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G E N E R A L A N D C O M P A R A T I V E E N D O C R I N O L O G Y 76, 53-62 (1989)
Corticosteroid Biosynthesis in the Interrenal Cells of the Teleost
Fish, Oreochromis mossambicus
P. H. M.
B a l m ,*
J. D. G.
L a m b e r t ,t a n d
S. E.
W endelaar
Bon ga*
* Department o f Animal Physiology, University o f Nijmegen, 6525 ED Nijmegen, and tDepartment o f
Experimental Zoology, State University o f Utrecht, 3584 CH Utrecht, The Netherlands
Accepted June 5, 1989
Applying high-performance liquid chromatography and thin-layer chromatography to sep­
arate corticosteroids, vve studied the biosynthesis o f steroids by the interrenal cells o f the
head kidneys (the adrenocortical homolog) of Oreochromis mossambicus. Intact head kid­
neys con ve rte d exogenous
17a-hydroxyprogesterone into m a in ly c o rtiso l, but
11-
deoxycortisol, cortisone, and androstenedione were also recovered from the medium . In ­
cubation of intact tissue with pregnenolone in addition resulted in the formation o f large
amounts of an unidentified product, which was absent in incubations of tissue homogenates
with pregnenolone.
©
1989 Academic Press. Inc.
There is substantial literature on the bio­
synthesis of steroids by the teleost interrenal cells, the adrenocortical homolog of
Ish. In vitro, these cells seem capable of
synthesizing every corticosteroid identified
in the vertebrates (Butler, 1973). Despite
i his apparent in vitro versatility of the tele)st interrenal cells, to date cortisol is con­
sidered the most important corticosteroid
n fish, partly because of the quantities prouced and the range of biological activities
eported, but also because searches for bi>logical activities of some of the other verebrate corticosteroids (corticosterone, alosterone) in teleosts have been unfruitful
Henderson and Garland, 1981; Henderson
and K im e , 1987; Mayer-Gostan et a l.,
1987).
In fo r m a tio n on steroid b io sy n th e tic
outes in teleosts generally has been obained from incubations of head kidney honogenates with labeled precursors (Sangaang et al., 1972; Colombo et al., 1972). A
problem associated with much of the earlier
work is that it ‘'should be examined with
¡he understanding that identifications are at
best tentative” (Butler, 1973) with the
methods available at that time. We there­
fore have reevaluated the biosynthesis of
steroids by the interrenal cells (the adreno­
cortical homolog of fish) of Oreochromis
mossambicus ( tilapia ) by applying highp e rfo rm a n c e liq u id c h ro m a to g ra p h y
(H P L C ) and thin-layer chrom atography
(TLC). This report is part of a study on the
control of synthesis and release of cortico­
steroids by the interrenal cells of teleost
fish.
MATERIALS AND METHODS
Animals
Sexually mature female Oreochromis mossambicus
(formerly Tilapia mossambica and Sarotherodon mos­
sambicus, Cichlidae) were used. They were kept iso­
lated from males at least 2 months prior to the exper­
iments to reduce reproductive activity (Silverm an,
1978). W hen sacrificed, the animals usually were 7
months old and sexually mature, with an average body
weight of 20 g, unless indicated otherwise.
Fish were kept in 120-liter freshwater aquaria under
constant aeration and filtration (E h e im a q u a riu m
pumps). The water (for composition, see W endelaar
and van der Meij, 1981) was renewed regularly. The
temperature was maintained at 28°. The fish were kept
under a 12L/12D regimen (light on: 7:00
a m
)
and were
fed Tetramin tropical fish food.
Anim als received their last meal 24 hr before sacri­
0016-6480/89 $1.50
Copyright © 1989 by Academic Press, Inc.
