AMER. ZOOL., 13:839-879 (1973).
Structure and Function of the Adrenal Gland of Fishes
DAVID GORDON BUTLER
Department of Zoology, University of Toronto, Toronto 181, Ontario, Canada
SYNOPSIS. The structure and distribution of the adrenal gland in fishes is reviewed.
Studies on the in vitro and in vivo biosynthesis of adrenocortical steroids and their
occurrence in the blood of fishes are evaluated in the light of modern techniques for
the identification and quantification of steroids. There follows an appraisal of some
literature dealing with the adrenocortical control of intermediary metabolism and ion
transport in fishes.
and Mazzocchi, 1970). Recently Seiler et al.,
(1970) examined the presumptive adrenoLiterature on the adrenal gland of the cortical tissue in Lampetra planeri and P.
Cyclostomata is scanty, and a complete de- marinus. The cells contained saturated lipid,
scription of the adrenocortical and chro- phospholipid, and cholesterol, but there
maffin tissue of jawlessfishesis lacking. Gia- was no evidence for A-5-3/J-hydroxysteroid
comini (1902) reported that adrenocortical dehydrogenase, a finding later confirmed
tissue of Petromyzon marinus comprised by Hardisty (1972) and Youson (1972). The
small groups of cells scattered near and in cytoplasm contains lipid droplets, smooth
the mesonephros from the cardiac region to endoplasmic reticulum, and vesticular to
the cloaca. These small lobules comprised tubular cristae (Hardisty and Baines, 1971;
polyhedral cells which were osmiophilic and Youson, 1972). In general, these features
contained lipid droplets, both generally typify the adrenocortical steroid-secreting
characteristic of the adrenocortical cells of cells, not only of higher bony fishes (Yamahigher vertebrates. Chromaffin (medullary) moto and Onozato, 1965; Ogawa, 1967), but
cells were set apart from the adrenocortical also of higher vertebrates (for amphibians
cells and distributed near the aorta and see Berchtold, 1969; Picheral, 1970; for repcardinal veins (see also Hardisty, 1972). Gas- tiles see Dufaure, 1970; for mammals see
kell (1912) confirmed Giacomini's account Rhodin, 1971).
of the form and distribution of chromaffin
The only report of presumptive adrenocells in Petromyzontia. Sterba (1955) showed
that in P. planeri, the presumptive adreno- cortical cells in the other extant suborder
cortical cells are derived from coelomic of the jawless fishes, the Myxinoidea, is that
epithelium and that they are responsive to of Chester Jones et al. (1962). These authors
mammalian ACTH, a finding that was not did a preliminary investigation by cutting
confirmed by Hardisty (1972). Nevertheless serial sections of whole Myxine glutinosa L.
Youson (personal communication) has ob- and reported that "cells similar in appearserved that treatment with mammalian ance to these in Lampetra were invested in
ACTH is followed by a decrease in cyto- the walls of the cardinal veins."
plasmic lipid droplets and an increase in
In elasmobranch fishes the adrenocortical
numbers of mitochondria and amount of tissue becomes organized into a single comsmooth endoplasmic reticulum in adreno- pact gland which is located on and between
cortical cells from P. marinus, changes the kidneys and is therefore referred to as
which typify hyperadrenocorticalism in, the interrenal. It can, on the basis of form,
for example, rat adrenal cells (Nussdorfer be assigned to one of three groups, the rodshaped type (e.g., Scyllium catulus), the
The author gratefully acknowledges the help of horse-shoe type (e.g., Raja batis) or the comMr. Hilary Ko in preparing the manuscript. This
work was supported by the National Research Coun- pact type (e.g., Torpedo marmorata) (Kisch,
1928; Chester Jones, 1957). On closer examcil of Canada.
