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Effect of cortisol on the growth of chicks
GENER4L
ASD
COMl’4K.4TIVE
5, 402-410
ESlJOC‘KISOL00Y
Effect
of Cortisol
II. BELLAMY
Received
(1965)
on the Growth AND
RUTH
November
of Chicks
A. LEONARD
12, 1964
Daily injections of cortisol (1.5 mg/loO gm body weight) into week-old male chicks inhibited the gr0wt.h of most tissues. From measurements of tissue RNA and DNA it did not appear that t,he inhibition of growth was related to a decrease in the concentration of nucleic acids. In general, low doses of cortisol resulted in biochemical changes similar to those observed in hypophysectomized mammals. With high doses there was also a specific hrrakdown of muscle tissue with the loss of intrat*ellular cat,ions and protein.
The injection of cortisol and similar corticosteroids into various mammalian species, or the inclusion of these substances in the diet, inhibits growth and at high concentrations may cause the animals to lose weight (Ingle, 1950; Antopol, 1950; Follis, 1951; Bodansky and Money, 1954; Cannon et al., 1956; Faludi et al., 1964). These effects have also been noted in birds during cortisol treatment (Kowalewski, 1962). This paper deals with the changes in tissue growth and alterations in muscle composition brought about by the repeated injection of cortisol into young chicks. In view of the apparent antagonism between corticosteroids and anabolic agents (Szirmai, 1962)) the action of anabolic steroids and pituitary preparat.ions has been examined in cortisol-t,reated chicks. MSTERIALS
AND
17a-ethyl-19-nor-testosterone were suspended in 0.9% NaCl containing 1% carboxymethylcellulose as described previously (Bellamy and Leonard, 1964). Chicks were injected subcutaneously in the left leg or breast with 0.2 ml of suspension. Rats were inject.ed subcutaneously in the back of the neck. Control groups received the NaCl/carboxymethylcrllulose solution. The amount of steroid injected into each animal in an experimental group was the same and based on the mean initial body weight. of each group. It was given as a single daily dose. Ovine growth (SIH) and 9a-fluoro-l&zhormone, prolactin methylprrdnisolonr-21-phosphate were dissolved in 0.9
METHODS
Animals. Day-old male inbred chicks (Rhode Island Red X Light Sussex, obtained from Hans Hall Farm, Willaston, Cheshire) were fed “chick crumbs” (British Oil and Cnkr Mills Ltd., London). They were maintainrd under const.ant illumination at 25°C. Male rals werp ohtained from an inbred colony maintained in the Department of Zoology, University of Shrffield. They were fed Oxoid Diet No. 86 (0x0 Ltd., London). Injections. Cortisol. trptosterone propionate and 402
COltTISOL
ASD
over a dose range of between 0.14 and 2.0 mg/lOO gm initial body weight. Complete inhibition was observed with 1.5 mg cortisol/100 gm; a further increase in dose was always observed to produce a slight drop in mean body weight (Table 1). In this respect chicks were nearly twice as sensitive to cortisol as male rats of equivalent body weight (3-4 weeks old). The inhihition of growth was not due to irreversible changes in the tissues and normal growth was attained 24 hours after stopping the injection (see also Winter et al. [1950] for rats). Another glucocorticoid, Sa-fluoro16a-methylprednisolone-21-phosphate (Betnelan phosphate, Glaxo Ltd.), given over the same period (0.05-1.0 mg/lOO gm body weight), was found to be about twice RS potent in inhibiting growth as cortisol. Cortisol, from its effect. on muscle, may be termed a catabolic steroid (Bellamy and Leonard, 1964). It was interesting, therefore, to examine the effect of this steroid in the presence of substances having predominantly anabolic actions. The daily administration of cortisol and testosterone propionate in equal amounts (1 mg each/ 100 gm body weight per 9 clays) did not alter the growth-inhibitory action of cortisol. However, when testosterone was replaced by the same amount of 17wethyl19-nor-testosterone (Nilevar, Searle Ltd.} for the same period, a 5070 reduction in the inhibitory action of cortisol was observed. Nilevar alone did not affect body growth although it, had a marked andro-
determined by the method of Berenblum and Chain (1938) on heparinized plasma ohtained after decapitation. A771ino Acids and Protein. Amino acid nitrogen was determined on 5% trichloroacetic acid (TC.4) rstracts of the tissues by the method of Rosen ( 1957). “Alkali soluble” protein in muscle was determined by the method of Rarusen and Johnstone (1961). Strcleic Acids. About 500 mg muscle was broken up in 10 ml ice-cold TCA and the mixture left in ice for 30 minutes with occasional stirring. The solid matter was cent,rifuged down and washed twice on the centrifuge with 5 ml icecold 5Oh TCA, allowing the tissue to remain in contact with the fresh solution for at least 30 minutes before centrifuging. The washed residue was heated for 15 minutes in a boiling water bath with 2 ml 5% TCA and then centrifuged down again. Samples of the supernatant solution were used for the calorimetric determination of deoxyribonucleic acid (DNA) and ribonucleic acid (RSA) by the method of Schneider (1957). Calibration curves were prepared using purified RNA (British Drug Houses Ltd., Poole), and DKA (Worthington Biochemical Co., New Jersey, G.S.A.). Ahline Phosphatase. Plasma alkaline phosphatase was measured by the method of Binkley et al. (1944). RESULTS
Body Growth. Normal week-old chicks under laboratory conditions gained weight at a rate of between 3-8 gm/day. During the third week of post-embryonic life the growth rate increased to about 11-12 gm/ day. The administration of cortisol suspension in the second week inhibited growth TABLE EFFECT
OF CORTISOL
ON
THE
1 GROWTH Weight
Dose of cortisol (mg/100
g m initial
_--wt.)
0
103
GROWTH
0.4
OF CHICKP of animalsh (am) 0.6
2.0
1.5
Time (days) 1
2 4 S
59.9 64.5 75.5 101.0
f 2k f *
2.4 2.3 3.4 3.3
a Chicks (l-week old) were injected rortisol and weighed at intervals over b &leans f SE for 6 animals.
63.2 64.6 70.4 93.2
+ + 5 +
2.9 2.3 2.9 3.0
64.2 66.0 67.0 66.9
* + + 31
2.5 2.4 2.7 3.1
60.0 61.9 60.9 60.4
subcutaneously as described in the text with 8 days. Injections were started on Day 1.
