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The diel rhythm of circulating acth titer in the goldfish (Carassius auratus L.)
Comp. Biochem. Physiol.. 1976. Vol. 53A. pp. 291 to 293. Pergamon Press. Printed in Great Britain
THE DIEL RHYTHM OF CIRCULATING ACTH TITER IN THE GOLDFISH
(CARASSIUS AURATUS L.) JOHN A. SINGLEY AND WALTER CHAVIN Department of Biology, Wayne State University, Detroit. MI 48202, U.S.A. (Received 17 March 1975)
Abstract--l. A diel rhythm of circulating ACTH titer was demonstrated in goldfish maintained on a 12 hr light, 12 hr dark photoperiod (light 8.00L20.00 hr). 2. The rhythm is bimodal, having two peak ACTH titers. The first peak occurs at 10.00--I1.00 hr (2-3 hr post light onset). The second peak occurs at 13.00-16.00 hr (5-9 hr post light onset). 3. The mean peak ACTH titer is 101.5 + 28.7 pg/ml and the mean low ACTH titer 42.1 + 13.7 pg/ml. The total mean ACTH titer is 56"9 _ 3-6 pg/ml. 4. The peaks ofACTH titer precede the peak cortisol levels by 1 hr.
INTRODUCTION
METHODS AND MATERIALS
THE CmCADIAN rhythm of circulating ACTH is accompanied by a similar rhythm of circulating glucocorticoids in mammals. Diurnal mammals show peak ACTH and cortisol levels in the early hours after the onset of the light period followed by a decline in activity throughout the remaining 24 hr period (Hatwood & Mason, 1956; Peterson, 1957). As the time intervals examined have been 4-6 hr apart, other peaks of pituitary-adrenal activity may exist, in fact, many small bursts of ACTH secretion occur throughout a 24 hr period (Krieger, 1974). Nocturnal mammals show a different timing of ACTH and corticosterone titers with the peak hormone levels occurring prior to the onset of darkness (McCarthy et al., 1960). Episodic release of both ACTH and cortisol have been reported in mammals with the ACTH increases preceding the cortisol increases by a small but finite interval (McNatty et al., 1972; Gallagher et al., 1973; Krieger, 1974). Thus, episodic secretion of both ACTH and cortisol occur within the framework of a true circadian rhythm showing generally high and low circulating hormone titers. While circadian rhythms of adrenocorticoids in teleosts have been reported (Boehlke et a l., 1966; Singley & Chavin, 1971 ; Srivastava & Meier, 1972; Garcia & Meier, 1973; Redgate, 1974; Singley & Chavin, 1975a), information dealing with the circadian rhythm of ACTH is not present in lower vertebrates. In goldfish, the diel cortisol rhythm consists of two peak periods of cortisol titer (Singley & Chavin, 1971, 1975a); killifish are similar in this regard (Garcia & Meier, 1973). The peak period of goldfish adrenocortical activity occurs at 4 hr and 7-9 hr after the onset of light. The present study was undertaken to determine whether the diel ACTH rhythm is present, and if so, whether a temporal relationship between goldfish ACTH and cortisol secretion is present.
A total of 144 common goldfish (Carassius auratus L.) 4.2-8.9 g (6.4 + 0.2 g; ,X + S.E.M.), were maintained, acclimated and net captured as previously described (Singley & Chavin, 1975a). Blood was obtained by terminal cardiac puncture using non-heparinized microhematocrit tubes and centrifuged (Chavin & Young, 1970). The serum was immediately separated and stored at -27°C until analyzed. ACTH was determined by radioimmunoassay as previously described (Singley & Chavin, 1975b) and the results expressed as pg/ml _+ S.E.M. Six fish were sacrificed at hourly intervals during the 24 hr cycle. During the dark period (20.00-8.00 hr) one group was sacrificed per night, the fish captured in total darkness and bled under a shielded 25 W red light, rheostatically set to the lowest possible setting (Singley & Chavin, 1975a). The data were analyzed using one way analysis of variance and Student t-test (Bliss, 1967).
