Adrenocortical Function: Corticosterone Levels in Female BALB/c and C3H Mice under Various Conditions EDWARD F. HAWKINS,’ PATRICIA N. YOUNG, ANDREA M. C. HAWKINS AND HOWARD A. BERN Department of Zoology and Cancer Research Laboratory, University of California, Berkeley, C a l f o r n i a 94720, U . S . A .

ABSTRACT A diurnal rhythmicity in plasma corticosterone levels was demonstrated in female BALB/cCrgl and C3H/Crgl mice, with and without mammary tumor virus. Removal of the adrenals followed by metopirone treatment reduced circulating corticoid to non-detectable levels in C3H but not in BALB/c mice. Dexamethasone reduced but did not abolish corticosteroid secretion in both strains, Neonatal exposure to exogenous hormones failed to cause any obvious change in corticoid levels. Bilateral ovariectomy of these neonatally treated mice at 40 days of age resulted in a subsequent lowering of plasma corticoid levels when compared with intact animals.

The importance of corticosteroid hormones in the initiation and maintenance of secretory activity of the mammary gland is well known. Although the adrenal cortex secretes corticosteroid in response to a variety of exteroceptive and enteroceptive stimuli or “stresses,” it additionally maintains a stress-independent basal corticoid secretion. This report is concerned with the collection of data on this latter phenomenon in two strains of mice frequently used in mammary tumor studies. Included also are data on experimental manipulations (metopirone and dexamethasone administration; bilateral adrenalectomy) which might be expected to alter or inhibit the secretion of corticosterone, the principal steroid produced by the adrenal cortex of the mouse (Triller and Birmingham, ’65). The effects of neonatal exposure to steroid hormones and of ovariectomy are also reported. MATERIALS AND METHODS

Blood sampling Blood was withdrawn from the orbital sinus into a heparinized hematocrit tube. The hematocrit was recorded and duplicate 5- or 1 0 - ~ 1plasma samples taken for corticosterone analysis. Diurnal rhythm experiment The animals used for this study were all adult females. Four groups were examined: J. ExP. ZOOL., 194: 479-484

BALB/cCrgl, BALB/cfC3H/Crgl, C3H/Crgl, and C3Hf/Crgl, each containing 54 mice housed three to a cage. All animals were ear-tagged to facilitate rapid identification. In case such a procedure should alter the final steroid values, additional groups of untagged and tagged BALB/c females were established as controls, to be bled concurrently with the diurnal rhythm group. Cages were placed at similar levels in the animal quarters to ensure that all animals received a similar intensity of illumination. Food and water were provided ad libitum and cages were inspected at the same time of day twice weekly and cleaned as necessary. Artificial lighting was provided for 12 hours per day, the period of illumination commencing at 7 A.M. The temperature was maintained at 21 2 1“C. After an acclimation period of four weeks, nine animals from each group were bled from the orbital sinus every four hours for 24 hours. Vaginal smears and individual hematocrits were also recorded. Duplicate 5- or 10-pl plasma samples were stored in redistilled absolute ethanol prior to corticosterone determination. Blood samples were also obtained from an additional group of 54 BALBlc females (BALB/c-X) housed in a separate building where they were subjected to high noise levels during the period of illumination. 1 Present address: Institut Jules Bordet, 1 rue HegerBordet, 1000 Bruxelles, Belgium.

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E. HAWKINS, P. YOUNG, A. HAWKINS AND H. BERN

Bilateral adrenalectomy and metopirone administration Bilateral adrenalectomies were performed on some animals from each group following the diurnal rhythm study. The operations were performed under nembutal anesthesia, and the immediate areas from which the glands were removed were cauterized. The excised glands were examined for signs of damage under a binocular dissecting microscope. Following the operation, the diet was supplemented with 5% glucose/saline solution. After a 2-week recovery period, blood samples were taken (4 P.M.). The same mice were subsequently injected intraperitoneally with 30 pg/gm body weight of the llp-hydroxylase inhibitor, metopirone (as the ditartrate; Ciba Pharmaceutical Co., N. J.) in isotonic saline. Three injections were given, 12 hours apart, and blood samples taken at 4 P.M., eight hours after the final injection.

1,2-H3 corticosterone (48 Ci/mM, 98% pure; New England Nuclear, Boston, Mass.) per 100 ml distilled water, was added to each tube. Following five minutes agitation at 45°C and ten minutes at O’C, unbound steroid was removed with florisil (60-80 mesh, MCB Chemicals; 60 mg per tube). Four hundred microliters of each supernatant were taken for scintillation counting (Packard 2425 scintillation spectrometer). The scintillation fluid contained 7 gm PPO and 600 mg POPOP per 1 liter toluene and 500 ml 2-ethoxyethanol. Counting efficiency was 28 1% . The standard curve was reproducibly linear between 0.1 and 4.0 ng corticosterone (1.0-40 p g / l O O ml for a 1 0 - ~ plasma 1 sample). Values below 0.1 ng are expressed as undetectable. The specificity of the method was considered adequate since cortisol and progesterone added to plasma pools at up to 20 times the concentration of endogenous corticosterone were not detected by the assay.