+ + + +
2.3 2.4 2.9 2.2
57.7 57.6 56.5 55.6
* + f A
various
amounts
2.0 2.2 2.5 2.S of
1.60
8
b Means f SE for 5 animals. 0 p > 0.06 compared with control
0 Normal week-old chicks were tiesues of normal animals (N).
ztO.04
1.33 zto.05 1.42~~1~0.07 1.49 ho.06
N
1 2 4
Time (dwd
value.
cortisol
0.952
nith
ckO.08
1.44~
injected
0.250 0.500 0.801
C
1.47 f0.04 1.36czkO.06 1.30”: f0.03
Brain
(1.5 mg/lOO
3~0.09
zto.03 +0.02 kO.04
N
EFFECT
rto.02 ho.04 ho.03
C
g m body
weight).
0.234’z!cO.O4
0.230 0.239c 0.228e
Heart
OF CORTISOL
Animals
0.860 were
f0.15
;t 0.03 fO.O1 f0.09
N
Weight
TISSUE
TABLE
0.255 0.304 0.611
ON
killed
f0.04
~1~0.06 z!zO.O7 ztO.07
C
11.502
0.504 3.214 5.600
of 5) and
f1.04
&to.07 fO.09 AO.17
N
CHICKS
Muscle
(groups
THE
Skeletal
(gm)b
IN
at intervals
0.234~
0.230 0.2390 0.228~
Lee:
of tissues
GROWTH
2
C
the tissue
weights
0.468’ztO.16
0.574 f0.04 0.49% f 0.10 0.460’~0.12
Pectoral
determined
0.850
0.311 0.417 0.546
compared
with
ho.04 f 0.09 ztO.ll
C
0.321’&0.08
0.326 0.314~ 0.309c
(C) and
Eto.15
f0.06 f 0.08 ztO.11
N
Tibia
6
2
R
5 d
Fj
F
E
CORTISOL
AKiD
GROWTH
405
That is to say, cortisol brought the growth process to a standstill. The exceptional tissue was liver, which appeared to continue growing at its initial rapid rate (Fig. 1). Because the rate of liver growth normally decreased during the third week of post embryonic life, cortisol apparently stimulated liver growth when the experiment was carried out for a period of several days. No selective action of cortisol on bone growth was observed in the present work (Table 2 ; cf. Kowalewski, 1962). Chicks injected with a dose of cortisol greater than that sufficient to inhibit growth completely looked thin, and examination showed that these animals had a low skeletal muscle mass. In chicks injected daily for 8 days with 5 mg cortisol, the proportion of pectoral muscle relative to body weight was 50% smaller than in normal animals of the same age. The fall in tissue mass appeared to be specific for skeletal muscle, there being no significant difference in the relative weight of the heart 12compared with normal animals. One of the most characteristic effects of cortisol administration in mammals is a rapid decrease in the weight of lymphoid tissue. In the case of rat thymus the weight loss was particularly noticeable 24 hours after the administration of a single dose of cortisol. The “thymolytic” activity of cortisol was also well marked under similar circumstances in the chick (Table 3). In addition to the thymus, birds possess a secondary, easily accessible mass of lymphoid tissue, the bursa of Fabricius. The function of the ‘(bursa” is believed to be similar to that of the thymus of mamI I mals and it appears to play a prominent I6 8 role in antibody formation (Glick et al., Time (days) 1956). The weight of the “bursa” fell after FIG. 1. Effect of cortisol on liver growth. treatment with cortisol, but the percentage Chicks were injected daily with 1.5 mg cortisol/ loss of weight 24 hours after one injection 100 gm body weight and killed at intervals over was smaller than observed with thymus a &day period. The liver weights (U) were (Table 3). Repeated injections caused both compared with the saline-injected controls (0). Each point is the mean of six animals. tissues to lose about 950/O of their mass in 10 days. injection of cortisol at a level which just DNA and RXd Content of Tissues. The inhibited general growth did not affect the DNA content of the pectoral and leg musweight relat,ionships of the various tissues. cle of normal animals increased at a con-
genie action (lo-fold increase in comb weight in 9 days; not observed when given together with cortisol). Ovine growth hormone (2 mg/lOO gm body weight per day for 5 days) did not affect the cortisol inhibition of growth, nor did ovine prolactin (2 mg/lOO gm per day for 5 days), aIthough this substance has been reported t.o restore the growth of hypophysectomized birds (Hijhn, 1961). Organ Growth. In normal chicks the initial post-embryonic growth rate of a number of tissues was found to be constant (Table 2). Brain, heart, various leg muscles, and the lymphoid tissues increased in weight at a rate of 0.03 to 0.08 gm/day. The pectoral muscle was exceptional in having a high rate of growth (average of 1.5 gm/day, Table 2). Liver growth was not linear. Initially, the latter tissue gained weight at a rate of about 0.6 gm/day, but this- was not maintained over the experimental period (Fig. 1). In general, the
406
BELLAMY
AKD
TABLE EFFECT
OF CORTISOL
ON LYMPHOID
LEONARD
3 TISSUES
OF KIT
Weight of tissues Rat
.\NI)
CHICK”
(gm)h Chick
-_-__---___-
N
c
--~.~ c
K
~~ -... ._ ._ ___.
Thymes Bursa Body
0.310
*
0.037
0.133c
&- 0.014
67.1
* 3.1
64.3
f 0.6
a Six male rats (l-month old) and five chicks (lo-days old) cortisol in 0.2 ml 0.9% NaCI. The weight of selected lymphoid compared with animals injected with 0.9% XaCl. b Means + SE. c p < 0.05 compared with control value.
stant rate over the experimental period of 8 days. The tissue volume increased considerably in this time. For example, in the case of pect,oral muscle there was a 30fold change in tissue weight (Table 2). This resulted in a rapid fall in the concentration of DNA during the first 6-8 days of life (Fig. 2). Cortisol, at a dose which st,opped growth, appeared not to inhibit 4.9 .
ID
Muscle
wt. ( g.)
2. Effect of cortisol on the DNA concentration in the pectoral muscle. Week-old chicks were inject,ed daily with 1.5 mg cortisol for 10 days, and the DNA concentration in the right pectoral muscle (m) compared with that of saline-injected controls (0). FIG.