* Contribution number 341, Department of Biology. 291
RESULTS The circulating ACTH titers ranged from 16.3 + 2.5 to 147.9 + 36"9 pg/ml. The mean ACTH titer for the 24 hr period was 56"9 + 3"6 pg/ml. Goldfish serum ACTH levels show two peaks (Fig. I). The first ACTH peak titer (120"8 _+ 28"6 pg/rnl) occurred at I0.00-I 1.00 hr, 2-3 hr after the onset of the photoperiod. A second ACTH peak titer (91"8 + 28"6 pg/ml) occurred at 13.00-16.00 hr, 5-9 hr after the onset of the light period. These ACTH peak titers were not significantly different from each other but were significantly different (P < 0"05) from those at the other time intervals studied. Both peak titer periods were of short duration. The mean peak ACTH titer was 101"5 + 28"6 pg/ml. The remaining portions of the cycle consisted of two periods of low ACTH titers (Fig. 1). The first (33.7 + 5.5 pg/ml) was an extremely short period (1 hr) following the early 10.1X~--I1.00 hr ACTH peak. The second low ACTH titer (42-5_ 1~8 pg/ml) period began at 17.00 hr, continued through the entire dark period and extended 1 hr into the light portion
292
J.A. SINGLEY AND W. CHAVIN
1 I0j-
150-
gO" 70SO-
SO" IOLIGHT
2'4' ; ' :, " ~ ' ~ ' ,~" ,h" ,~" ,~" ,h'/o'2~'z'4 HOUR
Fig. 1. Diel rhythm of serum ACTH in normal fasted goldfish, Carassius auratus L., during 24 hr period. Maximal ACTH titers occur at two peak periods, 10.00-I 1.00 hr and 13.00-16.00 hr. The shaded bars at the bottom of the figure represent the dark portion of the photoperiod; the open bar represents the light portion of the photoperiod. Each point represents the mean ACTH titer of 6 fish. The vertical line at each point represents the S.E.M. of the cycle (9.00 hr). No statistically significant difference in ACTH titers was present during this extended period. Further, the two low periods of ACTH titer were not significantly different. The mean low ACTH titer was 42.1 + 13.7 pg/ml or 41.5~ of the mean peak ACTH titer. D~CU~ION
The diel rhythm of circulating ACTH titer in goldfish in a general way is comparable to that of diurnal mammals (Matsuyama et al., 1971; Krieger, 1974). However, the bimodal rhythmicity of peak ACI'H activity in normal goldfish has not been described previously in a vertebrate. This ACTH bimodality conforms to that of the cortisol diel rhythm in the goldfish (Singley & Chavin, 1975a). As anticipated from the established hypophyseal-adrenocortieal relationship, a slight lag is present in goldfish circulating cortisol titers when compared with the ACTH titers. Peak ACTH titers in goldfish precede the peak cortisol periods by 1 hr, the shortest interval sampled. The circulating ACTH and cortisol concentrations in the goldfish show a correlated cycle over a 24 hr period. The amplitude of this cycle is large 2.6-4.9 gg/dl per g cortisol and 33.7--101.5 pg/ml ACTH. The major peak activities occur during the hours of light. The mechanism controlling the goldfish pituitaryadrenal diel rhythm is not clear. Such is also the case in mammals where cycles have received considerable attention. A number of variables have been suggested to affect the cycle including sleep. However, despite the relation of growth hormone secretion to the 24 hr sleep--wake period, ACTH has not been demonstrated to follow this pattern in man (Krieger, 1974). Studies of sleep in teleosts are difficult so that information dealing with this potential factor are not available. Mammalian motor activity and adrenocorticoid titer appear to be correlated (Seabloom, 1965; Andrews, 1970) but the relationship of locomotor activity cycle in the teleosts utilized in the reported hormone studies remains to be explored. Light also has been suggested as a control of diel ACTH--corticoid rhythms (Andrews, 1970; Matsuyama et al., 1971).