All rights of reproduction in any form reserved
54
BALM, LAMBERT, A N D W E N D E L A A R BONGA
fice. Fish were netted rapidly and killed by spinal dis­
Thin-Layer Chromatography
section. In view o f the reported daily rhythmicities in
plasma cortisol levels in fish (Spieler, 1979; Spieler
Before sample extraction, 25 fig of each o f the fol­
and Noeske, 1981; Ranee et al., 1982; Pickering and
lowing carriers was added: pregnenolone (P5), proges­
Pottinger. 1983; Nichols and Weisbart, 1984) care was
terone (P4), 17a-hydroxypregnenolone (17a-Ps), 17a-
taken to sacrifice all animals at 9:00
hydroxyprogesterone (17a-P4), 11-deoxycorticoste­
a m
.
Head kidneys
were excised and processed for incubation.
rone, corticosterone, 11-deoxycortisol (“ co m po un d
S ” ), cortisol, and cortisone. The steroids were ex­
tracted from the sample o f m edium or homogenate
with dichloromethane (3 x 10 ml). To remove the ex­
Incubations
cess nonsteriodal lipids, residues were extracted with
0.5 ml hexane and then with acetone (6 x 1 ml). The
Homogenates of head kidneys were prepared in a
extracts were stored overnight (- 2 0 °) to precipitate
sucrose buffer (0.25 M sucrose, 0.1 M sodium phos­
apolar lipids. These lipids were centrifuged (10 min,
phate buffer, pH 7.4; 1/2 w/v; 0°) with an all-glass
800g, 0°), washed twice with acetone (- 2 0 °), and then
homogenizer. The homogenate was centrifuged (10
discarded. The combined supematants were evapo­
min, 800 g, 2°, Beckman TJ-6) to prepare a cell-free
rated and the residue (dissolved in a few drops of
system. The supernatant (I ml) was transferred to a
d ic h lo ro m e th a n e - m e th a n o l 9/1) was subjected to
glass vial containing [3H)pregnenoIone (3 p.Ci in 0.5 ml
T L C . Thin-layer chromatography was carried out on
propyleneglycol), 2 m M N A D , 2 mM N A D P H , and
precoated plates with silica gel F 254 (Merck) in satu­
sodium phosphate buffer (0.1 M, pH 7.4). Incubation
rated tanks. Three systems were used. In system 1,
volume was 4.5 ml. During the incubations, which
plates were developed in toluene-cyclohexane (1/1;
lasted 3 hr, samples were taken and dichloromethane
3 x ) to separate apolar com pounds (triglycerides) from
was added to terminate steroid bioconversion. Tissue
steroids. In system 2, steroids were separated in ben-
incubations, which lasted 4 hr, were carried out with
zene-ethyl acetate (3/1; 4 x ); and in system 3, in chlo-
various amounts o f tissue (25—400 mg; see legends) in
roform-ethanol (95/5; 2 x ). Carrier and reference ste­
2 ml Dulbecco's modified Eagle medium (M D M ), con­
roids were detected by u'' absorption (in the case of
taining 4 jiCi pHJpregnenolone or 4 p.Ci 17a-|^H]-
3-keto-4-steroids) or by spray ing w ith p rim u lin e
hydroxyprogesterone, both dissolved in 0.2 ml propy­
(Wright, 1971) (other steroids). Radioactive areas on
leneglycol. No cofactors were added. All incubations
T L C plates were located by means o f a Berthold thin-
were carried out at 28° in an air atmosphere under
layer chromatogram scanner.
continuous shaking, and were terminated by adding
dichloromethane.
Reagents
The tritiated compounds were purchased from Am-
Hig h-Pe rforn ia n c e
Liquid Chromatography
ersham U .K .; the specific activity of pregnenolone
was 12.5 Ci/mmol and o f 17a-hydroxyprogesterone, 11
Ci/mmol. Reference steroids were from Steraloids or
Samples o f medium or homogenate (1-2 ml) were
extracted three times with dichloromethane (5 ml).
The organic phase was evaporated, redissolved in 0.5
M a k o r.