Dexamethasone injection Statistical analysis Dexamethasone sodium phosphate (“DecStatistical evaluation of the diurnal adron,” Merck, Sharp and Dohme, Pa.) rhythm data included analysis of variance was injected intraperitoneally into female and an assessment of all significant intraBALB/cfC3H mice. Two experiments were group differences by Duncan’s multiple performed. In the first, 8 pglgm dexameth- range test (Steel and Torrie, ’60). Analysis asone or its injection vehicle (isotonic sa- of covariance was performed with hematoline) were given for four days at 4 P.M. crit and vaginal smear data as the covariand the animals bled 15.5 hours after the ates. All other statistical comparisons were last injection. In the second, a single in- performed with the “t” test of Student. jection of 8 pg/gm was followed by blood RESULTS sampling ten hours later. Site of blood withdrawal Neonatal injection of hormones Table 1 includes corticosterone values Female BALB/cfC3H mice were injected obtained by blood sampling via the orbital subcutaneously once daily for the first five sinus and by decapitation. Although the days of life with hormones (table 5). Bilat- resultant means compare favorably, blood eral ovariectomy was performed on some was always withdrawn from thg orbital mice at 40 days of age. Blood samples were sinus in further experiments, thus pretaken just before sacrifice. cluding the possibility of contamination with other body fluids. Corticos terone assay Diurnal r h y t h m Total (free protein-bound) corticosterThe presence of a metal tag in the ear one was measured in duplicate 5- or 10-111 plasma samples following the scheme of did not chronically elevate adrenal steroid Murphy (‘67). Steroid was extracted from secretion; plasma corticoid levels were simplasma with 2 X 400-pl redistilled abso- ilar in BALB/c mice whether or not such 2%). Etha- tags were present (table 1). lute ethanol (recovery = 97 All sub-groups had a diurnal rhythmicity nol extracts were dried under filtered nitrogen, and 1 ml binding solution, containing of basal corticoid values (table 2). With 350-p1 rhesus monkey serum and 5 pCi our sampling schedule, mean values were

+

*

48 1

MOUSE CORTICOSTERONE LEVELS TABLE 1

maximal at midnight for BALB/c-X and BALB/cfC3H, at 8 P.M. for C3H and C3Hf and at 4 P.M. for BALB/c. Lowest levels were at noon for BALB/c, BALB/cfC3H, C3H and C3Hf (not significantly different from 4 A.M. in the last group) and at 8 P.M. for BALB/c-X. Analysis of covariance with hematocrit or vaginal smear data did not result in significant alterations in any of the observed values. It is of interest that the steroid levels observed in the BALB/c and BALB/c-X groups have different periodicities with respect to the occurrence of the peaks and troughs, although the mean values fall within similar ranges.

A . Route of blood sampling: comparison of plasma corticoid figures from blood samples obtained f r o m the orbital sinus or by decapitation of female BalblcfCSH mice Plasma corticosterone (pgflD0 ml) ( n ) Route of sampling

1

Orbital sinus Decapitation

Intraperitoneal saline (4 X 1 O O p 1 ) injection

Intraperitoneal dexamethasone (4 X 8 p g / g m ) injection

36.7 t 5.54 (6) 35.5k 5.54 (6)

6 . 9 k 2.26 (6) 9.5k 3.26 (6)

B . E@ct of ear tagging on plasma corticosterone; Balblc females bled f r a the orbital sinus at 4 P.M. Treatment

Controls (not tagged) Tagged 2 1 2

Bilateral adrenalectomy and metopirone Following bilateral adrenalectomy, residual plasma corticosterone was still detectable in both BALB/c and BALB/cfC3H mice (table 3). It was reduced, but not abolished, by subsequent metopirone treatment.

Plasma corticosterone (pg1100 ml) ( n )

12.0 k 2.16 (9) 10.9 2 3.24 (8)

Animals bled 15.5 hours after last idection. Animals tagged about six weeks before bleeding.

TABLE 2

Plasma corticosterone levels

1:

diurnal rhythms and statistical evaluation (Duncan’s multiple range test)

C3HlCrgl Time of sampling

Midnight 4 A.M. 8 A.M. Noon 4 P.M. 8 P.M.

C3HlfCrgl

B alblcfC3HCrgl

BalblcCrgl

BalblcCrgl-X

n

mean*S.E.

n

mean2S.E.

n

mean_tS.E.

n

mean2S.E.

n

mean*S.E.

9 9 9 9 9 9

13.3k1.52 7.9&0.88 6.321.38 4.9k0.85 9.121.66 19.621.61

8

8.320.89 4.0k0.56 5.050.51 4.6k0.48 10.1t1.06 11.5t1.32

8 9 8 9 9 9

29.1 k 2 . 9 3 14.2k3.49 18.8k2.73 13.822.90 24.2k1.31 25.1k2.35

9 9 9 9 9 8

12.622.70 8.1k0.85 8.121.58 7.7t1.10 17.6k2.91 15.2k1.50

9 9 9 9 9 9

21.7k3.97 21.3k3.59 9.8k2.10 9.4k1.97 8.8t1.46 8.3k1.38

9 9 9 9 7

Comparison of values ( p ) C3HlCrgl

all time 8 P.M. >periods

noon, 8 A.M.

4 A.M. 4 P.M.

noon Mldmght 8 A.M.

8 P.M. > m i d n i g h t

BalblcCrgl

noon, 4P.M.,>8A.M., 4 A.M. noon, 8 P.M. > 8 A.M. 4 A.M. 1 pgl100

m l plasma.

B alblcfC3HCrgl

C3HlfCrgl

4 A.M.

8 A.M.

< 0.05

4 P.M. > noon, 4 A.M.

c and C3H mice under various conditions.

A diurnal rhythmicity in plasma corticosterone levels was demonstrated in female BALB/cCrgl and C3H/Crgl mice, with and without mammary tumor virus. R...
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