However, in goldfish neither the onset of light nor the onset of darkness appear to immediately affect the periodicity of peak ACTH and cortisol titers as the bimodal peaks of pituitary-adrenocortical activity occur several hours after the onset of light and several hours prior to the onset of darkness. Further, the other teleosts studied show peaks of adrenocortical activity which do not appear to be related to the onset of light or darkness. Studies of teleosts under different photoperiods will be necessary to clarify this point. The initiating stimuli of the observed hormonal rhythmicities, therefore, remain to be clarified in vertebrates generally. The small fluctuations of the circulating ACTH titer similar to those present in goldfish have been described in mammals (Matsuyama et al., 1971). The secretion of both ACTH and cortisol are episodic in mammals (Gallagher et al., 1973; Krieger, 1974). Such episodic A C r H secretion by the goldfish pituitary would account for the demonstrated variation in circulating ACTH titers with subsequent episodic secretion of cortisol as seen previously (Singley & Chavin, 1975a). The presence o f a diel rhythm of the goldfish pituitary-adrenocortieal axis suggests such to be a generalized phenomenon among vertebrates. REFERENCES
ANDREWSR. V. (1970) Circadian variations in adrenal secretion of lemmings, voles and mice. Acta endocr., Copnh. 65, 645-649. BLISSC. I. (1967) Statistics in Biology, Vol. I, p. 558. McGraw-Hill, New York. BOEHLKE K. W., CHURCH R. U, TtEMEIER O. W. & ELEF-
THERIOU B. E. (1966) Diurnal rhythm in plasma glucocorticoid levels in channel catfish (Ictalurus punctatus). Gen. comp. Endocr. 7, 18-21. CHAVINW. & YOUNGJ. E. (1970) Factors in the determination of normal serum glucose levels of goldfish, Carassius auratus L. Comp. Biochem Physiol. 33, 629-653. GALLAGHERT. F., YOSHIDA K., ROFFWANGH. O., FURCUSHIMA D. K., WEITZMAN E. D. • HELLMAN L. (1973)
ACTH and cortisol secretory patterns in man. J. clin Endocr. Metab. 36, 1058-1068. GARCIA L. E. & METER A. H. (1973) Daily rhythms in concentration of plasma cortisol in male and female killifish, Fundulus grandis. Biol. Bull. mar. biol. Lab., Woods Hole 144, 471-479. HARWOOD, C. T. & MASON J. W. (1956) Effect of intravenous infusion of autonomic agents on peripheral
blood 17-hydroxycorticosteroid levels in the dog. Am. J. Physiol. 186, 445-452.
KRtEGER D. T. (1974) Biorhythms in central nervous system disease (primarily those of pituitary-adrenal hormones). In Biorhythms and Human Reproduction (Edited by FERINM., HAl.BERGF., RICHARTR. M. & VANDEWIELE R. L.), pp. 629-649. Wiley, New York. MATSUYAMA H., RUHMANN-WENNHOLD A. & NELSON D.
H. (1971) Radioimmunoassay of plasma ACTH in intact rats. Endocrinology 88, 692-695. MCCARTHY J. L., CORLEV,R. C. & ZARROWM. X. (1960) Diurnal rhythm in plasma eorticosterone and* lack of diurnal rhythm in plasma compound F-like material in the rat. Prec. Soc. exp. Biol. Med. 104, 787-789. MCNATTYK. P., CASHMEREM. & YOUNGA. (1972) Diurnal variation in plasma cortisol levels in sheep. J. Endocr. 54, 361-362. Pl:'rEgSON R. E. (1957) Plasma corticosterone and hydrocortisone levels in man. J. clin. Endocr. Metab. 17, 1150-I 157.
ACTH diel rhythm REDGATE E. S. (1974) Neural control of pituitary-adrenal activity in Cyprinus carpio. Gen. comp. Endocr. 22, 35-41. SEABLOOMR. W. (1965) Daily motor activity and corticosterone secretion in the meadow vole. J. Mammal. 46, 286-295. SINGLEY J. A. & CnAVIN W. (1971) Cortisol levels of normal goldfish, Carassius auratus L.. and response to osmotic change. Am. Zoologist 11, 653.° SINGLEYJ. A. ,~r CHAVINW. (1975a) Serum cortisol in normal goldfish, Carassius auratus L. Comp. Biochem. Physiol. 50A, 77-82.
293
SI~OLEYJ. A. & CHAVI~ W. (1975b) The adrenocortical-hypophyseal response to saline stress in the goldfish, Carassius auratus L. Comp. Biochem. Physiol. 51A, 749-756. SRIVASTAVAA. K. & MEIER A. H. (1972) Daily variation in concentration of cortisol in plasma in intact and hypophysectomized gulf killiiish. Science, N.Y 177, 185-187.