NADPH
and N A D
were o b ta in e d from
B o e h r in g e r - M a n n h e im . C h e m ic a ls a n d
s o lv e n ts
(Baker) were o f analytical grade.
ml dichloromethane, and analyzed by H P L C (Spectra
Physics SP 8000, Eindhoven, The Netherlands), which
RESULTS
was equipped with a LiChroSorb Si-60-5 column (250
x 4.6 mm, Chrom pack Middelburg, The Netherlands).
S t e r o id s
w e re
e lu t e d
w it h
d ic h lo r o m e th a n e -
ethanol-water (94/5/1) at a flow rate of 0.9 ml/min. One
hundred fractions (0.2 min) were collected with an
L K B Redirac fraction collector. Time between runs
was 15 min. Four milliliters of scintillation fluid was
added to the fractions and radioactivity was deter­
mined with an L K B Wallac 1216 Rackbeta liquid scin­
tillation counter. Elution times for several reference
corticosteroids were analyzed using com m ercially
available tritiated com pounds. Results are expressed
as the percentage o f total radioactivity for every frac­
tion (% 3H).
The H P L C elution profiles of the steroid
standards (Fig. 1) show that under the con­
ditions employed the method is suitable for
separation of corticosteroids derived from
17a-hydroxyprogesterone: deoxycortisol,
cortisol, and cortisone. When C H 2CL ex­
traction and H P L C were used for steroid
separation, total recovery (radioactivities
in the water phase + H P L C fractions) was
calculated as 92 ± 3% (mean ± S E M for six
samples).
55
C O RTI COST E R O I D O G EN ES IS IN tilapia
% radioactivity
80
17c, P5
P5
17,, P 4
P4
40
h
I
o
•
9
•
"1
9
12
12
6
T -
9
cortlson«
11 d*oxycortl»ol
r—
12
---- *-----
3
6
"y
9
■
1
12
cortisol
40
I
0J
i
\
3
8
9
12
6
I .
12
6
9
12
15
elution tim e
F
ig
.
- mm -
1. H P L C elution profiles for com m ercially available corticosteroid standards:
17aP5,
17a-hydroxypregnenolone; P5, pregnenolone; 17aP4, 17a-hydroxyprogesterone; P4, progesterone.
Tissue Homogenates Supplied
with [' H]Pregnenolone
Steroid H P L C profiles of incubation of
head kidney homogenate with tritiated
pregnenolone for 10, 90, and 180 min are
given in Fig. 2. The peak eluting at 7 min
likely consists of a mixture of steroids
(pregnenolone, 17a-hydroxypregnenolone;
see Fig. 1) and is termed precursor peak. It
should be noted here that products eluting
after 9 min tended to shift to shorter elution
times with consecutive runs. To identify
cortisol, and especially cortisone, stan­
dards were run between sample runs. It is
clear from Fig. 2 that homogenates of tila­
pia head kidneys converted pregnenolone
into one major end product which coeluted
with cortisol. In addition to the two uniden-
tified minor peaks eluting at 17 and 19 min,
the product eluting from the column at 10
min might also qualify as an end product.
Cortisone serves as a likely candidate for
this peak and the rate at which it was pro­
duced seems to decline after 90 min. The
unidentified compounds eluting at 9.2 and
\\g m in apparently represent intermediates of steroidogenesis.
Intact Tissue Supplied
with [3H ]Pregnenolone
Incubation of intact head kidney tissue
with tritiated pregnenolone resulted in a
strikingly different picture (Fig. 3a). Preg­
nenolone again was converted into cortisol
(16.1 min) and cortisone (10.3 min) but the
larger part of the radioactivity was recov-
56
BALM, LAMBERT, AND W E N D E L A A R BONGA
% ra d io a ctivity
8
n
180 min
90 min
10 mm
COMiSO»*
/
COMiSOl
\A
\ ;\
\M.