0.223 0.167 69.2
-+ 0.011 + 0.014 It 1.1
0.096” 0.09ic 62.4
..-
i 0. r11:; * 0.012 f
0 s
were injerted subcutaneously with 2.6 mg organs was determined 24 hours later nntl
net DNA synthesis completely; and the smallest non-growing injected birds had on the average a greater amount of DNA for a particular muscle weight than the normal animals. This was particularly noticeable in small muscles from animals weighing about 35 gm (Fig. 2) where a slight change in DNA formation would be expected to be more noticeable. The RNA content of leg muscle increased in a linear fashion while the net formation of RNA in pectoral muscle decreased with time. Cortisoi appeared to inhibit the net, synthesis of RNA complet,ely. There was no correlation between the nucleic acid concentration in various muscles and their growth rate (Table 4; cf. Table 2). Cortisol at a dose sufficient t.o bIock gr0wt.h in large animals did not noticeably affect the concentration of either DNA or RNA in a range of muscle tissues (Table 4). Tissue Electrolytes, Protein, and A mine Acids. Cortisol treat’ment in excess of that necessary to inhibit growth completely did not affect the electrolyte content of live1 (Table 5). Since nearly 40% of this tissue is occupied by extracellular fluid (D. Bellamy, unpublished observations), it may also be concluded that cortisol did not affect the electrolyte content of the plasma through changes in kidney function. However, there was a marked difference in the electrolyte composition of the pectoral muscle, which is characterized by having a much lower cxtraccllular dpace. In this
CORTISOL
EFFECT
AND
407
GROWTH
TABLE 4 ON THE NUCLEIC ACID CONTENT
OF CORWSOL
OF VARIOUS
MUSCLES=
Nucleic rtcid content mg/gm wet wtb DNA
RNA
N
Pectoral Leg Wing Heart
1.26 1.51 1.29 2.92
N
C
k f * A
0.10 0.21 0.13 0.24
1.25 1.37 1.54 2.66
+ f 5 *
0.20 0.17 0.15 0.20
2.94 5.42 6.04 6.25
C
+ + + +
0.23 0.66 0.78 0.92
3.66” 5.25 5.88 4.96
zk f 2 +
a Week-old chicks (mean weight 61.4 gm) were injected daily with 1.5 mg cortisol/lOO gm body 10 days and the nucleic acid content of various muscles (C) compared with that of control-injected
0.14 0.42 0.33 0.30 weight for animals
6’). b Means * SE for 6 animals. c p < 0.05 compared with control
value.
TABLE EFFECT
OF CORTISOL
ON THE
SODIUM,
5
POTASSIUM,
.~ND PEcTOR.~L
AND
Electrolyte (mm&s/kg
Liver Normal Cortisol Muscle Normal Cortisol u Liver and muscle used for the estimation 5 animals. b p < 0.05 rompared
content of tissue tissue water)
24.8 24.4
* 0.5 f 0.3
55.2 59.9
+ 2.2 + 2.7
121.6 119.1
+ 6.7 * 7.1
treated
23.5 18.16
+ 0.4 + 0.3
45.6 67.2b
f 2.6 f 3.1
14S.6 82.26
+ 5.2 * 4.4
samples were taken from animals injected for 8 days with 2 mg cortisol/lOO of sodium and potassium as described in the text. Figures refer to means with
control
Amino acids mm&s/kg
k 0.27 f 0.34
OF CORTISOL
Phosphate mm&s/kg
0.34 0.21d
gm and f SE for
value.
TABLE ON PL.ISUA
6 :LND
MUSCLE
Plasma
5.00 8.22d
OF LIVER
treated
EFFECT
Normal Cortisol
CONTENT
WATER
MUSCLE"
+ 0.02 + 0.01
COMPOSITION* Pectoral
Blkalineb Phosphatase (units/100 ml)
S.5 3.9d
f 0.62 + 0.20
Amino acids mm&s/kg
17.0 18.2
+ 4.4 + 5.3
Muscle
Alk;;O;z;;ble (mg/gm
wet wt.)
51.7 37.2d
* 2.2 + 2.5
(mg/lOO
54.3 103.3”
Fate gm body
wt.)
+ 5.8 It 12.4
a Week-old chicks were injected daily for 10 days with 2 mg cortisol/lOO gm body weight and a selection of plasma and muscle constituents compared with those of control-injected animals. Figures refer to means + SE for 6 animals. b One unit of enzyme liberated 1 mg phenol from disodium phenylphosphate per hotlr at 37”. c The strip of adipose tissue along the posterior edge of the pectoral muscle. d p < 0.05 compared with control value.
