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Relationship between temperature, ovarian recrudescence, and plasma cortisol level in Tilapia aurea (Cichlidae, Teleostei)
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
40, 143- 148 (1980)
Relationship between Temperature, Ovarian Recrudescence, and Plasma Cortisol Level in Tilapia aurea (Cichlidae, Teleostei) AVIVA *Department Israel, and
TERKATIN~HIMONY,* of Zoology, tDepartment
The George of Biological
Z. ILAN,*
Z. YARON,*
S. Wise Center for Sciences, Murray
AND D. W. JOHNSON?
Life Sciences, Tel-Aviv University, State University, Murray. Kentuchy
Tel-Aviv, 42071
Accepted August 17, 1979 An RIA procedure for measuring cortisol in the circulation of Tilapia aurea is described and evaluated. Cortisol, chromatographically purified from the fish plasma, accounted for its total immunoreactivity. The correlation between observed and expected cortisol values in the accuracy test was r = 0.977. The within-assay variance was 1% and the between-assays variance was 18.9%. The sensitivity of the whole RIA procedure was 0.24 nglml when the dilution was 1: 100 and 1.2 rig/ml when the dilution was 1:500. Female T. aurea collected in winter and kept at 17” had regressed ovaries, refused food, and their plasma cortisol levels at rest, measured after 1 and 2 months in captivity, were 74 + 3.5 and 65 2 6.1 rig/ml, respectively (mean f SEM). Two weeks after their exposure to 28” the fish resumed feeding, the GSI had doubled, and the plasma resting cortisol level had decreased to 40 ‘- 4.0 &ml. The cortisol level remained low in the subsequent 11 days. Fish collected in the breeding season (April) had a resting cortisol level of 35 2 4.0 rig/ml (mean 2 SEM, n = 28). Nine days following ovariectomy no change could be found in plasma cortisol. Cortisol level in ovariectomized fish injected with estradioL17fi did not ‘differ from that of oil-injected controls. In this nonmigratory, freshwater fish there is no relation between either the state of the ovary, or ovarian hormone, and the level of cortisol. The high level of plasma cortisol in fish exposed to low temperature may be attributed either to the slow rate of cortisol clearance or to the high activity of the interrenal tissue in the fasting fish.
Hyperactivity of the interrenal gland coincides with ovarian development in several species of migratory teleost. This is reflected in the hyperplasia and hypertrophy of the interrenal tissue in Oncorhynchus tshawytscha, in Salmo gairdnerii (Robertson and Wexler, 1959), and in 0. nerka (McBride and van Overbeeke, 1969) and in a high cortisol concentration in the plasma of 0. nerka during the spawning migration (Schmidt and Idler, 1962). Sexual maturation in 0. nerka is accompanied by an increase in the volume of distribution and metabolic clearance rate of cortisol, as well as by the secretion rate of this steroid (Donaldson and Fagerlund, 1970). The mechanism underlying the interrenal-ovarian relationship was attributed on one hand to the possible direct effect of estrogen on the interrenal tissue (McBride and van Overbeeke, 1969). Ovariectomy of
maturing salmon was followed by a decrease in the secretion rate and plasma levels of cortisol in resting fish, while estradiol administration reversed the process (Donaldson and Fagerlund, 1969, 1970). On the other hand the increase in plasma cortisol in maturing salmon was attributed to a reduced clearance rate of cortisol rather than to an increase of interrenal activity (reviewed by Idler and Truscott, 1972). In this salmon “migration is accompanied by rapid gonad development and severe depletion of fat and protein and involves sustained as well as vigorous activity. Interpretation of steroid studies on these animals is often difficult because so many things happen at once” (Idler and Truscott, 1972). We have chosen, therefore, a nonmigratory, freshwater fish-Tilapia aurea-as a subject for the study of the relationship 143 00166480/80/020143-06$01.00/0 Copyright All rights
@ 15’80 by Academic Press. Inc. of reproduction in any form reserved.
TERKATIN-SHIMONY
144
between temperature change, ovarian crudescence, and cortisol level. MATERIALS
AND Fish
ET AL.
re-
ing to Bassett and Hinks (1969). RIA buffer, 100 ~1, containing BGG was added to each column. Rabbit anti-cortisol-21-bovine serum albumin (BSA) serum, diluted with the RIA buffer to the concentration required to bind 35% of radioactive cortisol, was also added to each column (100 ~1). The serum was a gift from Professor H. Lindner and Dr. Fortune Kohen, Department of Hormone Research, The Weizmann Institute of Science. Rehovot, Israel. After 10 min equilibration at room temperature, [ 1,2,6,7(n)3H]cortisol (The Radiochemical Centre, Amersham, sp act 82 Ciimmol) was added to each column (10 nCi/lOO /*-I buffer). Elution of the bound fraction and counting of radioactivity were as described previously (Yaron et al., 1977).
METHODS
Tilapia a~rea Steindachner were taken from the fish ponds of Ma’ayan Zvi, South of Haifa. The details of fish maintenance and surgical procedures were as described previously (Yaron et al., 1977).
