mice studied under the same lighting regimen has revealed a noon “high” and a night “low” for mitoses in pinnal and interscapular epidermis (Chaudhry et al., ’56) and in liver parenchyma (Jardetzky et al., ’56), it may be concluded that daily periodic mitotic activity in these tissues is well over 90” and, perhaps, up to 180” out of phase with the mitotic periodicity of the adrenal cortex. Similar phase differences among mitotic rhythms also characterize rats studied under standardized conditions. In the rat, mitoses, while “high” at night and “low” by day in the adrenal cortex, are “low” at night and “high” by day in pinnal epidermis, oral mucosa, periodontal membrane (Halberg et al., ’54) and thyroid (Miihlemann et al., ’ 5 5 ) . Similar phase differences among mitotic rhythms were reported by Blumenfeld (’42) on the basis of a comparison of mitotic activity in epidermis, renal cortex and submaxillary gland of rats. Obviously, such differences in the timing of mitotic rhythms must be considered in designing studies of mitotic activity within its physiologic range : without checking f o r possible phase differences, a given time of day, chosen in order t o encounter peak mitotic activity in a given tissue may not be expected t o be necessarily associated with mitotic peaks in all other tissues chosen for study. But a more important aspect of studies on mitotic periodicity also comes to mind. The description ADRENAL CORTICAL MITOSES 353 of a mitotic rhythm under standardized circumstances consitutes part of a n exploration of the scqucntial order and of the time relations of functional and mitotic activity in cells of various organs. If then, in a given organ, maximal mitotic and maximal functional activities are dissociated along the 34-hour time scale, the determination of the time of daily mitotic c c l ~ ~may ’ 7 serve as a first approximation of the daily functional “high,” where functional activity is not conveniently evaluated by a more direct approach. Indeed, evidence has accumulated to suggest that cells undergo division upon reduction or cessation of functional activity. Politzer ( ’28) found that moving cells do not divide and Peter (’29) observed that increased activity in the tubular epithelium of the renal cortex inhibits mitosis and that decreased activity stimulates it. Furthermore, from studies on the pituitary Hu n t ( ’49, ’51) suggested that the amount of secretory product in the cells of the tissue where division occurs constitutes in itself an important control of mitotic activity. Accordingly, “division of cells does not occur if there is an adequate amount of secretory substance and, conversely, mitotic activity is increased when the secretion falls below a certain level due to depletion.” If this suggestion applies to the adrenal, mitotic activity should decrease when corticoid manufacture proceeds and it should increase again when corticoid release from the gland occurs. It is from this point of view that the time relations of the rhythms in cortical mitoses and in blood eosinophils gain particular interest. But a comparison of these rhythms must be based in this study upon mitotic counts on adrenals of mice 5 weeks of age and upon eosinophil counts on mice 8 weeks of age. Despite this difference in age, such a comparison may be justifiable since earlier work in this laboratory showed a roughly comparable timing of eosinophil rhythm in mice of the 5-week and 8-week age groups. It may be seen from figure 1that eosinophils were “high” when cortical mitoses were ‘ilow,’’ and vice versa. Daily eosinopenia in the mouse depends, in turn, critically (though not exclusively) upon daily changes in hormone release from the cortex (Halberg et al., ’53, Brown and Dougherty, ’56for the corresponding data on human beings see Halberg et al., ’51b; Kaine et al., ’55). Study of time relations between rhythms in eosinophils and cortical mitoses may thus shed some light on time relations of mitotic activity in the cortex to hormone release from it. If allowance is made for a time lag between the starts of (1) corticoid release into the blood, 354 F. HALBERG, M. J. FRANTZ AND J. J. BITTNER and of (2) blood eosinophil depression, the timing of the latter may serve as an approximation of the timing of the former. It also is pertinent that such a time lag may not involve more than a few hours, while sampling in this study was done at a 12-hour interval. Thus, the data on the timing of eosinophil rhythm in relation to cortical mitotic rhythm, while probably affected by the occurrence of such a lag, are almost certainly not reversed thereby. If, then, a peak of mitoses in the adrenal cortex occurs at the daily time of eosinopenia it may be inferred that, normally, mitotic activity in this gland is enhanced at a time when its secretion is depleted. Obviously, this inference based upon the results of a morphologic approach must await further study by appropriate chemical methods. It seems established, however, that in the mouse important differences in phase characterize the timing of the daily rhythms in (1) blood eosinophils and in cortical adrenal mitoses, respectively, and in (2) epidermal and hepatic mitoses on one hand and cortical adrenal mitoses, on the other hand (for data on hepatic mitoses see Halberg, ’57). SUMMARY Counts of cortical adrenal mitoses and of tail blood eosinophils were done on C (Bagg albino) mice, of both sexes, kept on a standardized regimen, including provisions for light from 6 A.M. t o 6 P.M. and for darkness from 6 P.M. to 6 A.M. Separate groups of mice were used for such counts at noon and at midnight. In males, as well as in females, significant “within-day ’ changes were recorded for cortical mitoses and f o r blood eosinophils, the counts of mitoses being higher at night than at noon, and those of eosinophils higher at noon than at night. Since daily eosinophil depression depends significantly upon hormone release from the cortex, it is concluded that, ordinarily, mitotic activity in the adrenal cortex is enhanced at a time when corticoids have been released from the gland. Furthermore, since under the same conditions epidermal and hepatic mitoses of mice are more frequent at noon than at night, it is further concluded that ADRENAL CORTICAL MITOSES 355 a significant difference in phase exists between the mitotic rhythms in epidermis and liver on one hand and in the adrenal cortex on the other. LITERATURE CITED BACHMANN,R. 1954 Nehenniere (Blutgefasse, Lymphgefasse, Innersekretorische Driisen) zn v. Mollendorff 's Handbuch der mikroskopischen Anatomie des Mcnschen, Springer, Berlin, 6P: 1-952. BANDER,A. 1950 Die Beziehungen des 24-Stunden-Rliythmus vegetativer Funktionen zum histologischen Funktionsbild endokriner Driisen. Z. ges. cxper. Med., 115: 229-250. C. 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