THE DIEL RHYTHM OF CIRCULATING ACTH TITER IN THE GOLDFISH
(CARASSIUS AURATUS L.) JOHN A. SINGLEY AND WALTER CHAVIN Department of Biology, Wayne State University, Detroit. MI 48202, U.S.A. (Received 17 March 1975)
Abstract--l. A diel rhythm of circulating ACTH titer was demonstrated in goldfish maintained on a 12 hr light, 12 hr dark photoperiod (light 8.00L20.00 hr). 2. The rhythm is bimodal, having two peak ACTH titers. The first peak occurs at 10.00--I1.00 hr (2-3 hr post light onset). The second peak occurs at 13.00-16.00 hr (5-9 hr post light onset). 3. The mean peak ACTH titer is 101.5 + 28.7 pg/ml and the mean low ACTH titer 42.1 + 13.7 pg/ml. The total mean ACTH titer is 56"9 _ 3-6 pg/ml. 4. The peaks ofACTH titer precede the peak cortisol levels by 1 hr.
INTRODUCTION
METHODS AND MATERIALS
THE CmCADIAN rhythm of circulating ACTH is accompanied by a similar rhythm of circulating glucocorticoids in mammals. Diurnal mammals show peak ACTH and cortisol levels in the early hours after the onset of the light period followed by a decline in activity throughout the remaining 24 hr period (Hatwood & Mason, 1956; Peterson, 1957). As the time intervals examined have been 4-6 hr apart, other peaks of pituitary-adrenal activity may exist, in fact, many small bursts of ACTH secretion occur throughout a 24 hr period (Krieger, 1974). Nocturnal mammals show a different timing of ACTH and corticosterone titers with the peak hormone levels occurring prior to the onset of darkness (McCarthy et al., 1960). Episodic release of both ACTH and cortisol have been reported in mammals with the ACTH increases preceding the cortisol increases by a small but finite interval (McNatty et al., 1972; Gallagher et al., 1973; Krieger, 1974). Thus, episodic secretion of both ACTH and cortisol occur within the framework of a true circadian rhythm showing generally high and low circulating hormone titers. While circadian rhythms of adrenocorticoids in teleosts have been reported (Boehlke et a l., 1966; Singley & Chavin, 1971 ; Srivastava & Meier, 1972; Garcia & Meier, 1973; Redgate, 1974; Singley & Chavin, 1975a), information dealing with the circadian rhythm of ACTH is not present in lower vertebrates. In goldfish, the diel cortisol rhythm consists of two peak periods of cortisol titer (Singley & Chavin, 1971, 1975a); killifish are similar in this regard (Garcia & Meier, 1973). The peak period of goldfish adrenocortical activity occurs at 4 hr and 7-9 hr after the onset of light. The present study was undertaken to determine whether the diel ACTH rhythm is present, and if so, whether a temporal relationship between goldfish ACTH and cortisol secretion is present.
A total of 144 common goldfish (Carassius auratus L.) 4.2-8.9 g (6.4 + 0.2 g; ,X + S.E.M.), were maintained, acclimated and net captured as previously described (Singley & Chavin, 1975a). Blood was obtained by terminal cardiac puncture using non-heparinized microhematocrit tubes and centrifuged (Chavin & Young, 1970). The serum was immediately separated and stored at -27°C until analyzed. ACTH was determined by radioimmunoassay as previously described (Singley & Chavin, 1975b) and the results expressed as pg/ml _+ S.E.M. Six fish were sacrificed at hourly intervals during the 24 hr cycle. During the dark period (20.00-8.00 hr) one group was sacrificed per night, the fish captured in total darkness and bled under a shielded 25 W red light, rheostatically set to the lowest possible setting (Singley & Chavin, 1975a). The data were analyzed using one way analysis of variance and Student t-test (Bliss, 1967).