Blood Sampling Blood was sampled from the caudal vessels. Fish were housed singly for 4 hr before bleeding, in order to avoid elevating cortisol levels in the other fish due to disturbance. Plasma cortisol levels increased within 30 min after transfer from tank to tank (70- 105 @ml) but were lower and stable 3, 4, and 4.5 hr thereafter (42.34 ? 3.56, n = 9; 37.73 2 4.66, n = 9: and 45.56 ? 4.26 t&ml, n = 8, respectively). Blood was taken around 1 PM since it was found that around that time cortisol levels in the plasma of this fish were low and stable, while during the night the levels fluctuated in a variable manner.
Cortisol Determination
Evaluation Comparison graphed and
by RIA
Correlation and CPBR.
each into tisol was the other following
TABLE OFCORTISOL
DETERMINATIONS
NONCHROMATOGRAPHED
Fish No. 1
2 3 4 5 6
f tt _t 2 k
2.2” 1.2 2.9 7.2 9.9 14.0
bernaeen
cortisol
determinations
by RIA
Plasma samples of six fish were divided two subsamples. In one subsample the cordetermined directly in duplicates by RIA. In subsample cortisol was measured by CPBR, extraction, chromatography, and elution ac-
1 BY
PL.ASMA
Cortisol determined in plasma without chromatography (nghl) 23.7 49.1 79.8 132.2 150.2 205.7
of cortisol determination in chromatononchromatographed samples. Plasma
samples of six fish were used for comparison of cortisol concentrations before and after chromatography. The plasma sample of each fish was divided into two subsamples. Eight aliquots of one subsample were used for dcrect measurement by RIA. The other subsample was extracted, chromatographed on silica gel sheets with chloroform:ethanol(12:1), and the cortisol was eluted according to Ban and Yaron (1976). Eight aliquots of each eluate were assayed as above. The values obtained from whole plasma samples did not differ from those determined in the cortisol fraction of the respective chromatographed subsamples (Table 1). The correlation coefficient between the two sets of data was Y = 0.977.
All glassware was washed in methanol before use. Plasma samples (0.1 ml) were diluted 1: 10 with borate buffer (0.22 g boric acid, 6 ml methanol, and glassdistilled water up to 60 ml, adjusted to pH 8.0 with NaOH). After thorough mixing, the samples were shaken in a bath at 60” for 30 min. A standard curve was prepared over the range O-1000 pg using standard cortisol (Ikapharm, Israel) dissolved in the RIA buffer containing 1% (w/v) bovine y-globulin (BGG-Sigma). The RIA buffer contained 0.05 M Tris, 0.1 M NaCl, 0.015 M NaN,. and 0.001 M EDTA. The buffer was adjusted to pH 8.0 with HCI. Triplicates (20, 50, or 100 ~1) from the diluted plasma or standard cortisol were placed for assay on small Sephadex G-25 (fine) columns prepared accord-
COMPARISON
of the RIA for Cortisol
Specificity
RIA
IN CHROMATOGRAPHEDAND
OF Tilapia
ourea
Cortisol determined in cortisol fraction after tic (&ml)
11.1 42.6 80.0 160.7 178.6 199.2
? 5 -c i 5 t-
2.4 3.8 3.7 10.0 10.7 12.4
0 Results are expressed as means of 8 determinations -e SEM. Correlation coefficient between the two sets of data. r = 0.977.
CORTISOL cording to Ilan and Yaron (1976) using 1% male rabbit serum as a binding protein. The correlation coefficient calculated for the values obtained by the two methods was r = 0.869 (Fig. 1).
IN
145
Tilapia
used. Since the recovery was lOO%, the sensitivity of the assay was 0.24 and 1.2 nglml in lOO- and 20-~1 aliquots, respectively.
RESULTS
Accuracy The accuracy of the RIA was evaluated by measuring known amounts of standard cortisol (O-200 &ml) added to Tilupia plasma. The correlation coefftcient between the observed and expected values was r =
Plasma Cortisol Level during Temperature-Induced Ovarian Recrudescence (Experiment I)
0.974.
Thirty fish were brought into the laboratory on January 25. The gonadosomatic inThe precision of the assay was determined accorddex (GSI = (ovarian weight x loo/body ing to Abraham (1974). The within-assay variance was weight)) was measured in a sample of 7 fish. 10% or less and the between-assay variance was The rest were kept at 17” on a photoperiod 18.9%. of 12W12D until May 7. During this period Sensitivity fish fed poorly, most of the food offered The sensitivity of the standard curve (s), defined as remaining untouched. In the following 2 the smallest amount of cortisol standard that is signitidays the temperature was raised gradually cantly different from zero at the 95% confidence limit to 28” in all tanks except one with 5 control (Abraham, 1974), was 2.4 pg. The sensitivity of the assay (S) was calculated ac- fish. The fish kept at 28” resumed feeding cording to the equation: within a few days. The GSI was measured in samples of 2-3 fish on May 7, 12, 20, s=sRxF x 100 and 31. The reamining fish were killed on June 1 and their GSI was also measured. where s = the sensitivity of the standard curve, R = percentage recovery, and F = fraction of the sample Plasma cortisol level was determined in samples of 10 fish each, twice while the fish were kept at 17” (February 23 and March 24) and three times after raising the temperature (May 12, 20, and 31, Fig. 2). No change in the plasma cortisol could be detected during the “cold” period, and also 5 days after raising the temperature. Eight days later the plasma cortisol had decreased significantly (P = 0.0002). The level was still low after a further 11 days. An increase in GSI was noted in fish kept at 28” compared with the control fish kept at 17” (Fig. 2). Precision
d
,
Effects of Ovariectomy and Estrogen Administration on Cortisol Level (Experiment II)
1
100
200
ma/ml
RIA
FIG. 1. Correlation between cortisol determinations in the plasma of Tilapia aurea by RIA and by CPBR. CPBR was performed using 1% male rabbit serum in eluates of the cortisol fractions from plasma extract following tic. The correlation coefftcient between the two sets of data was r = 0.869.