HISTOLOGY OF THE ADRENAL GLAND OF FISH
839
840
DAVID GORDON BUTLER
ination, one finds that the adrenocortical
cells of mesoblastic origin are loosely arranged in cords. Groups of cords are often
organized into lobules which, in turn, are
surrounded by connective tissue and separated by capillaries and sinusoids that course
between them. The adrenocortical cells are
either round or polygonal, possessing large
nuclei and basophilic chromatin (Chester
Jones, 1957). Cytoplasmic lipoidal droplets
are present as in adrenocortical cells of
higher vertebrates (Aboim, 1939, 1944,
1946). As in cyclostomes, the chromaffin tissue is separated from the adrenocortical
tissue (Vincent, 1922) and is organized to
form chromaffin bodies which are closely
associated with the sympathetic ganglia
(Sacarrao, 1944).
In the Class Osteicthyes (higher bony
fishes), members of the superorders Chondrostei and Holostei have received only
superficial treatment as regards the adrenal.
Idler and O'Halloran (1970) reported that
the adrenocortical tissue in the Atlantic
sturgeon (Acipenser oxyrhynchus) forms
compact yellow bodies which are scattered
along the entire length of both kidneys and
along the postcardinal veins to their point
of entry to the heart. They are formed by
cords of cells and permeated by blood sinuses. Each cell possesses a large, round nucleus and granular cytoplasm and gives a
positive histochemical reaction for 3/J-hydroxysteroid dehydrogenase when dehydroepiandrosterone and pregnenolone were
employed as substrates. In the Holostean,
Amia calva the adrenocortical cells, arising
from coelomic epithelium, are not organized
into strands, but form compact cell groups
devoid of blood sinuses, as in the Acipenseridae, in which they are found along the
walls of the right and left postcardinal veins
(De Smet, 1962). Chromaffin cells of cubical
or slightly cylindrical shape and enlarged
nucleus were observed to form a layer one
or two cells in thickness under the endothelium which wraps the anterior veins,
especially the right anterior cardinal. These
cells were thought to be homologous with
the chromaffin cells of the fish of the superorder Teleostei (D'Ancona, 1955; De Smet,
1962).
Here, for the first time, adrenocortical
and chromaffin cells are accompanied by a
third group of cells which are derived from
the distal portion of the pronephric duct
as well as the mesonephric tubules and organized into the so-called corpuscles of Stannius (Stannius, 1839) which are from 40-50
in number and are imbedded in the kidney
(Garrett, 1942; De Smet, 1962).
In the superorder Teleostei, the cytology
of the adrenocortical and chromaffin tissue
has been relatively thoroughly examined
(Baecker, 1928; Callamand, 1943; Aboim,
1946; Rasquin, 1951; Olivereau and Fromentin, 1954; Oguri and Hibiya, 1957; Pickford and Atz, 1957; Chester Jones, 1957;
Oguri, 1960a,fc; van Overbeeke, 1960;
Chavin and Kovacevic, 1961; Chavin, 1966;
Olivereau, 1966; Hanke and Chester Jones,
1966).
Adrenocortical cells are often arranged
in cords which form masses or layers which
are invested in the walls of the cardinal
veins and scattered throughout the hemopoietic tissue of the anterior head kidney.
The chromaffin cells are closely associated
with the adrenocortical cells and are very
often interdispersed (Krauter, 1951). Earlier
histochemical tests for cytoplasmic lipid
droplets and for ascorbic acid in teleost
adrenocortical cells were generally negative
(Rasquin, 1951, Pickford, 1953a; Olivereau
and Fromentin, 1954; Oguri, 1960&). However, chemical methods (Hatey, 1952; Fontaine and Hatey, 1954&) did show the presence of ascorbic acid in the adrenocortical
tissue from European eels and rainbow
trout. Chavin (1966), using histochemical
methods, has reported that adrenocortical
cells in a number of fresh-water and seawater teleosts contain significant amounts
of lipid droplets, cholesterol, and ascorbic
acid. Moreover, European and conger eel
adrenocortical cells give a positive histochemical test for 3-/?-ol-dehydrogenase (Chieffi and Botte, 1963).