; %
\
l\
m
•
l\
15
17
\ /'
\
l\
\
o
11
15
19
23
11
15
19
23
11
23
time -minF i g . 2. Head kidney homogenate incubation with [3H)pregnenolone. Head kidney tissue from 4 F W
females (average weight 55 g) was homogenized and prepared for incubation as outlined under M a ­
terials and M ethods. W eight o f the head kidney tissue: 284 mg. In c u b a tio n volum e: 4.5 ml;
[3H]pregnenolone: 3 fxCi. At 180 min, 6.6% of the total radioactivity could be recovered from the water
phase. Arrows indicate elution positions of cortisone and cortisol standards.
ered from a peak eluting at 11.5 min. We
subjected the sample of medium to T LC
and the resulting radiochromatogram is
shown in Fig. 3b. Apparently the H P L C
“ precursor peak" (8.7 min; Fig. 3a) con­
sisted mainly of pregnenolone. No proges­
terone was present. Chromatography of the
peak marked with an asterisk in chloroform-ethanol 95/5 (system 3; l x ) resulted
in separation of 17a-hydroxypregnenolone
( 8 0 % o f th e t o t a l p e a k d p m ) a n d
17a-hydroxyprogesterone (20%). The com ­
pound with the same Rr as deoxycortisol
(Fig. 3b) still behaved as deoxycortisol in
system 3 (not shown). The peak containing
cortisol and cortisone was also redeveloped
in system 3 (2x) and the radiochromato­
gram is in Fig. 3c. We conclude on the basis
of the relative peak areas that the larger
peak from Fig. 3c probably is identical to
the H P L C peak eluting at 11.5 min (Fig.
3a).
After the incubation the remaining tissue
was homogenized and extracted; subse-
quent H P L C analysis (not shown) revealed
that very small amounts (2%) of the newly
synthesized cortisol (m edium + tissue)
were tissue bound, indicating that storage
was negligible, the more since this value
was not corrected for extracellular spaces.
Neither were conjugated steroids stored.
The thin-layer chromatogram of the homog­
enate (not shown) illustrated that preg­
nenolone was the principal intracellular ste­
roid, while 17a-hydroxypregnenolone and
17a-hydroxyprogesterone were present in
much smaller amounts. No progesterone
could be demonstrated in the tissue.
Intact Tissue Supplied
with 17a-[3H]Hydroxyprogesterone
Figure 4 depicts the H P L C profile and
radiochromatograms of the medium after
incubation of head kidney tissue with tritiated 17a-hydroxyprogesterone. Judging
from the H P L C profile it seems that tilapia
interrenal cells converted 17a-hydroxypro-
C O R T I C O S T E R O I D O G E N E S I S IN tilapia
57
% radioactivity
n
8
-
4-
2-
;\
\
\
••••
O11
15
19
23
tim e -m in F i g . 3. Head kidney incubation with [3H]pregnenoIone (240 min), (a) H P L C elution profile of the
medium, (b) T L C radiochromatogram of the medium. T L C was carried out in systems l (3 x ) and in
2 (4 x ). *See Text, (c) T L C radiochromatogram of the shaded area from (b) after chromatography in
system 3 (2x ). At 240 min, 8.5% o f the radioactive products were water soluble and 2.1% o f the newly
synthesized cortisol was in the tissue. The tissue did not contain any conjugated steroids; 25 mg head
kidney tissue was incubated (material from four females, weighing 10 g on average). F, cortisol; E,
cortisone; S, 11-deoxycortisol; T, testosterone; 17aP4, 17a-hydroxyprogesterone; P5, pregnenolone.
gesterone almost exclusively into cortisol,
vvhereas only small amounts of the steroid
were processed to cortisone (10.5 min).