In the previous experiment, a decrease in cortisol level coincided with both the increase of water temperature and the growth of the ovary. The present experiment was designed to clarify whether the ovary or the ovarian estrogen in this fish was involved in the decrease of plasma cortisol during the
146
TERKATIN-SHIMONY
ET AL.
al., 1977). Plasma cortisol level did not alter significantly even 64 days after ovariectomy (P > 0.05). In the same fish plasma estradiol was almost undetectable (Yaron et al., 1977). Cortisol levels in the plasma of females treated with estradiol and consequently with elevated estradiol levels (Terkatin-Shimony and Yaron, 1978) did not differ significantly throughout the experiment from those of females injected with the vehicle only (Fig. 3). DISCUSSION The GSI of female Tilapia aurea exposed
FIG. 2. Effect of temperature on GSI and plasma cortisol level in female Tilnpia aurea. Results are expressed as group means or as mean f SEM. Numbers in parentheses = n. (A) GSI of fish kept at 17” (open circle) or following the transfer to 28” (black circles). Initial GSI (open square). (B) Cortisol level in the plasma of fish kept at 17” and following their transfer to 28”.
temperature-induced ovarian recrudescence. Female fish (130-180 g body wt) were collected at the beginning of the breeding season in April and acclimatized to the laboratory for 1 month or more on a 12L:l2D photoperiod at a temperature of 25”, which allows ovarian growth (Fishelson, 1966). Blood was sampled from 28 females on Day 0 at 1 PM. Fish were ovariectomized later on the same day. Blood was sampled again, at the same hour, on Day 9. Estradiol-17p (0.5 mg&sh in 0.2 ml of sesame oil) was injected ip on Day 14 to 13 fish which were separated from 13 oil-injected control fish. The injections were repeated in the afternoon of Days 22, 30, 36, and 43 following ovariectomy. Blood was sampled again at 1 PM on Days 22, 36, 50, and 64. Plasma cortisol level in these females with developed ovaries (GSI = 3.25 r 0.19), i.e., prior to ovariectomy, was 35.0 -+ 3.5 r&ml while estradiol plasma level in these fish was 3.1 t 0.75 &ml (Yaron et
to 28” for 2 or 3 weeks increased significantly compared with that of control fish kept at 17” (Fig. 2A). It is reasonable to assume that estradiol levels have also increased in these fish since a positive correlation between GSI and circulating estradiol occurs in this species (Yaron et al., 1977). The plasma cortisol level, however, decreased in these fish 2 weeks following the exposure to the high temperature. This
0
9
22
36 Days
50
64
FIG. 3. Plasma cortisol level in ovariectomized Tilupiu aurea. Fish were ovariectomized after blood sampling on Day 0. Estradiol-17/3 (0.5 mg in sesame oil) or the vehicle was injected ip on the days marked with arrows. Numbers in parentheses = n. Plasma level of cortisol (mean 2 SEW before ovariectomy stippled bar; following ovariectomy, vertically hatched bar; in estradiol-injected, ovariectomized fish, obliquely hatched bars; and in oil-injected ovariectomized fish, open bars.