The Stannius corpuscles which first appeared in the chondrosteans are also found
in the Teleostei (Garrett, 1942; Bauchot,
1953). They vary in number and originate
as protrusions from the pro- and mesonephric ducts (Huot, 1898; Giacomini, 1933;
ADRENAL GLAND OF FISHES
Ford, 1959; De Smet, 1962; Krishnamurthy
and Bern, 1969) and finally become lodged
in the ventral surface of the mesonephric
kidney. These compact, ductless glands are
formed of a number of ovoid and polymorphic lobules consisting of radially oriented
columnar secretory cells. Each of these cells
is supplied with as many as two (Krishnamurthy and Bern, 1969), types of round
cytoplasmic granules and rough-surfaced
endoplasmic reticulum (Oguri, 1966; Fujita
and Honma, 1967; Ogawa, 1967; Krishnamurthy and Bern, 1969). These cells are of
the type that may synthesize and/or secrete
protein, and they have some features in common with cells of the exocrine pancreas
(see Palade, 1956; Ristow and Piepho, 1963).
They do not, however, appear to be similar
to the adrenocortical cells of higher vertebrates in which one finds a smooth endoplasmic reticulum, numerous mitochondria
with tubular cristae (Berchtold, 1969; Dufaure, 1970; Rhodin, 1971; Nussdorfer and
Mazzocchi, 1970). Earlier histochemical
studies failed to demonstrate the presence
of steroid 3/?-ol-dehydrogenase in the Stannius corpuscles of European (Anguilla anguilla) and conger {Conger conger) eels
(Chieffi and Botte, 1963). One must not ignore the fact that corpuscular cells sometimes contain sudanophilic droplets (Bobin,
1949; Ogawa, 1963; Krishnamurthy, 1968)
and ascorbic acid (Fontaine and Hatey,
1955; Krishnamurthy, 1968), both traditional criteria for identifying adrenocortical cells in higher vertebrates.
ADRENOCORTICOSTEROIDS IN FISH
841
Phillips et al. (1962) later reported that the
serum of Atlantic hagfish (M. glutinosa L.)
contained corticosterone (27 fig/100 ml) and
cortisol (10 fig/100 ml). Weisbart and Idler
(1970), employing the improved technical
methods that had been developed in the
intervening period, re-examined the problem. They found that the Atlantic hagfish
plasma contained not more than 0.022 fig
cortisol/100 ml and 0.013 yug corticosterone/
100 ml and that the plasma of marine lampreys contained 0.005 fig cortisol, 0.002 fig
corticosterone, and 0.002 fig cortisone/100
ml. These authors concluded that "the
amount of radioactivity associated with
these nanogram levels is too low to accept
these values as representing authentic corticosteroids." However, in the same year (Idler et al., 1971) became more confident that
cortisol, cortisone and corticosterone were
actually present in hagfish serum when they
observed that serum cortisone and cortisol
concentrations (double isotope derivative
assay) increased following the injection of
a total of 60 IU of porcine ACTH over a
period of 10 days. 11-deoxycortisol, not detected in serum from the saline controls, was
present in hagfish injected with ACTH
(0.27/ig/100 ml).
Presumptive adrenocortical tissue from
Atlantic hagfish and marine lampreys failed
to convert labelled progesterone to cortisol,
cortisone, or corticosterone in vitro; however, the incubates did yield some labelled
17 a-hydroxy progesterone. The presumptive
adrenocortical cells in M. glutinosa did
not produce corticosteroids in vitro (Weisbart and Idler, 1970).
Class Agnatha (jawless fishes):
Class Chondricthyes (cartilaginous fishes)
Myxinoidea (Subclass Cyclostomata, Orders
In cartilaginous fishes, adrenocortical
Myxiniformes and Petromyzonttform.es)
tissue is organized into a relatively compact
The first attempt to identify adrenocor- gland whose shape varies according to the
tical steroids in blood of any representative species. It is normally located between the
of the Class Agnatha was made by Phillips two lobes of the posterior kidney and for
(1959). He reported that Pacific hagfish this reason was named the interrenal (Bal(Polistrema stouti) plasma contained 9.6 ^.g four, 1878). This term should only be used
of cortisol/100 ml and 14.0 ^g of cortico- for the adrenocortical homologue in cartisterone/100 ml. Fresh-water P. marinus laginous fishes because, in the jawless fishes
plasma contained 47 /jg cortisol/100 ml and and the higher bony fishes, the adrenocor4.4 //.g of corticosterone/100 ml (male fish). tical tissue is distributed randomly through-
842
DAVID GORDON BUTLER
out the kidney and near the postcardinals
and is not actually interrenal tissue (see also
Fontaine, 1963).