408
BELLAMY
AND
tissue cortisol brought about an increase in the concentration of sodium and a decrease in the potassium concentration (Table 5). Sodium did not replace all of the lost, potassium and the sum of sodium and potassium was reduced by almost 20%. The sum of sodium and potassium may be taken as an approximate measure of the total non-diffusible anionic groups within the muscle fibers (Boyle and Conway, 1941), so that these differences indicate the loss of cellular material of high molecular weight, possibly protein. In agreement with this there was a reduction in both the percentage and total dry weight of the pectoral muscle, and a 30% drop in the concentration of alkali soluble protein (Table 6). Rather surprisingly this marked catabolic effect of cortisol on pectoral muscle was associated with an increased deposition of fat (Table 6; see also Winter et al., 1950). No changes were found in the level of free amino acids in muscle even though there was a considerable rise in plasma amino acids (Table 6). Plasma phosphate decreased, possibly as a consequence of the fall in alkaline phosphatase (Table 6). DISCUSSION Cortisol was found to inhibit the growth of a number of tissues in the chick. The continued growth of liver in the cortisoltreated animals could be the result of an increased load placed upon this organ OWing to an abnormally high rate of amino acid deamination (Goodlad and Munro, 1959; Bellamy and Leonard, 1964). HOWever, liver cells may be resistant to the actions of cortisol and to a certain extent may be able to grow independently of other organs. Thus the continued growth of liver at its initial rate would give the high liver-body weight ratio observed in cortisol-treated chicks. In the present work the net synthesis of DNA, RNA and protein was prevented and the growth process in the majority of tissues came to a standstill. The almost complete block of growth by cortisol raises the possibility that the effect of this steroid is mediated through t.he loss or inhibition
LIWh‘AHD
of the action of some general growth factor. In addition to hormones of the “glucocorticoid” type, esogcnous estrogens arc also known to inhibit t.hc body growth of mammals. There is good evidence that the action of estrogens is mediated t,hrough an inhibition of growth hormone release (Gaarenstroom and Levie, 1939; Noble, 1939; Reere and Leonard, 1939). However, because of the possibility of a stimulation of corticostcroid secretion by estrogens (Zondek and Burstein, 1952), the effect of estrogens on growth may be due to the action of rndogenous adrenal steroids on the anterior pituitary gland (Kanematsu and Sawyer, 1963). Indeed, the changes in t.he various body constituents of the chick (Table 6) and the alterations in urea synthesis and liver transaminase that result from cortisol injection in the rat (Bellamy and Leonard, 1964) are similar to the effects of hypophysectomy and opposite to the effects of growth hormone (Li and Evans, 1948; Gaebler, 1955). Furthermore, the administration of growth hormone to rats counteracts the inhibition of growth and other changes which follow cortisol and ACTH t.rcatment (Selye, 1951; Geschwind and Li, 1955). Against this, however, must be set the present experiments, which demonstrated that neither ovine growth hormone nor ovine prolactin (said to restore the growth of hypophysectomized birds (HShn, 1961) ) maintained growth in cortisol-injected chicks. Clearly, more basic information is required on the control of avian growth before any definite conclusion can be drawn as to the action of cortisol. The injection of cortisol in excess of that required to inhibit growth caused certain IDUSC~C tissues, particularly the pectoralis, to lose weight. That is, a general inhibition of growth occurred at lowcortisol levels, while higher doses brought about a specific breakdown of muscle tissue. The biological implications of a direct action of corticosteroids in mobilizing the muscle protein of non-growing animals during the growth of reproductive tissues have been discussed previously (Chester .Jonts and Bellamy, 1964).
CORTISOL
AND
The most marked change in muscle composition of cortisol-treated animals was a a reduction in the protein content. This may also account for the observed fall in the major muscle cations, partly by the loss of anionic proteins (Cole, 1950) and perhaps also by a decreased access of cations, particularly potassium, to binding sites. The latter process would also account for the net gain of muscle sodium, although in the case of this ion a change in the cell membrane that favored sodium diffusion into the tissue cannot be ruled out. From the measurements of nucleic acids in the present work, it is clear that the absolute concentration of these substances in muscle does not determine the growth rate. For example, the pectoral muscle of the week-old chick grew some twenty times faster than heart and leg muscle yet cont,ained up to 50% less nucleic acid per unit weight. Also, the daily weight increment of normal muscles was constant over the experimental period although the size, nucleic .acid content, and rate of RNA formation might change considerably. There was no evidence that the inhibition of growth by cortisol was due to a specific reduction in nucleic acid metabolism or t.hat the inhibitory action was linked with either the amount of nucleic acid in the tissues or the rate of muscle growth. It may well be that only a small fraction of muscle RNA participates in the growth process and that the remainder serves some other role, perhaps as an important structural component of the tissue. If this is so it is possible that relatively small alterations in “template-nucleic acid” concentration were associated with the cortisolinhibition of growth but went undetected in the present work. ACKNOWLEDGMENTS The authors thank the Endocrinology Study Section, National Institutes of Health, Bethesda, Maryland, U.S.A. for the gift of ovine growth hormone and prolactin; and Messrs. Searle Ltd. for the gift o’f Nilevar. The work was largely financed by grants to the Department of Zoology, University of Sheffield from the Agricultural Research Counciland the Department of Scientific and Industrial Research.
409
GROWTH
REFERENCES W. (1950). Anatomic changes produced in mice treated with excessive doses of cortisone. Proc. Sot. Exptl. Biol. Med. 73, 262-265. BELLAMY, D., ASD LEONARD, R. A. (1964). The effect of cortisol on the activity of glutamicpyruvic transaminase and the formation of glycogen and urea in starved rats. Rio&em. J. 93, 331336. BERENBLUM, I., AND CHAIN, E. (1938). An improved method for the calorimetric determination of phosphate. Biochem. J. 32, 29S298. BINKLET, F., SHANK, R. E., AND HOACLAND, C. L. (1944). Modification of the King-Armstrong method for the determination of phosphatase. ANTOPOL,
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156, 253-256. MONEY,
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W. L. (1954). Rates and regression of changes prolonged cortisone adminiEndocrinology 55, 173-185. BOYLE, P. J., AND C,ONWAY, E. J. (1941). Potassium accumulation in muscle and associated changes. f. Physiol. 100, l-63. CANNON, P. R., FRAZIER, 1,. E., AKD HUGHES, R. H. (1958). The influence of cortisone upon prot,ein metabolism. Arch. Pnthol. 61, 271-279. CHESTER JONES, I., AND BELL.IMT, D. (1964). Hormonal mechanisms in the homeostatic rrgulation of the vertebrate body with special reference to the adrenal cortex. Sot. Esptl. Biol. Med. Symp. 18, 195-236. COLE, D. F. (1950). Chemical changes in the tissues of t.he rat after adrenalectomy. J. EndoFaludi, G., Mills, L. C., .~SD CHATES, Z. W. (1964). Effect of steroids on muscle. Actn Eatlocrirlol. 45, 68-78. FOLLIH, R. H. (1951). Non-effect of cortisone on growing bones of mice, guinea pigs, and rabbits. Proc. Sot. Exptl. Biol. Med. 78, 723-724. GAARESSTROOM, J. H., AND LEVIE, L. H. (1939). Disturbance of growth by diethylstilboestrol and oestrone. J. Endocrinol. 1, 42a29. G:jssLsR, 0. H. (1955). Effect of growth hormone on t,ransaminasrs and other enzyme systems. 1,~ “The Hypophyseal Growth Hormone, Nature and Actions (R. W. Smith, 0. H. Gaebler, and C. N. H. Long, eds.), pp. 383-393. McGraw Hill, New York. GESCHWIND, I. I., AND LI, C. H. (1955). The tibia test for growth hormone. ILL “The Hypophyseal Growth Hormone, Nature and Actions” (R. W. Smith, 0. H. Gaebler, and C. N. H. Long, eds.), pp. 2853. McGraw Hill, New York.