CORTISOL
change in cortisol level could be attributed to either the effect of the high temperature per se or to the resulting recrudescence of the ovary. A relationship between the ovary, or ovarian hormone, and cortisol level and dynamics has been reported in several migratory teleosts. The concentration of 17hydroxycorticosteroids in the plasma of spawning Oncorhynchus tshawytscha was considerably higher than in the prespawning fish (Hane and Robertson, 1959). The plasma cortisol of the Chilko-Lake sockeye salmon with GSI of 15.9 was 31.7 pg/lOO ml, more than ninefold the level found in fish with smaller ovaries (GSI = 9.1; Schmidt and Idler, 1962). The cortisol secretion rate of sexually mature sockeye salmon at rest was more than double the rate before the breeding season (Donaldson and Fagerlund, 1970). The administration of estradiol to ovariectomized 0. nerka resulted inter alia in the increase of both cortisol secretion rate and plasma concentration in resting fish (Donaldson and Fagerlund, 1969). Histological observations confirmed the positive correlation between ovarian maturation and the activity of the interrenal tissue in 0. nerka (McBride and van Overbeeke, 1969). Moreover, injection of estradiol to ovariectomized fish resulted in morphological characteristics of interrenal hyperfunction (van Overbeeke and McBride, 1971). In Tifapia aurea, however, neither ovariectomy nor the administration of estradiol, the ovarian estrogen of this species (Yaron et al., 1977; Terkatin-Shimony and Yaron, 1978), to ovariectomized fish has resulted in any significant change in the plasma cortisol concentration (Fig. 3). It seems reasonable, therefore, to assume that the change in cortisol level during the temperature-induced ovarian growth was not due to the growing ovary nor to the presence of estradiol. Unpublished experiments in our labora-
IN
147
Tilapia
tory on cortisol clearance rates in T. aurea show that the T,,, of injected [3H]cortisol, estimated from total radioactivity in plasma ethyl-acetate extracts, was 208 min in fish at 17”, but only 132 min in fish at 28”. The difference in plasma cortisol concentrations following transfer of fish from 17 to 28” in Experiment I can be attributed to the different clearance rates of the steroid at these temperatures, provided however, that the secretion rate of cortisol did not change. It should be emphasized that the fish which were kept at 17” and had high cortisol plasma levels were feeding quite sparingly and in this respect were in a similar situation to the migrating salmonids and eels. High cortisol levels in fasting fish probably reflect a “physiological stress during which an increased secretion of corticosteroids would be expected, related to the mobilization and metabolism of protein and fats and their conversion to carbohydrates” (Woodhead, 1975; see also Butler, 1973; Chan and Woo, 1978). In the migrating salmonids and eels (see Donaldson and Fagerlund, 1969) it is quite difficult to separate the effect of the growing ovary on interrenal activity from that of fasting during active migration. In T. aurea, however, fasting occurs at low temperature while ovarian growth is stimulated in warm water. In this freshwater fish and also in the marine fish, Pleuronectes platessa (Wingfield and Grimm, 1977), cortisol levels seem to be independent of ovarian development and are possibly associated with the state of nutrition. Nevertheless, the possibility that the clearance rate of the steroid plays a major role in controlling cortisol plasma level cannot be excluded. ACKNOWLEDGMENTS We wish to thank Professor H. R. Lindner and Dr. Fortune Kohen, Department of Hormone Research, The Weizmann Institute of Science, Israel, for the gift of the anti-cortisol serum used in this study. We also wish to thank Dr. A. S. Grimm and Dr. R. T. Duggan, Department of Zoology, University College of North Wales, for reading the manuscript.
TERKATIN-SHIMONY
148
This research was supported by a grant from the United States-Israel Binational Science Foundation (BSF) Jerusalem, Israel
REFERENCES Abraham, G. E. (1974). Radioimmunoassay of steroids in biological materials. Acfa Endocrinologica 75, (Suppl. 183), 23-28. Bassett, J. M., and Hinks, N. T. (1969). Micro determination of corticosteroids in ovine peripheral plasma: Effect of venipuncture, corticotrophin, insulin and glucose. J. Endocrinol. 44, 387-403. Butler, D. G. (1973). Structure and function of the adrenal gland of fishes. Amer. Zool. 13, 839-879. Chan, D. K. O., and Woo, N. Y. S. (1978). Effect of cortisol on the metabolism of the eel, Anguilla japonica.
Gen.
Comp.
Endocrinol.
35, 205-215.
Donaldson, E. M., and Fagerlund, U. H. M. (1969). Cortisol secretion rate in gonadectomized female sockeye salmon (Oncorhynchus nerka): Effects of estrogen and cortisol treatment. J. Fish. Res. Bd. Canad.
26, 1789-
1799.
Donaldson, E. M., and Fagerlund, U. H. M. (1970). Effect of sexual maturation and gonadectomy at sexual maturity on cortisol secretion rate in sockeye salmon Bd. Canad.
(Oncorhynchus 27, 2287-2296.
nerkat.
J. Fish.
Res.
Fishelson, L. (1966). Cichlidae of the genus Tilapia in Israel. Bamidgeh Bull. Fish Cult. Isr. 18, 67-80. Hane, S., and Robertson, 0. H. (1959). Changes in plasma 17-hydroxy-corticosteroids accompanying sexual maturation and spawning of the Pacific salmon (Oncorhynchrts tshawytscha) and rainbow trout (Salmo gairdnerii). Proc. Nat. Acad. Sci. USA 45, 886-893. Idler, D. R., and Truscott, B. (1972). Corticosteroids in fish. In “Steroids in Nonmammalian Vertebrates” (D. R. Idler, ed.), pp. 126-252. Academic Press, New York/London.
ET AL.
Ilan, Z., and Yaron, Z. (1976). Stimulation of carp interrenal function by adrenocorticotrophin. J. Endocrinol. 68, 13-20. McBride, J. R.. and van Overbeeke. A. P. (1969). Hypertrophy of the interrenal tissue in sexually maturing sockeye salmon (Oncorhynchus nerka) and the effect of gonadectomy. J. Fish. Rex Bd. Canad.
26, 2975-2985.