(Bern et al., 1962). Idler and Truscott (1968)
reported that the average concentration of
la-hydroxycorticosterone in the blood of
the thorny skate (Raja erinacea) was 2.1 ±
0.5 /tg/100 ml. Significant concentrations of
Subclass Elasmobranchi (sharks, skates,
this steroid were found in the perivisceral,
and rays)
pericardial, and cranial fluids. In evaluating
Phillips and Chester Jones (1957) found the earlier reports of the presence of 17that a pooled plasma sample from male and hydroxylated steroids in elasmobranch fishes
female Raja clavata contained corticoste- (Phillips and Chester Jones, 1957; Phillips,
rone (8.0 /j.g/100 ml), whereas a pooled sam- 1959) which have been extensively reported
ple from both sexes of Scylliorhinus canicula in reviews (Chester Jones et al., 1959, 1969;
contained cortisol (2.5 /j.g/100 ml) but no Chester Jones and Phillips, 1960), one must
cortisone or corticosterone. Two years later, bear in mind that (1) 17-hydroxylated stePhillips (1959) reported the presence of roids have not been identified in the plasma
from any elasmobranch when rigorous chemcortisol and corticosterone in seven of
eight species of elasmobranchs including ical proofs have been applied and (2) intermembers of the orders Selachii (sharks) and renal tissue from a number of species inBatoidei (skates and rays). Plasma of T. cluding Squalus acanthius L., S. canicula L.,
marmorata was assayed for steroids during and Galeus melastomus (Simpson and
various stages of the sexual cycle (Lupo di Wright, 1970) convert labelled precursors
Prisco et al., 1967). These authors found, to la-hydroxycorticosterone, corticosterone,
for example, that cortisol (55.7 ^g/100 ml), and 11-deoxycorticosterone, but not to 17cortisone (17.5 /ig/lOO ml), corticosterone OH steroids. Improvements in experimental
(21.8 /tg/100 ml), and deoxycorticosterone design have not kept pace with the im(5.8 jug/100 ml) were present in the plasma proved methods for detection of steroids in
of immature fish. The high concentrations plasma. Quantitative comparison between
of steroids reported here may, in part, be species are of little value because methods
due to the treatment of plasma samples of capture and condition of the fish at the
with glucuronidase prior to extraction. time of sampling vary considerably.
Values would then represent both unconMore effort has been directed toward injugated and conjugated steroids. A frozen vestigations of the biosynthesis of adrenosample of T. marmorata plasma was shipped cortical steroids in vitro than to their identifrom that laboratory to Dr. Idler. Following fication in blood. Macchi and Rizzo (1962)
extraction, purification, and crystallization incubated the interrenal glands from -R.
of the unknowns to constant isotope ratios, erinacea and reported that mammalian
it was found that the Torpedo plasma con- ACTH increased the production of material
tained only 0.082 /xg of la-DH corticoste- which absorbed UV light (240 m^,) and
rone/100 ml and no detectable amounts of which reacted with blue tetrazolium. Bern
corticosterone or 17-hydroxylated steroids et al. (1962) incubated interrenal tissue
(Truscott and Idler, 1972). These authors from dogfish (S. acanthius), skates (R. rhina)
also failed to detect 17-hydroxylated steroids and ratfish (Hydrolagus colliei) and rein the plasma of three species of selachians ported the presence of aldosterone and corand four species of batoids (Truscott and ticosterone in dogfish incubates and aldoIdler, 1972); however, la-hydroxycortico- sterone, cortisol, and corticosterone in skate
sterone was present in the blood of all of incubates. A double isotope dilution derivathese species, and corticosterone and 11- tive assay was used, but the "unknown"
deoxycorticosterone were present either compounds were not purified to constant
singly or together. No attempt was made to isotope ratios, so identification is tentative.