I%, CHANG, 'I.. S., ASD JA~P, R. G. (1956). Bursa of Fahricius and antibody prodmPoultry Sci. 35, 224-225. GOODLAD, G. A. J., AND Mrrsso, H. N. (1959). Diet and action of cortisone on protein mctabolism. Biochem. d. 73, 343-348. H~~HIY, E. 0. (1961). Endocrine glands, thymus and pincal body. In “The Biology and Comparative Physiology of Birds” (A. J. Marshall. ed.) Vol. 2, pp. 87-114. Academic Press, Srw York. ISGLE, D. J. (1959). The biologic properties of cortisone. A review. J. Clin. Enrlocrinol. ASletnh. 10, 1312-1354. I~ANEMATSU, S., .~XD Sawrs~, C. H. (1963). Oestrogen stimulated synthesis and release of prolactin. Eutlooinology 72, 243-252. KOWALEWSKI, K. (1962). Effect of steroids on bone formation. 111 “Protein metabolism” (F. Gross, cd.), pp. 238-262. Springer, Berlin. LI, C. H., ASD Ev.~Ns, H. M. (1948). Thr biochemistry of pituitary growth hormone. Rec. Prop-. Hormor~e Res. 3, 3-44. NOBLE, R. I,. (1939). Effects of synthetic estrogens and carcinogens when administered to rats In subcutaneous implantation of crystals or tablets. J. Endocrirtd. 1, 216-229. R.~CVSEN, D., ASD JOHNSTOTE, D. B. (1961). EstiGLICB,
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R. P., ASD ikOX.4HD, 8. I,. (1939). b-lll’tllc~l evidence for a mammogenic factor in 1he rst hypophysis. Proc. Sot. E.rptL. Bid. ,KfV/. 43, 200-202. Kosns, H. (1957). A modified c~olorimetric~ :m;dysis for amino acicls. drrh. RiocheW. Hio/~h (1.~. 67, 10-15. SCHNEIDEK, W. C. (1957). Determination of nucleic acids in tissues by pmtose amtlysis. 111 “Mrthods in Enzymology” Vol. 3. (S. I’. Colowick & K. D. Kaplan, edi;.). t>l,. 680-684. Acadrmic Press, New York. SELYE, H. (1951). The influence of STH, .%CTH and cortisone upon resistance to infection. c’ro/. dlerl. Assoc. J. 64, 489-492. Smnx41, J. A. (1962). Histological aspects of the action of oest,rogens. In “Protein Metabolism” (F. Gross, ed.), pp. 4574. Springer, Berlin. MTISTER, C. A., SILBER, IX. H., AXD STOEHK, H. C. (1959). Product,ion of reversible hyprradrenocortinism in rats by prolonged administration of c*ort.inone. R~!rLocrirtolog~ 47, 60-72. ZOSDEK. B.. AXD BITRSTEIN. S. (1952). Thr rcl:ttionship of rort,icoid excretion to ovarian hormones in the guinea pig. Endocrirrology 50, 419428. &ECE,
ASD
COMl’4K.4TIVE
5, 402-410
ESlJOC‘KISOL00Y
Effect
of Cortisol
II. BELLAMY
Received
(1965)
on the Growth AND
RUTH
November
of Chicks
A. LEONARD
12, 1964
Daily injections of cortisol (1.5 mg/loO gm body weight) into week-old male chicks inhibited the gr0wt.h of most tissues. From measurements of tissue RNA and DNA it did not appear that t,he inhibition of growth was related to a decrease in the concentration of nucleic acids. In general, low doses of cortisol resulted in biochemical changes similar to those observed in hypophysectomized mammals. With high doses there was also a specific hrrakdown of muscle tissue with the loss of intrat*ellular cat,ions and protein.
The injection of cortisol and similar corticosteroids into various mammalian species, or the inclusion of these substances in the diet, inhibits growth and at high concentrations may cause the animals to lose weight (Ingle, 1950; Antopol, 1950; Follis, 1951; Bodansky and Money, 1954; Cannon et al., 1956; Faludi et al., 1964). These effects have also been noted in birds during cortisol treatment (Kowalewski, 1962). This paper deals with the changes in tissue growth and alterations in muscle composition brought about by the repeated injection of cortisol into young chicks. In view of the apparent antagonism between corticosteroids and anabolic agents (Szirmai, 1962)) the action of anabolic steroids and pituitary preparat.ions has been examined in cortisol-t,reated chicks. MSTERIALS
AND
17a-ethyl-19-nor-testosterone were suspended in 0.9% NaCl containing 1% carboxymethylcellulose as described previously (Bellamy and Leonard, 1964). Chicks were injected subcutaneously in the left leg or breast with 0.2 ml of suspension. Rats were inject.ed subcutaneously in the back of the neck. Control groups received the NaCl/carboxymethylcrllulose solution. The amount of steroid injected into each animal in an experimental group was the same and based on the mean initial body weight. of each group. It was given as a single daily dose. Ovine growth (SIH) and 9a-fluoro-l&zhormone, prolactin methylprrdnisolonr-21-phosphate were dissolved in 0.9
METHODS
Animals. Day-old male inbred chicks (Rhode Island Red X Light Sussex, obtained from Hans Hall Farm, Willaston, Cheshire) were fed “chick crumbs” (British Oil and Cnkr Mills Ltd., London). They were maintainrd under const.ant illumination at 25°C. Male rals werp ohtained from an inbred colony maintained in the Department of Zoology, University of Shrffield. They were fed Oxoid Diet No. 86 (0x0 Ltd., London). Injections. Cortisol. trptosterone propionate and 402
COltTISOL
ASD
over a dose range of between 0.14 and 2.0 mg/lOO gm initial body weight. Complete inhibition was observed with 1.5 mg cortisol/100 gm; a further increase in dose was always observed to produce a slight drop in mean body weight (Table 1). In this respect chicks were nearly twice as sensitive to cortisol as male rats of equivalent body weight (3-4 weeks old). The inhihition of growth was not due to irreversible changes in the tissues and normal growth was attained 24 hours after stopping the injection (see also Winter et al. [1950] for rats). Another glucocorticoid, Sa-fluoro16a-methylprednisolone-21-phosphate (Betnelan phosphate, Glaxo Ltd.), given over the same period (0.05-1.0 mg/lOO gm body weight), was found to be about twice RS potent in inhibiting growth as cortisol. Cortisol, from its effect. on muscle, may be termed a catabolic steroid (Bellamy and Leonard, 1964). It was interesting, therefore, to examine the effect of this steroid in the presence of substances having predominantly anabolic actions. The daily administration of cortisol and testosterone propionate in equal amounts (1 mg each/ 100 gm body weight per 9 clays) did not alter the growth-inhibitory action of cortisol. However, when testosterone was replaced by the same amount of 17wethyl19-nor-testosterone (Nilevar, Searle Ltd.} for the same period, a 5070 reduction in the inhibitory action of cortisol was observed. Nilevar alone did not affect body growth although it, had a marked andro-