Robertson, 0. H., and Wexler, B. C. (1959). Hyperplasia of the adrenal cortical tissue in Pacific salmon (Genus Oncorhynchus) and rainbow trout (Salmo gairdnerii) accompanying sexual maturation and spawning. Endocrinology 65, 225 -238. Schmidt, P. J., and Idler, D. R. (1962). Steroid hormones in the plasma of salmon at various states of maturation. Gen. Camp. Endocrinol. 2,204-214. Terkatin-Shimony, A., and Yaron, Z. (1978). Estrogens and estrogenic effects in Tilapiu aurea (Cichlidae, Teleostei). Ann. Biol. Anim. Biochem. Biophys. 18, 1007- 1012. van Overbeeke, A. P.. and McBride, J. R. (1971). Histological effects of 1 I-ketotestosterone, 17 a-methyltestosterone, estradiol, estradiol cypionate, and cortisol on the interrenal tissue, thyroid gland, and pituitary gland ofgonadectomized sockeye salmon (Oncorhynchus nerka). 1. Fisk. Res. Bd. Canad.
28, 477-484.
Wingfield. J. C.. and Grimm, A. S. (1977). Seasonal changes in plasma cortisol, testosterone and oestradiol-17P in the plaice, Pleuronectes platessa L. Gen. Camp. Endocrinol. 3 1, I - 11. Woodhead, A. D. (1975). Endocrine physiology of fish migration. Oceanogr. Mar. Biol. Annu. Rev. 13, 287-382. Yaron. Z.. Terkatin-Shimony, A., Shaham, Y., and Salzer, H. (1977). Occurrence and biological activity of estradiol-17P in the intact and ovariectomized Tilapiu aurea (Cichlidae, Teleostei). Gen. Comp.
Endocrinol.
33, 45-52.
AND
COMPARATIVE
ENDOCRINOLOGY
40, 143- 148 (1980)
Relationship between Temperature, Ovarian Recrudescence, and Plasma Cortisol Level in Tilapia aurea (Cichlidae, Teleostei) AVIVA *Department Israel, and
TERKATIN~HIMONY,* of Zoology, tDepartment
The George of Biological
Z. ILAN,*
Z. YARON,*
S. Wise Center for Sciences, Murray
AND D. W. JOHNSON?
Life Sciences, Tel-Aviv University, State University, Murray. Kentuchy
Tel-Aviv, 42071
Accepted August 17, 1979 An RIA procedure for measuring cortisol in the circulation of Tilapia aurea is described and evaluated. Cortisol, chromatographically purified from the fish plasma, accounted for its total immunoreactivity. The correlation between observed and expected cortisol values in the accuracy test was r = 0.977. The within-assay variance was 1% and the between-assays variance was 18.9%. The sensitivity of the whole RIA procedure was 0.24 nglml when the dilution was 1: 100 and 1.2 rig/ml when the dilution was 1:500. Female T. aurea collected in winter and kept at 17” had regressed ovaries, refused food, and their plasma cortisol levels at rest, measured after 1 and 2 months in captivity, were 74 + 3.5 and 65 2 6.1 rig/ml, respectively (mean f SEM). Two weeks after their exposure to 28” the fish resumed feeding, the GSI had doubled, and the plasma resting cortisol level had decreased to 40 ‘- 4.0 &ml. The cortisol level remained low in the subsequent 11 days. Fish collected in the breeding season (April) had a resting cortisol level of 35 2 4.0 rig/ml (mean 2 SEM, n = 28). Nine days following ovariectomy no change could be found in plasma cortisol. Cortisol level in ovariectomized fish injected with estradioL17fi did not ‘differ from that of oil-injected controls. In this nonmigratory, freshwater fish there is no relation between either the state of the ovary, or ovarian hormone, and the level of cortisol. The high level of plasma cortisol in fish exposed to low temperature may be attributed either to the slow rate of cortisol clearance or to the high activity of the interrenal tissue in the fasting fish.
Hyperactivity of the interrenal gland coincides with ovarian development in several species of migratory teleost. This is reflected in the hyperplasia and hypertrophy of the interrenal tissue in Oncorhynchus tshawytscha, in Salmo gairdnerii (Robertson and Wexler, 1959), and in 0. nerka (McBride and van Overbeeke, 1969) and in a high cortisol concentration in the plasma of 0. nerka during the spawning migration (Schmidt and Idler, 1962). Sexual maturation in 0. nerka is accompanied by an increase in the volume of distribution and metabolic clearance rate of cortisol, as well as by the secretion rate of this steroid (Donaldson and Fagerlund, 1970). The mechanism underlying the interrenal-ovarian relationship was attributed on one hand to the possible direct effect of estrogen on the interrenal tissue (McBride and van Overbeeke, 1969). Ovariectomy of
maturing salmon was followed by a decrease in the secretion rate and plasma levels of cortisol in resting fish, while estradiol administration reversed the process (Donaldson and Fagerlund, 1969, 1970). On the other hand the increase in plasma cortisol in maturing salmon was attributed to a reduced clearance rate of cortisol rather than to an increase of interrenal activity (reviewed by Idler and Truscott, 1972). In this salmon “migration is accompanied by rapid gonad development and severe depletion of fat and protein and involves sustained as well as vigorous activity. Interpretation of steroid studies on these animals is often difficult because so many things happen at once” (Idler and Truscott, 1972). We have chosen, therefore, a nonmigratory, freshwater fish-Tilapia aurea-as a subject for the study of the relationship 143 00166480/80/020143-06$01.00/0 Copyright All rights
@ 15’80 by Academic Press. Inc. of reproduction in any form reserved.