identify aldosterone even though this comIdler and Truscott (1966) found that expound had earlier been tentatively identi- tracts of plasma from R. radiata Donovan
fied in chondricthyean interrenal incubates and R. ocellata, when separated by thin
ADRENAL GLAND OF FISHES
layer chromatography, contained both cortisol and corticosterone at concentrations of
about 0.1 /xg/100 ml and, in addition, an unknown compound more polar than cortisol.
Interrenal tissue from both species, when
incubated with corticosterone, yielded lahydroxycorticosterone. Truscott and Idler
(1968) incubated fresh interrenal tissue from
R. radiata, R. erinacea Mitchell, R. laevis
Mitchell, S. acanthius L., R. clavata L.,
Scyllorhinus stelleris L., frozen-thawed interrenal tissue from Dasyatis violaces Bonaparte, and five species of sharks, Prionace
glauca L., Isurus oxyrhinchus, Carcharhinus
falciformis (Muller and Hanle), Carcharhius obscurus, and Sphyrna lewini (Griffith),
with radio-inert corticosterone or radioactive corticosterone and identified la-hydroxycorticosterone and 11-dehydrocorticosterone in the incubation media. The latter
compound was positively identified in hammerhead shark (S. lewini) incubates. Significant quantities of 11-deoxy cor ticosterone
and corticosterone were identified as metabolites of endogenous precursors in P. glauca.
Methods for detection of sub-microgram
quantities of 17-hydroxylated steroids were
not applied to extracts of the 3-hr pre-incubation fluids for the interrenals of any of
the species studied. These experiments do
not, therefore, exclude the possibility that
17-OH steroids were synthesized in vitro
from endogenous precursors, although this
now seems unlikely in view of the recent
failure to detect 17-OH corticosteroids in
plasmas from a number of elasmobranch
species including some which were used for
these incubation studies (Truscott and Idler,
1972). The first experiments to clearly show
that the interrenal biosynthetic pathways
actually involve transformations through
pregnenolone, progesterone, corticosterone,
11-deoxycorticosterone, and la-hydroxycorticosterone in vitro were done by Simpson
and Wright (1970). Interrenal tissue from
S. acanthius L., S. canicula L. and G. melastomus Rafinesque all produced the above
steroids in vitro, but there was no evidence
for the biosynthesis of either 17-hydroxylated steroids or aldosterone.
843
Subclass Holocephali (Order
Chimaeridae—ratfishes)
Bern et al. (1962) first reported that interrenal tissue from a species of the order
Chimaeridae, the rat fish H. colliei, converted endogenous precursors to cortisol and
aldosterone. It was possible that even if
17-hydroxylating enzymes were not present
in the elasmobranch interrenal, they would
be found in holocephalians. Idler et al.
(1969) incubated interrenal tissue from H.
colliei and tentatively identified cortisol and
corticosterone in the pre-incubation medium. After incubation with 4-14C progesterone, transformation products were tentatively identified as 17-OH progesterone,
cortisol, ll-deoxycortisol, and corticosterone.
When 4-"C corticosterone was added to interrenal incubates, there was no evidence
for conversion of the labelled substrate to
la-OH corticosterone although 32% of the
radioactivity was incorporated into authentic radioactive cortisol. Hydrolagus, one of
the few living members of the holocephalians, apparently lacks la-hydroxylase, an
enzyme which is present in the interrenal
of a large number of elasmobranch species
(Idler etal., 1969).