determined by the method of Berenblum and Chain (1938) on heparinized plasma ohtained after decapitation. A771ino Acids and Protein. Amino acid nitrogen was determined on 5% trichloroacetic acid (TC.4) rstracts of the tissues by the method of Rosen ( 1957). “Alkali soluble” protein in muscle was determined by the method of Rarusen and Johnstone (1961). Strcleic Acids. About 500 mg muscle was broken up in 10 ml ice-cold TCA and the mixture left in ice for 30 minutes with occasional stirring. The solid matter was cent,rifuged down and washed twice on the centrifuge with 5 ml icecold 5Oh TCA, allowing the tissue to remain in contact with the fresh solution for at least 30 minutes before centrifuging. The washed residue was heated for 15 minutes in a boiling water bath with 2 ml 5% TCA and then centrifuged down again. Samples of the supernatant solution were used for the calorimetric determination of deoxyribonucleic acid (DNA) and ribonucleic acid (RSA) by the method of Schneider (1957). Calibration curves were prepared using purified RNA (British Drug Houses Ltd., Poole), and DKA (Worthington Biochemical Co., New Jersey, G.S.A.). Ahline Phosphatase. Plasma alkaline phosphatase was measured by the method of Binkley et al. (1944). RESULTS
Body Growth. Normal week-old chicks under laboratory conditions gained weight at a rate of between 3-8 gm/day. During the third week of post-embryonic life the growth rate increased to about 11-12 gm/ day. The administration of cortisol suspension in the second week inhibited growth TABLE EFFECT
OF CORTISOL
ON
THE
1 GROWTH Weight
Dose of cortisol (mg/100
g m initial
_--wt.)
0
103
GROWTH
0.4
OF CHICKP of animalsh (am) 0.6
2.0
1.5
Time (days) 1
2 4 S
59.9 64.5 75.5 101.0
f 2k f *
2.4 2.3 3.4 3.3
a Chicks (l-week old) were injected rortisol and weighed at intervals over b &leans f SE for 6 animals.
63.2 64.6 70.4 93.2
+ + 5 +
2.9 2.3 2.9 3.0
64.2 66.0 67.0 66.9
* + + 31
2.5 2.4 2.7 3.1
60.0 61.9 60.9 60.4
subcutaneously as described in the text with 8 days. Injections were started on Day 1.
+ + + +
2.3 2.4 2.9 2.2
57.7 57.6 56.5 55.6
* + f A
various
amounts
2.0 2.2 2.5 2.S of
1.60
8
b Means f SE for 5 animals. 0 p > 0.06 compared with control
0 Normal week-old chicks were tiesues of normal animals (N).
ztO.04
1.33 zto.05 1.42~~1~0.07 1.49 ho.06
N
1 2 4
Time (dwd
value.
cortisol
0.952
nith
ckO.08
1.44~
injected
0.250 0.500 0.801
C
1.47 f0.04 1.36czkO.06 1.30”: f0.03
Brain
(1.5 mg/lOO
3~0.09
zto.03 +0.02 kO.04
N
EFFECT
rto.02 ho.04 ho.03
C
g m body
weight).
0.234’z!cO.O4
0.230 0.239c 0.228e
Heart
OF CORTISOL
Animals
0.860 were
f0.15
;t 0.03 fO.O1 f0.09
N
Weight
TISSUE
TABLE
0.255 0.304 0.611
ON
killed
f0.04
~1~0.06 z!zO.O7 ztO.07
C
11.502
0.504 3.214 5.600
of 5) and
f1.04
&to.07 fO.09 AO.17
N
CHICKS
Muscle
(groups
THE
Skeletal
(gm)b
IN
at intervals
0.234~
0.230 0.2390 0.228~
Lee:
of tissues
GROWTH
2
C
the tissue
weights
0.468’ztO.16
0.574 f0.04 0.49% f 0.10 0.460’~0.12
Pectoral
determined
0.850
0.311 0.417 0.546
compared
with
ho.04 f 0.09 ztO.ll
C
0.321’&0.08
0.326 0.314~ 0.309c
(C) and
Eto.15
f0.06 f 0.08 ztO.11
N
Tibia
6
2
R
5 d
Fj
F
E
CORTISOL
AKiD
GROWTH
405
That is to say, cortisol brought the growth process to a standstill. The exceptional tissue was liver, which appeared to continue growing at its initial rapid rate (Fig. 1). Because the rate of liver growth normally decreased during the third week of post embryonic life, cortisol apparently stimulated liver growth when the experiment was carried out for a period of several days. No selective action of cortisol on bone growth was observed in the present work (Table 2 ; cf. Kowalewski, 1962). Chicks injected with a dose of cortisol greater than that sufficient to inhibit growth completely looked thin, and examination showed that these animals had a low skeletal muscle mass. In chicks injected daily for 8 days with 5 mg cortisol, the proportion of pectoral muscle relative to body weight was 50% smaller than in normal animals of the same age. The fall in tissue mass appeared to be specific for skeletal muscle, there being no significant difference in the relative weight of the heart 12compared with normal animals. One of the most characteristic effects of cortisol administration in mammals is a rapid decrease in the weight of lymphoid tissue. In the case of rat thymus the weight loss was particularly noticeable 24 hours after the administration of a single dose of cortisol. The “thymolytic” activity of cortisol was also well marked under similar circumstances in the chick (Table 3). In addition to the thymus, birds possess a secondary, easily accessible mass of lymphoid tissue, the bursa of Fabricius. The function of the ‘(bursa” is believed to be similar to that of the thymus of mamI I mals and it appears to play a prominent I6 8 role in antibody formation (Glick et al., Time (days) 1956). The weight of the “bursa” fell after FIG. 1. Effect of cortisol on liver growth. treatment with cortisol, but the percentage Chicks were injected daily with 1.5 mg cortisol/ loss of weight 24 hours after one injection 100 gm body weight and killed at intervals over was smaller than observed with thymus a &day period. The liver weights (U) were (Table 3). Repeated injections caused both compared with the saline-injected controls (0). Each point is the mean of six animals. tissues to lose about 950/O of their mass in 10 days. injection of cortisol at a level which just DNA and RXd Content of Tissues. The inhibited general growth did not affect the DNA content of the pectoral and leg musweight relat,ionships of the various tissues. cle of normal animals increased at a con-
genie action (lo-fold increase in comb weight in 9 days; not observed when given together with cortisol). Ovine growth hormone (2 mg/lOO gm body weight per day for 5 days) did not affect the cortisol inhibition of growth, nor did ovine prolactin (2 mg/lOO gm per day for 5 days), aIthough this substance has been reported t.o restore the growth of hypophysectomized birds (Hijhn, 1961). Organ Growth. In normal chicks the initial post-embryonic growth rate of a number of tissues was found to be constant (Table 2). Brain, heart, various leg muscles, and the lymphoid tissues increased in weight at a rate of 0.03 to 0.08 gm/day. The pectoral muscle was exceptional in having a high rate of growth (average of 1.5 gm/day, Table 2). Liver growth was not linear. Initially, the latter tissue gained weight at a rate of about 0.6 gm/day, but this- was not maintained over the experimental period (Fig. 1). In general, the
406
BELLAMY
AKD
TABLE EFFECT
OF CORTISOL
ON LYMPHOID
LEONARD
3 TISSUES
OF KIT
Weight of tissues Rat
.\NI)
CHICK”
(gm)h Chick
-_-__---___-
N
c
--~.~ c
K
~~ -... ._ ._ ___.