Thin-layer chromatography (systems 2 and
.*), however, revealed the presence of sub­
stantial amounts of deoxycortisol and androstenedione in the medium (Fig. 4b). Tes­
tosterone could not be identified in both
medium and tissue. Important in this re­
spect is that the tissue incubated with preg­
nenolone did not release androstenedione
to the medium as could be evaluated from
TLC of the pregnenolone peak (Fig. 3b) in
system 3 (data not shown). The peak con­
taining cortisol and cortisone could be
shown to contain mainly cortisol (Fig. 4c).
The tissu e a g a in c o n t a in e d m in i m a l
amounts of newly synthesized cortisol (not
shown). Head kidneys incubated with preg­
nenolone as exogenous substrate released
58
BALM, LAMBERT, AND W E N D E L A A R BONGA
% radioactivity
10
origin I
0-
Carrier
steroids
T17o P4
4 -
i
I
origin
\
I
I .A
••
\
t
oJ
• f4
— 1—
15
11
19
23
Carrier
steroids
time-min-
I
E
F i g . 4. Head kidney tissue incubation with l7a-I3H]hydroxyprogesterone (240 min), (a) H P L C
elution profile of the medium, (b) T L C radiochromatogram of the medium. T L C was carried out in
systems l (3 x ) and 2 (4x). (c) T L C radiochromatogram o f the shaded area from (b) after chrom atog­
raphy in system 3 (2x). At 240 min, 1.0% of the radioactive products were water soluble and 1.0% of
the newly synthesized cortisol was tissue bound. The tissue did not contain any conjugated steroids;
26 mg head kidney tissue was incubated (material from four females, weighing 10 g each). F, E, S, T,
17aP4, and P5, as in Fig. 3; A 4, androstenedione.
markedly more conjugated steroids (8.5%
of the total medium radioactivity) than tis­
sue s u p p l ie d w ith 17 a - h y d r o x y p r o gesterone (1.0%).
DISCUSSION
Steroid Biosynthesis
✓
Several authors who incubated teleost
head kidney tissue or homogenates with or
without added substrates in vitro have dem­
onstrated the capacity of the interrenal cells
to synthesize corticosteroids. Reviewing
the older literature (Butler, 1973) it appears
that, although species differences exist, the
teleost interrenal cells synthesize many of
the corticosteroids found in mammals. Be­
cause of the arrangement of the interrenal
C O R T IC O S T E R O ID O G E N E S IS IN tilapia
cells in the head kidneys of most fish it is
not possible to incubate the cell without re­
nal elements (glomeruli and tubules), pig­
ment cells, chromaffin tissue, and hemopoi­
etic elements. In tilapia the picture is rela­
tively simple due to the absence of renal
elements and the predominantly myeloid
nature of the hemopoietic tissue. Nandi and
Bern (1965) incubated tilapia head kid­
neys with or without A C T H and reported
the presence of cortisol, cortisone, 17ahydroxy-, 11-deoxycorticosterone ( = 11deoxycortisol), and 11-dehydrocorticosterone. Seven years later C o lo m b o et
al. (1972) investigated corticosteroidogenesis by tilapia head kidneys from
[l4C]progesterone and identified 17a-hydroxyprogesterone, cortisol, cortisone,
11-deoxycorticosterone, corticosterone,
11-dehydrocorticosterone, 1 lp-hydroxyprogesterone, and 21-deoxycortisol. In this
latter study Colombo et al. used radioactive
progesterone as precursor because their
preliminary results, utilizing [l4C]acetate,
had indicated that progesterone might be an
ntermediate in tilapia head kidney corticosteroidogenesis. Since the data from these
'»reliminary experiments were unconvincng (they obtained only 0.002% conversion
)f [14C]acetate to progesterone), we de­
eded to use pregnenolone rather than pro­
gesterone as radioactive substrate. Preglenolone is an obligatory intermediate in
:orticosteroidofenesis, whereas progester­
one is not.