* Contribution number 341, Department of Biology. 291
RESULTS The circulating ACTH titers ranged from 16.3 + 2.5 to 147.9 + 36"9 pg/ml. The mean ACTH titer for the 24 hr period was 56"9 + 3"6 pg/ml. Goldfish serum ACTH levels show two peaks (Fig. I). The first ACTH peak titer (120"8 _+ 28"6 pg/rnl) occurred at I0.00-I 1.00 hr, 2-3 hr after the onset of the photoperiod. A second ACTH peak titer (91"8 + 28"6 pg/ml) occurred at 13.00-16.00 hr, 5-9 hr after the onset of the light period. These ACTH peak titers were not significantly different from each other but were significantly different (P < 0"05) from those at the other time intervals studied. Both peak titer periods were of short duration. The mean peak ACTH titer was 101"5 + 28"6 pg/ml. The remaining portions of the cycle consisted of two periods of low ACTH titers (Fig. 1). The first (33.7 + 5.5 pg/ml) was an extremely short period (1 hr) following the early 10.1X~--I1.00 hr ACTH peak. The second low ACTH titer (42-5_ 1~8 pg/ml) period began at 17.00 hr, continued through the entire dark period and extended 1 hr into the light portion
292
J.A. SINGLEY AND W. CHAVIN
1 I0j-
150-
gO" 70SO-
SO" IOLIGHT
2'4' ; ' :, " ~ ' ~ ' ,~" ,h" ,~" ,~" ,h'/o'2~'z'4 HOUR
Fig. 1. Diel rhythm of serum ACTH in normal fasted goldfish, Carassius auratus L., during 24 hr period. Maximal ACTH titers occur at two peak periods, 10.00-I 1.00 hr and 13.00-16.00 hr. The shaded bars at the bottom of the figure represent the dark portion of the photoperiod; the open bar represents the light portion of the photoperiod. Each point represents the mean ACTH titer of 6 fish. The vertical line at each point represents the S.E.M. of the cycle (9.00 hr). No statistically significant difference in ACTH titers was present during this extended period. Further, the two low periods of ACTH titer were not significantly different. The mean low ACTH titer was 42.1 + 13.7 pg/ml or 41.5~ of the mean peak ACTH titer. D~CU~ION
The diel rhythm of circulating ACTH titer in goldfish in a general way is comparable to that of diurnal mammals (Matsuyama et al., 1971; Krieger, 1974). However, the bimodal rhythmicity of peak ACI'H activity in normal goldfish has not been described previously in a vertebrate. This ACTH bimodality conforms to that of the cortisol diel rhythm in the goldfish (Singley & Chavin, 1975a). As anticipated from the established hypophyseal-adrenocortieal relationship, a slight lag is present in goldfish circulating cortisol titers when compared with the ACTH titers. Peak ACTH titers in goldfish precede the peak cortisol periods by 1 hr, the shortest interval sampled. The circulating ACTH and cortisol concentrations in the goldfish show a correlated cycle over a 24 hr period. The amplitude of this cycle is large 2.6-4.9 gg/dl per g cortisol and 33.7--101.5 pg/ml ACTH. The major peak activities occur during the hours of light. The mechanism controlling the goldfish pituitaryadrenal diel rhythm is not clear. Such is also the case in mammals where cycles have received considerable attention. A number of variables have been suggested to affect the cycle including sleep. However, despite the relation of growth hormone secretion to the 24 hr sleep--wake period, ACTH has not been demonstrated to follow this pattern in man (Krieger, 1974). Studies of sleep in teleosts are difficult so that information dealing with this potential factor are not available. Mammalian motor activity and adrenocorticoid titer appear to be correlated (Seabloom, 1965; Andrews, 1970) but the relationship of locomotor activity cycle in the teleosts utilized in the reported hormone studies remains to be explored. Light also has been suggested as a control of diel ACTH--corticoid rhythms (Andrews, 1970; Matsuyama et al., 1971).