Thymes Bursa Body
0.310
*
0.037
0.133c
&- 0.014
67.1
* 3.1
64.3
f 0.6
a Six male rats (l-month old) and five chicks (lo-days old) cortisol in 0.2 ml 0.9% NaCI. The weight of selected lymphoid compared with animals injected with 0.9% XaCl. b Means + SE. c p < 0.05 compared with control value.
stant rate over the experimental period of 8 days. The tissue volume increased considerably in this time. For example, in the case of pect,oral muscle there was a 30fold change in tissue weight (Table 2). This resulted in a rapid fall in the concentration of DNA during the first 6-8 days of life (Fig. 2). Cortisol, at a dose which st,opped growth, appeared not to inhibit 4.9 .
ID
Muscle
wt. ( g.)
2. Effect of cortisol on the DNA concentration in the pectoral muscle. Week-old chicks were inject,ed daily with 1.5 mg cortisol for 10 days, and the DNA concentration in the right pectoral muscle (m) compared with that of saline-injected controls (0). FIG.
0.223 0.167 69.2
-+ 0.011 + 0.014 It 1.1
0.096” 0.09ic 62.4
..-
i 0. r11:; * 0.012 f
0 s
were injerted subcutaneously with 2.6 mg organs was determined 24 hours later nntl
net DNA synthesis completely; and the smallest non-growing injected birds had on the average a greater amount of DNA for a particular muscle weight than the normal animals. This was particularly noticeable in small muscles from animals weighing about 35 gm (Fig. 2) where a slight change in DNA formation would be expected to be more noticeable. The RNA content of leg muscle increased in a linear fashion while the net formation of RNA in pectoral muscle decreased with time. Cortisoi appeared to inhibit the net, synthesis of RNA complet,ely. There was no correlation between the nucleic acid concentration in various muscles and their growth rate (Table 4; cf. Table 2). Cortisol at a dose sufficient t.o bIock gr0wt.h in large animals did not noticeably affect the concentration of either DNA or RNA in a range of muscle tissues (Table 4). Tissue Electrolytes, Protein, and A mine Acids. Cortisol treat’ment in excess of that necessary to inhibit growth completely did not affect the electrolyte content of live1 (Table 5). Since nearly 40% of this tissue is occupied by extracellular fluid (D. Bellamy, unpublished observations), it may also be concluded that cortisol did not affect the electrolyte content of the plasma through changes in kidney function. However, there was a marked difference in the electrolyte composition of the pectoral muscle, which is characterized by having a much lower cxtraccllular dpace. In this
CORTISOL
EFFECT
AND
407
GROWTH
TABLE 4 ON THE NUCLEIC ACID CONTENT
OF CORWSOL
OF VARIOUS
MUSCLES=
Nucleic rtcid content mg/gm wet wtb DNA
RNA
N
Pectoral Leg Wing Heart
1.26 1.51 1.29 2.92
N
C
k f * A
0.10 0.21 0.13 0.24
1.25 1.37 1.54 2.66
+ f 5 *
0.20 0.17 0.15 0.20
2.94 5.42 6.04 6.25
C
+ + + +
0.23 0.66 0.78 0.92
3.66” 5.25 5.88 4.96
zk f 2 +
a Week-old chicks (mean weight 61.4 gm) were injected daily with 1.5 mg cortisol/lOO gm body 10 days and the nucleic acid content of various muscles (C) compared with that of control-injected
0.14 0.42 0.33 0.30 weight for animals
6’). b Means * SE for 6 animals. c p < 0.05 compared with control
value.
TABLE EFFECT
OF CORTISOL
ON THE
SODIUM,
5
POTASSIUM,
.~ND PEcTOR.~L
AND
Electrolyte (mm&s/kg
Liver Normal Cortisol Muscle Normal Cortisol u Liver and muscle used for the estimation 5 animals. b p < 0.05 rompared
content of tissue tissue water)
24.8 24.4
* 0.5 f 0.3
55.2 59.9
+ 2.2 + 2.7
121.6 119.1
+ 6.7 * 7.1
treated
23.5 18.16
+ 0.4 + 0.3
45.6 67.2b
f 2.6 f 3.1
14S.6 82.26
+ 5.2 * 4.4
samples were taken from animals injected for 8 days with 2 mg cortisol/lOO of sodium and potassium as described in the text. Figures refer to means with
control
Amino acids mm&s/kg
k 0.27 f 0.34
OF CORTISOL
Phosphate mm&s/kg
0.34 0.21d
gm and f SE for
value.