TERKATIN-SHIMONY
144
between temperature change, ovarian crudescence, and cortisol level. MATERIALS
AND Fish
ET AL.
re-
ing to Bassett and Hinks (1969). RIA buffer, 100 ~1, containing BGG was added to each column. Rabbit anti-cortisol-21-bovine serum albumin (BSA) serum, diluted with the RIA buffer to the concentration required to bind 35% of radioactive cortisol, was also added to each column (100 ~1). The serum was a gift from Professor H. Lindner and Dr. Fortune Kohen, Department of Hormone Research, The Weizmann Institute of Science. Rehovot, Israel. After 10 min equilibration at room temperature, [ 1,2,6,7(n)3H]cortisol (The Radiochemical Centre, Amersham, sp act 82 Ciimmol) was added to each column (10 nCi/lOO /*-I buffer). Elution of the bound fraction and counting of radioactivity were as described previously (Yaron et al., 1977).
METHODS
Tilapia a~rea Steindachner were taken from the fish ponds of Ma’ayan Zvi, South of Haifa. The details of fish maintenance and surgical procedures were as described previously (Yaron et al., 1977).
Blood Sampling Blood was sampled from the caudal vessels. Fish were housed singly for 4 hr before bleeding, in order to avoid elevating cortisol levels in the other fish due to disturbance. Plasma cortisol levels increased within 30 min after transfer from tank to tank (70- 105 @ml) but were lower and stable 3, 4, and 4.5 hr thereafter (42.34 ? 3.56, n = 9; 37.73 2 4.66, n = 9: and 45.56 ? 4.26 t&ml, n = 8, respectively). Blood was taken around 1 PM since it was found that around that time cortisol levels in the plasma of this fish were low and stable, while during the night the levels fluctuated in a variable manner.
Cortisol Determination
Evaluation Comparison graphed and
by RIA
Correlation and CPBR.
each into tisol was the other following
TABLE OFCORTISOL
DETERMINATIONS
NONCHROMATOGRAPHED
Fish No. 1
2 3 4 5 6
f tt _t 2 k
2.2” 1.2 2.9 7.2 9.9 14.0
bernaeen
cortisol
determinations
by RIA
Plasma samples of six fish were divided two subsamples. In one subsample the cordetermined directly in duplicates by RIA. In subsample cortisol was measured by CPBR, extraction, chromatography, and elution ac-
1 BY
PL.ASMA
Cortisol determined in plasma without chromatography (nghl) 23.7 49.1 79.8 132.2 150.2 205.7
of cortisol determination in chromatononchromatographed samples. Plasma
samples of six fish were used for comparison of cortisol concentrations before and after chromatography. The plasma sample of each fish was divided into two subsamples. Eight aliquots of one subsample were used for dcrect measurement by RIA. The other subsample was extracted, chromatographed on silica gel sheets with chloroform:ethanol(12:1), and the cortisol was eluted according to Ban and Yaron (1976). Eight aliquots of each eluate were assayed as above. The values obtained from whole plasma samples did not differ from those determined in the cortisol fraction of the respective chromatographed subsamples (Table 1). The correlation coefficient between the two sets of data was Y = 0.977.
All glassware was washed in methanol before use. Plasma samples (0.1 ml) were diluted 1: 10 with borate buffer (0.22 g boric acid, 6 ml methanol, and glassdistilled water up to 60 ml, adjusted to pH 8.0 with NaOH). After thorough mixing, the samples were shaken in a bath at 60” for 30 min. A standard curve was prepared over the range O-1000 pg using standard cortisol (Ikapharm, Israel) dissolved in the RIA buffer containing 1% (w/v) bovine y-globulin (BGG-Sigma). The RIA buffer contained 0.05 M Tris, 0.1 M NaCl, 0.015 M NaN,. and 0.001 M EDTA. The buffer was adjusted to pH 8.0 with HCI. Triplicates (20, 50, or 100 ~1) from the diluted plasma or standard cortisol were placed for assay on small Sephadex G-25 (fine) columns prepared accord-
COMPARISON
of the RIA for Cortisol
Specificity
RIA
IN CHROMATOGRAPHEDAND
OF Tilapia
ourea
Cortisol determined in cortisol fraction after tic (&ml)
11.1 42.6 80.0 160.7 178.6 199.2
? 5 -c i 5 t-
2.4 3.8 3.7 10.0 10.7 12.4
0 Results are expressed as means of 8 determinations -e SEM. Correlation coefficient between the two sets of data. r = 0.977.
CORTISOL cording to Ilan and Yaron (1976) using 1% male rabbit serum as a binding protein. The correlation coefficient calculated for the values obtained by the two methods was r = 0.869 (Fig. 1).
IN
145
Tilapia
used. Since the recovery was lOO%, the sensitivity of the assay was 0.24 and 1.2 nglml in lOO- and 20-~1 aliquots, respectively.