High-performance liquid chromatogra­
phy has been used to separate and analyze
teroids from the mammalian adrenal cor­
ex (Kessler, 1982; Cavina et al., 1979;
<ose and Jusko, 1979) and teleost plasma
md testis (Huang et al., 1983; MacFarlane,
984). The results of our incubation of 77apia head kidney homogenate with tritiited pregnenolone illustrated the rapid con­
version of pregnenolone into cortisol and
cortisone. After 3 hr, 26 and 13% of the
radioactivity were associated with cortisol
md cortisone, respectively. Other species
59
appear less effective in this respect: a com ­
parable incubation of rainbow trout head
kidney homogenate (using approximately
ten times as much tissue as in the present
study) with pregnenolone yielded only
2.5% cortisol after 150 min (Arai et al.,
1969). Sandor et al. (1967) also observed
only 4% conversion of pregnenolone into
cortisol after a 3-hr incubation of eel head
kidney homogenate.
Intact tilapia head kidney tissue con­
verted pregnenolone not only into cortisol
and cortisone but also into a product eluting
between these two steroids. Thin layer
chromatography of the incubation medium
confirmed the presence of this product with
a polarity between those of cortisol and
cortisone. It was present in the tissue as
well and therefore probably does not repre­
sent a modification of cortisol or cortisone
associated with secretion. Subsequent T LC
in other systems did not reveal the nature of
this product. In relation to this, Patino et al.
(1987) observed that head kidneys of coho
salmon, when supplied with added sub­
strate in vitro, secrete large amounts of an
unidentified steroid. The authors further re­
ported that this peak was absent in medium
when head kidney tissue of coho salmon
was incubated without added substrate.
Goodman (1980), perfusing the head kid­
neys of the eel with tritiated pregnenolone
in situ, also observed an unknown steroid
in the perfusate. Whether or not these prod­
ucts are related to our unidentified steroid
remains to be answered. The fact that this
product was not present in media of incu­
bations with 17a-hydroxyprogesterone in­
dicates that it must have been derived from
either p re g n e n o lo n e or 17a-hydroxypregnenolone. We suggest that its forma­
tion might be due to the large amounts of
pregnenolone (100 nM) supplied to the tis­
sue. Since in vivo the conversion of choles­
terol into pregnenolone is the rate-limiting
step in corticosteroidogenesis, the interrenal cells normally do not experience high
pregnenolone concentrations. This sugges­
60
BALM, LAMBERT, AND W E N D E L A A R BONGA
tion is corroborated by the data on coho
salmon cited earlier (Patino et al., 1987).
In our study it was established that the
principal pathway to cortisol and cortisone
in tilapia proceeds via p re g n e n o lo n e ,
17a-hydroxypregnenolone, and 17a-hydroxyprogesterone rather than via proges­
terone since this steroid was shown to be
virtually absent in both tissue and medium.
Preference for this route has also been es­
tablished for the herring and the Atlantic
salmon (Sangalang et al., 1972). The for­
mation of tritiated cortisol, cortisone, and
11-deoxycortisol by tilapia head kidneys
supplied with labeled 17a-hydroxyprogesterone confirms results obtained by C o­
lombo et al. (1972). Our rate of cortisol pro­
duction, however, was higher than that re­
ported by these authors. Using 26 mg head
kidney tissue we observed some 30% con­
version after 4 hr, whereas Colombo et al.,
incubating 600 mg tissue with approxi­
m a te ly the c o n c e n t r a t io n o f 17a-hydroxyprogesterone we used, obtained a
17% yield of cortisol after 6 hr. They did not
report the formation of any steroids other
than cortisol, cortisone, and 11-deoxycortisol from 17a-hydroxyprogesterone. However, we found that tilapia
head kidneys in vitro apparently are able to
convert 17a-hydroxyprogesterone into androstenedione by cleaving the C-20 and C21 side chains of 17a-hydroxyprogesterone.
No subsequent androgen formation (testos­
terone) could be demonstrated. Arai et al.