TABLE ON PL.ISUA
6 :LND
MUSCLE
Plasma
5.00 8.22d
OF LIVER
treated
EFFECT
Normal Cortisol
CONTENT
WATER
MUSCLE"
+ 0.02 + 0.01
COMPOSITION* Pectoral
Blkalineb Phosphatase (units/100 ml)
S.5 3.9d
f 0.62 + 0.20
Amino acids mm&s/kg
17.0 18.2
+ 4.4 + 5.3
Muscle
Alk;;O;z;;ble (mg/gm
wet wt.)
51.7 37.2d
* 2.2 + 2.5
(mg/lOO
54.3 103.3”
Fate gm body
wt.)
+ 5.8 It 12.4
a Week-old chicks were injected daily for 10 days with 2 mg cortisol/lOO gm body weight and a selection of plasma and muscle constituents compared with those of control-injected animals. Figures refer to means + SE for 6 animals. b One unit of enzyme liberated 1 mg phenol from disodium phenylphosphate per hotlr at 37”. c The strip of adipose tissue along the posterior edge of the pectoral muscle. d p < 0.05 compared with control value.
408
BELLAMY
AND
tissue cortisol brought about an increase in the concentration of sodium and a decrease in the potassium concentration (Table 5). Sodium did not replace all of the lost, potassium and the sum of sodium and potassium was reduced by almost 20%. The sum of sodium and potassium may be taken as an approximate measure of the total non-diffusible anionic groups within the muscle fibers (Boyle and Conway, 1941), so that these differences indicate the loss of cellular material of high molecular weight, possibly protein. In agreement with this there was a reduction in both the percentage and total dry weight of the pectoral muscle, and a 30% drop in the concentration of alkali soluble protein (Table 6). Rather surprisingly this marked catabolic effect of cortisol on pectoral muscle was associated with an increased deposition of fat (Table 6; see also Winter et al., 1950). No changes were found in the level of free amino acids in muscle even though there was a considerable rise in plasma amino acids (Table 6). Plasma phosphate decreased, possibly as a consequence of the fall in alkaline phosphatase (Table 6). DISCUSSION Cortisol was found to inhibit the growth of a number of tissues in the chick. The continued growth of liver in the cortisoltreated animals could be the result of an increased load placed upon this organ OWing to an abnormally high rate of amino acid deamination (Goodlad and Munro, 1959; Bellamy and Leonard, 1964). HOWever, liver cells may be resistant to the actions of cortisol and to a certain extent may be able to grow independently of other organs. Thus the continued growth of liver at its initial rate would give the high liver-body weight ratio observed in cortisol-treated chicks. In the present work the net synthesis of DNA, RNA and protein was prevented and the growth process in the majority of tissues came to a standstill. The almost complete block of growth by cortisol raises the possibility that the effect of this steroid is mediated through t.he loss or inhibition
LIWh‘AHD
of the action of some general growth factor. In addition to hormones of the “glucocorticoid” type, esogcnous estrogens arc also known to inhibit t.hc body growth of mammals. There is good evidence that the action of estrogens is mediated t,hrough an inhibition of growth hormone release (Gaarenstroom and Levie, 1939; Noble, 1939; Reere and Leonard, 1939). However, because of the possibility of a stimulation of corticostcroid secretion by estrogens (Zondek and Burstein, 1952), the effect of estrogens on growth may be due to the action of rndogenous adrenal steroids on the anterior pituitary gland (Kanematsu and Sawyer, 1963). Indeed, the changes in t.he various body constituents of the chick (Table 6) and the alterations in urea synthesis and liver transaminase that result from cortisol injection in the rat (Bellamy and Leonard, 1964) are similar to the effects of hypophysectomy and opposite to the effects of growth hormone (Li and Evans, 1948; Gaebler, 1955). Furthermore, the administration of growth hormone to rats counteracts the inhibition of growth and other changes which follow cortisol and ACTH t.rcatment (Selye, 1951; Geschwind and Li, 1955). Against this, however, must be set the present experiments, which demonstrated that neither ovine growth hormone nor ovine prolactin (said to restore the growth of hypophysectomized birds (HShn, 1961) ) maintained growth in cortisol-injected chicks. Clearly, more basic information is required on the control of avian growth before any definite conclusion can be drawn as to the action of cortisol. The injection of cortisol in excess of that required to inhibit growth caused certain IDUSC~C tissues, particularly the pectoralis, to lose weight. That is, a general inhibition of growth occurred at lowcortisol levels, while higher doses brought about a specific breakdown of muscle tissue. The biological implications of a direct action of corticosteroids in mobilizing the muscle protein of non-growing animals during the growth of reproductive tissues have been discussed previously (Chester .Jonts and Bellamy, 1964).
CORTISOL
AND
The most marked change in muscle composition of cortisol-treated animals was a a reduction in the protein content. This may also account for the observed fall in the major muscle cations, partly by the loss of anionic proteins (Cole, 1950) and perhaps also by a decreased access of cations, particularly potassium, to binding sites. The latter process would also account for the net gain of muscle sodium, although in the case of this ion a change in the cell membrane that favored sodium diffusion into the tissue cannot be ruled out. From the measurements of nucleic acids in the present work, it is clear that the absolute concentration of these substances in muscle does not determine the growth rate. For example, the pectoral muscle of the week-old chick grew some twenty times faster than heart and leg muscle yet cont,ained up to 50% less nucleic acid per unit weight. Also, the daily weight increment of normal muscles was constant over the experimental period although the size, nucleic .acid content, and rate of RNA formation might change considerably. There was no evidence that the inhibition of growth by cortisol was due to a specific reduction in nucleic acid metabolism or t.hat the inhibitory action was linked with either the amount of nucleic acid in the tissues or the rate of muscle growth. It may well be that only a small fraction of muscle RNA participates in the growth process and that the remainder serves some other role, perhaps as an important structural component of the tissue. If this is so it is possible that relatively small alterations in “template-nucleic acid” concentration were associated with the cortisolinhibition of growth but went undetected in the present work. ACKNOWLEDGMENTS The authors thank the Endocrinology Study Section, National Institutes of Health, Bethesda, Maryland, U.S.A. for the gift of ovine growth hormone and prolactin; and Messrs. Searle Ltd. for the gift o’f Nilevar. The work was largely financed by grants to the Department of Zoology, University of Sheffield from the Agricultural Research Counciland the Department of Scientific and Industrial Research.
409
GROWTH
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