RESULTS
Accuracy The accuracy of the RIA was evaluated by measuring known amounts of standard cortisol (O-200 &ml) added to Tilupia plasma. The correlation coefftcient between the observed and expected values was r =
Plasma Cortisol Level during Temperature-Induced Ovarian Recrudescence (Experiment I)
0.974.
Thirty fish were brought into the laboratory on January 25. The gonadosomatic inThe precision of the assay was determined accorddex (GSI = (ovarian weight x loo/body ing to Abraham (1974). The within-assay variance was weight)) was measured in a sample of 7 fish. 10% or less and the between-assay variance was The rest were kept at 17” on a photoperiod 18.9%. of 12W12D until May 7. During this period Sensitivity fish fed poorly, most of the food offered The sensitivity of the standard curve (s), defined as remaining untouched. In the following 2 the smallest amount of cortisol standard that is signitidays the temperature was raised gradually cantly different from zero at the 95% confidence limit to 28” in all tanks except one with 5 control (Abraham, 1974), was 2.4 pg. The sensitivity of the assay (S) was calculated ac- fish. The fish kept at 28” resumed feeding cording to the equation: within a few days. The GSI was measured in samples of 2-3 fish on May 7, 12, 20, s=sRxF x 100 and 31. The reamining fish were killed on June 1 and their GSI was also measured. where s = the sensitivity of the standard curve, R = percentage recovery, and F = fraction of the sample Plasma cortisol level was determined in samples of 10 fish each, twice while the fish were kept at 17” (February 23 and March 24) and three times after raising the temperature (May 12, 20, and 31, Fig. 2). No change in the plasma cortisol could be detected during the “cold” period, and also 5 days after raising the temperature. Eight days later the plasma cortisol had decreased significantly (P = 0.0002). The level was still low after a further 11 days. An increase in GSI was noted in fish kept at 28” compared with the control fish kept at 17” (Fig. 2). Precision
d
,
Effects of Ovariectomy and Estrogen Administration on Cortisol Level (Experiment II)
1
100
200
ma/ml
RIA
FIG. 1. Correlation between cortisol determinations in the plasma of Tilapia aurea by RIA and by CPBR. CPBR was performed using 1% male rabbit serum in eluates of the cortisol fractions from plasma extract following tic. The correlation coefftcient between the two sets of data was r = 0.869.
In the previous experiment, a decrease in cortisol level coincided with both the increase of water temperature and the growth of the ovary. The present experiment was designed to clarify whether the ovary or the ovarian estrogen in this fish was involved in the decrease of plasma cortisol during the
146
TERKATIN-SHIMONY
ET AL.
al., 1977). Plasma cortisol level did not alter significantly even 64 days after ovariectomy (P > 0.05). In the same fish plasma estradiol was almost undetectable (Yaron et al., 1977). Cortisol levels in the plasma of females treated with estradiol and consequently with elevated estradiol levels (Terkatin-Shimony and Yaron, 1978) did not differ significantly throughout the experiment from those of females injected with the vehicle only (Fig. 3). DISCUSSION The GSI of female Tilapia aurea exposed
FIG. 2. Effect of temperature on GSI and plasma cortisol level in female Tilnpia aurea. Results are expressed as group means or as mean f SEM. Numbers in parentheses = n. (A) GSI of fish kept at 17” (open circle) or following the transfer to 28” (black circles). Initial GSI (open square). (B) Cortisol level in the plasma of fish kept at 17” and following their transfer to 28”.
temperature-induced ovarian recrudescence. Female fish (130-180 g body wt) were collected at the beginning of the breeding season in April and acclimatized to the laboratory for 1 month or more on a 12L:l2D photoperiod at a temperature of 25”, which allows ovarian growth (Fishelson, 1966). Blood was sampled from 28 females on Day 0 at 1 PM. Fish were ovariectomized later on the same day. Blood was sampled again, at the same hour, on Day 9. Estradiol-17p (0.5 mg&sh in 0.2 ml of sesame oil) was injected ip on Day 14 to 13 fish which were separated from 13 oil-injected control fish. The injections were repeated in the afternoon of Days 22, 30, 36, and 43 following ovariectomy. Blood was sampled again at 1 PM on Days 22, 36, 50, and 64. Plasma cortisol level in these females with developed ovaries (GSI = 3.25 r 0.19), i.e., prior to ovariectomy, was 35.0 -+ 3.5 r&ml while estradiol plasma level in these fish was 3.1 t 0.75 &ml (Yaron et
to 28” for 2 or 3 weeks increased significantly compared with that of control fish kept at 17” (Fig. 2A). It is reasonable to assume that estradiol levels have also increased in these fish since a positive correlation between GSI and circulating estradiol occurs in this species (Yaron et al., 1977). The plasma cortisol level, however, decreased in these fish 2 weeks following the exposure to the high temperature. This
0
9
22
36 Days
50
64
FIG. 3. Plasma cortisol level in ovariectomized Tilupiu aurea. Fish were ovariectomized after blood sampling on Day 0. Estradiol-17/3 (0.5 mg in sesame oil) or the vehicle was injected ip on the days marked with arrows. Numbers in parentheses = n. Plasma level of cortisol (mean 2 SEW before ovariectomy stippled bar; following ovariectomy, vertically hatched bar; in estradiol-injected, ovariectomized fish, obliquely hatched bars; and in oil-injected ovariectomized fish, open bars.