However, in goldfish neither the onset of light nor the onset of darkness appear to immediately affect the periodicity of peak ACTH and cortisol titers as the bimodal peaks of pituitary-adrenocortical activity occur several hours after the onset of light and several hours prior to the onset of darkness. Further, the other teleosts studied show peaks of adrenocortical activity which do not appear to be related to the onset of light or darkness. Studies of teleosts under different photoperiods will be necessary to clarify this point. The initiating stimuli of the observed hormonal rhythmicities, therefore, remain to be clarified in vertebrates generally. The small fluctuations of the circulating ACTH titer similar to those present in goldfish have been described in mammals (Matsuyama et al., 1971). The secretion of both ACTH and cortisol are episodic in mammals (Gallagher et al., 1973; Krieger, 1974). Such episodic A C r H secretion by the goldfish pituitary would account for the demonstrated variation in circulating ACTH titers with subsequent episodic secretion of cortisol as seen previously (Singley & Chavin, 1975a). The presence o f a diel rhythm of the goldfish pituitary-adrenocortieal axis suggests such to be a generalized phenomenon among vertebrates. REFERENCES
ANDREWSR. V. (1970) Circadian variations in adrenal secretion of lemmings, voles and mice. Acta endocr., Copnh. 65, 645-649. BLISSC. I. (1967) Statistics in Biology, Vol. I, p. 558. McGraw-Hill, New York. BOEHLKE K. W., CHURCH R. U, TtEMEIER O. W. & ELEF-
THERIOU B. E. (1966) Diurnal rhythm in plasma glucocorticoid levels in channel catfish (Ictalurus punctatus). Gen. comp. Endocr. 7, 18-21. CHAVINW. & YOUNGJ. E. (1970) Factors in the determination of normal serum glucose levels of goldfish, Carassius auratus L. Comp. Biochem Physiol. 33, 629-653. GALLAGHERT. F., YOSHIDA K., ROFFWANGH. O., FURCUSHIMA D. K., WEITZMAN E. D. • HELLMAN L. (1973)
ACTH and cortisol secretory patterns in man. J. clin Endocr. Metab. 36, 1058-1068. GARCIA L. E. & METER A. H. (1973) Daily rhythms in concentration of plasma cortisol in male and female killifish, Fundulus grandis. Biol. Bull. mar. biol. Lab., Woods Hole 144, 471-479. HARWOOD, C. T. & MASON J. W. (1956) Effect of intravenous infusion of autonomic agents on peripheral
blood 17-hydroxycorticosteroid levels in the dog. Am. J. Physiol. 186, 445-452.
KRtEGER D. T. (1974) Biorhythms in central nervous system disease (primarily those of pituitary-adrenal hormones). In Biorhythms and Human Reproduction (Edited by FERINM., HAl.BERGF., RICHARTR. M. & VANDEWIELE R. L.), pp. 629-649. Wiley, New York. MATSUYAMA H., RUHMANN-WENNHOLD A. & NELSON D.
H. (1971) Radioimmunoassay of plasma ACTH in intact rats. Endocrinology 88, 692-695. MCCARTHY J. L., CORLEV,R. C. & ZARROWM. X. (1960) Diurnal rhythm in plasma eorticosterone and* lack of diurnal rhythm in plasma compound F-like material in the rat. Prec. Soc. exp. Biol. Med. 104, 787-789. MCNATTYK. P., CASHMEREM. & YOUNGA. (1972) Diurnal variation in plasma cortisol levels in sheep. J. Endocr. 54, 361-362. Pl:'rEgSON R. E. (1957) Plasma corticosterone and hydrocortisone levels in man. J. clin. Endocr. Metab. 17, 1150-I 157.
ACTH diel rhythm REDGATE E. S. (1974) Neural control of pituitary-adrenal activity in Cyprinus carpio. Gen. comp. Endocr. 22, 35-41. SEABLOOMR. W. (1965) Daily motor activity and corticosterone secretion in the meadow vole. J. Mammal. 46, 286-295. SINGLEY J. A. & CnAVIN W. (1971) Cortisol levels of normal goldfish, Carassius auratus L.. and response to osmotic change. Am. Zoologist 11, 653.° SINGLEYJ. A. ,~r CHAVINW. (1975a) Serum cortisol in normal goldfish, Carassius auratus L. Comp. Biochem. Physiol. 50A, 77-82.
293
SI~OLEYJ. A. & CHAVI~ W. (1975b) The adrenocortical-hypophyseal response to saline stress in the goldfish, Carassius auratus L. Comp. Biochem. Physiol. 51A, 749-756. SRIVASTAVAA. K. & MEIER A. H. (1972) Daily variation in concentration of cortisol in plasma in intact and hypophysectomized gulf killiiish. Science, N.Y 177, 185-187.