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Phase difference between 24-hour rhythms in cortical adrenal mitoses and blood eosinophils in the mouse.

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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.
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F. HALBERG, M. J. FRANTZ AND J. J. BITTNER
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