CORTISOL
change in cortisol level could be attributed to either the effect of the high temperature per se or to the resulting recrudescence of the ovary. A relationship between the ovary, or ovarian hormone, and cortisol level and dynamics has been reported in several migratory teleosts. The concentration of 17hydroxycorticosteroids in the plasma of spawning Oncorhynchus tshawytscha was considerably higher than in the prespawning fish (Hane and Robertson, 1959). The plasma cortisol of the Chilko-Lake sockeye salmon with GSI of 15.9 was 31.7 pg/lOO ml, more than ninefold the level found in fish with smaller ovaries (GSI = 9.1; Schmidt and Idler, 1962). The cortisol secretion rate of sexually mature sockeye salmon at rest was more than double the rate before the breeding season (Donaldson and Fagerlund, 1970). The administration of estradiol to ovariectomized 0. nerka resulted inter alia in the increase of both cortisol secretion rate and plasma concentration in resting fish (Donaldson and Fagerlund, 1969). Histological observations confirmed the positive correlation between ovarian maturation and the activity of the interrenal tissue in 0. nerka (McBride and van Overbeeke, 1969). Moreover, injection of estradiol to ovariectomized fish resulted in morphological characteristics of interrenal hyperfunction (van Overbeeke and McBride, 1971). In Tifapia aurea, however, neither ovariectomy nor the administration of estradiol, the ovarian estrogen of this species (Yaron et al., 1977; Terkatin-Shimony and Yaron, 1978), to ovariectomized fish has resulted in any significant change in the plasma cortisol concentration (Fig. 3). It seems reasonable, therefore, to assume that the change in cortisol level during the temperature-induced ovarian growth was not due to the growing ovary nor to the presence of estradiol. Unpublished experiments in our labora-
IN
147
Tilapia
tory on cortisol clearance rates in T. aurea show that the T,,, of injected [3H]cortisol, estimated from total radioactivity in plasma ethyl-acetate extracts, was 208 min in fish at 17”, but only 132 min in fish at 28”. The difference in plasma cortisol concentrations following transfer of fish from 17 to 28” in Experiment I can be attributed to the different clearance rates of the steroid at these temperatures, provided however, that the secretion rate of cortisol did not change. It should be emphasized that the fish which were kept at 17” and had high cortisol plasma levels were feeding quite sparingly and in this respect were in a similar situation to the migrating salmonids and eels. High cortisol levels in fasting fish probably reflect a “physiological stress during which an increased secretion of corticosteroids would be expected, related to the mobilization and metabolism of protein and fats and their conversion to carbohydrates” (Woodhead, 1975; see also Butler, 1973; Chan and Woo, 1978). In the migrating salmonids and eels (see Donaldson and Fagerlund, 1969) it is quite difficult to separate the effect of the growing ovary on interrenal activity from that of fasting during active migration. In T. aurea, however, fasting occurs at low temperature while ovarian growth is stimulated in warm water. In this freshwater fish and also in the marine fish, Pleuronectes platessa (Wingfield and Grimm, 1977), cortisol levels seem to be independent of ovarian development and are possibly associated with the state of nutrition. Nevertheless, the possibility that the clearance rate of the steroid plays a major role in controlling cortisol plasma level cannot be excluded. ACKNOWLEDGMENTS We wish to thank Professor H. R. Lindner and Dr. Fortune Kohen, Department of Hormone Research, The Weizmann Institute of Science, Israel, for the gift of the anti-cortisol serum used in this study. We also wish to thank Dr. A. S. Grimm and Dr. R. T. Duggan, Department of Zoology, University College of North Wales, for reading the manuscript.
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This research was supported by a grant from the United States-Israel Binational Science Foundation (BSF) Jerusalem, Israel
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Ilan, Z., and Yaron, Z. (1976). Stimulation of carp interrenal function by adrenocorticotrophin. J. Endocrinol. 68, 13-20. McBride, J. R.. and van Overbeeke. A. P. (1969). Hypertrophy of the interrenal tissue in sexually maturing sockeye salmon (Oncorhynchus nerka) and the effect of gonadectomy. J. Fish. Rex Bd. Canad.
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Wingfield. J. C.. and Grimm, A. S. (1977). Seasonal changes in plasma cortisol, testosterone and oestradiol-17P in the plaice, Pleuronectes platessa L. Gen. Camp. Endocrinol. 3 1, I - 11. Woodhead, A. D. (1975). Endocrine physiology of fish migration. Oceanogr. Mar. Biol. Annu. Rev. 13, 287-382. Yaron. Z.. Terkatin-Shimony, A., Shaham, Y., and Salzer, H. (1977). Occurrence and biological activity of estradiol-17P in the intact and ovariectomized Tilapiu aurea (Cichlidae, Teleostei). Gen. Comp.
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