(1969) identified small amounts of androstenedione (1% after 30 min) in incubations
of trout head kidney homogenates. Adding
androstenedione as a substrate to their in­
cubations yielded 22% testosterone after
150 min. The biological significance of an­
drogen formation by tilapia interrenal cells
remains questionable since it did not occur
when pregnenolone was provided as a sub­
strate and it might therefore be related to
the high concentration of 17a-hydroxyprogesterone in the in vitro preparation. In
line with this, Huang et al. (1983) did not
observe androstenedione in carp head kid­
neys which had not been supplied with ex­
ogenous substrates.
Storage of Steroids
The results presented indicate that Tila­
pia interrenal cells in vitro do not store
newly synthesized cortisol, either free or
conjugated. Huang et al. (1983) analyzed
plasma and unincubated head kidneys from
carp, and it can be deduced from their data
that in vivo the head ki dneys cont ai n
enough cortisol to raise the plasma concen­
tration by some 50% upon instantaneous to­
tal release. The occurrence and significance
of cortisol storage in teleosts in vivo remain
controversial. Singley and Chavin (1975)
argued that the rise in plasma cortisol they
observed 15 sec after subjecting fish to a
saline stress [although 15 sec probably must
be interpreted as 60 sec; see personal com ­
munication in Spieler (1974)] must have
originated from the release of stored hor­
mone. Ashcom (1979) also concluded that
during the first 15 min of acid stress, brook
trout increased their plasma cortisol levels
without appreciable de novo synthesis of
cortisol. It seems unlikely that the rapid
stress-induced rise in plasma cortisol can
be attributed exclusively to the release of
stored cortisol.
In summary, we conclude that cortisol
and cortisone probably are the only two
corticosteroids produced in significant
amounts by tilapia interrenal cells in vivo.
Although Colombo et al. (1972) identified
17-deoxycorticosteroids in incubation me­
dia, we suggest that this is due to their
choice of large amounts of progesterone as
exogenous substrate, a steroid we have
shown to be bypassed when pregnenolone
was converted by tilapia head kidneys.
Nevertheless, species differences may exist
and some teleosts might synthesize aldoste­
rone in vivo (Whitehouse and Vinson, 1975;
Reinking, 1983). Taking into account the
C O R T I C O S T E R O I D O G E N E S I S IN tilapia
low sensitivity of the gill and gut mucosal
corticosteroid receptor of fish to aldoste­
rone and cortisone (DiBattista et al., 1984),
we conclude that cortisol quantitatively and
qualitatively is the most important cortico­
steroid in tilapia. Suggestions have been
put forward that cortisone, although con­
sidered biologically inactive itself, may fa­
cilitate cortisol action at the target level
(Leloup-Hatey, 1974, 1979).
61
I. W . Henderson, Eds.), pp. 471-523. Academic
Press, New York.
Henderson, I. W ., and K im e, D. E. (1987). The adre­
nal cortical steroids. In “ Vertebrate E ndocrinol­
ogy: Fundamentals and Biomedical Im plications”
(P. K. T. Pang and M. P. Schreibman, Eds.), pp.
121-142. Academic Press, New York.
Huang, F-L., Ke, F-C., Hw ang, J-J., and Lo, T-B.
(1983). High-pressure liquid chromatographic sep­
aration of a mixture of corticosteroids, androgens
and progestins. Arch. Biochem. Biophys. 225,
512-517.
Kessler, M. J. (1982). Analysis o f steroids from nor­
ACKNOWLEDGMENTS
This study was supported by the Foundation for Bi­
ological Research (B IO N ), which is subsidized by the
Dutch Organization for the Advancem ent of Scientific
Research (N W O ). The authors are indebted to Mrs.
E.
M. Jansen-Hoorweg, Mrs. J. G rannem an, and Mr.
F. A. T. Spanings for secretarial and technical assis­
tance.
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