Class Osteicthyes (higher bony fishes)
Superorders Chondrostei (sturgeons,
paddlefish, Polypterus) and Holostei
(garpike, bowfins)
Sangalang et al. (1971) were the first to
identify adrenocorticosteroids in the blood
of a chondrostean fish, but when present,
the concentrations were relatively low compared with those of teleost fishes. A plasma
sample from sexually immature Atlantic
sturgeon (A. oxyrhynchus Mitchill) contained cortisol (0.181 ju.g/100 ml), cortisone
(0.016 /xg/100 ml), and corticosterone (0.007
^g/100 ml); however, there was no trace of
these steriods in a plasma sample from a
sexually mature Atlantic sturgeon. There
was no published account of the function of
the presumtive adrenocortical tissue in chondrostean fishes until Idler and Sangalang
(1970) incubated (25C) minced adrenocor-
844
DAVID GORDON BUTLER
ber of solvent systems, the following compounds were tentatively identified (^g/100
ml plasma): cortisol, 5.2; cortisone, 6.1;
corticosterone, 7.3; and aldosterone, 0.12;
together with four unidentified compounds.
Idler et al. (1959), using other methods for
purification and identification of steroids,
extracted a pooled plasma sample from migrating sockeye salmon. The following compounds were isolated (/ig/100 ml): cortisol,
11.0, and cortisone, 22.0, but there was no
corticosterone or aldosterone. These authors reported that "a compound with
approximately the same polarity as aldosterone did not exhibit the expected chromatographic behaviour nor biological activity"
(Idler, 1970).
A great many authors have measured
plasma or serum levels of total 17-OH corticosteroids as an index of adrenocortical
activity in teleost fishes (Schmidt and Idler,
1962; Fontaine and Hatey, 1954a; Idler et
al., 1964; Hane and Robertson, 1959; Robertson et al., 1961; Boehlke et al., 1966), but
the data will not be discussed here. In the
last few years the plasma of a number of
species of teleost fishes has been examined
using relatively exhaustive and rigorous
procedures for identification and quantifiSuperorder Teleostei
cation of adrenocortical steroids (see Table
Adrenocortical steroids in blood. A search 1). Weisbart and Idler (1971) analyzed halifor corticosteroids in the blood of teleost but (Hippoglossus hippoglossus L.) plasma
fishes was first performed by Phillips and and found that the cortisol concentration
Chester Jones (1957) who reported that cod ranged from 5.3 to 46.9 /j.g/100 ml and the
plasma contained 1.0 fj.g of cortisol/100 ml. cortisone concentration from 0.28 to 3.2
This work was expanded to include a num- /xg/100 ml. Methods included the DIDA
ber of fresh-water and marine species (Phil- followed by successive recrystallizations to
lips, 1959; Chester Jones et al., 1959; Bondy constant isotope ratios. Owen and Idler
et al., 1957), but the methods for identifica- (1972), using comparable methods, reported
tion and quantification of steroids were in that sea raven (Hemitripterus americanus
the early stages of development. Reviewed Gmelin) plasma also contained cortisol and
from time to time (Chester Jones, 1957; cortisone, but as in the halibut, there was
Chester Jones et al., 1959, 1969; Chester no evidence for aldosterone. Cortisol levels
Jones and Phillips, 1960), these data should ranged from 4.0 to 9.2 ^g/100 ml (four fish)
be examined with the understanding that and cortisone concentrations ranged from
0.7 to 1.5 /zg/100 ml. Fluorimetric, competidentifications are at best tentative.
Phillips et al. (1959) reported that the itive protein binding, and DIDA assays
blood from 364 spawning male sockeye sal- were employed to identify cortisol in killimon (Oncorhynchus nerka) yielded a total fish (Fundiilns heteroclitus) plasma; only
volume of 8,868 ml of plasma. Following the DIDA was used to assay corticosterone
extraction of the plasma and separations (Liversage et al., 1971). These authors found
employing paper chromatography in a num- that a pooled blood sample from intact
tical tissue (yellow bodies) from Atlantic
sturgeons (A. oxyrhynchus) with 16-3H pregnenolone and 4-"C progesterone and observed that cortisol was labelled with 54.3
and 55.1% of the 3 H and "C respectively.
Doubly labelled cortisone, corticosterone,
11-deoxycortisol, 17a-OH progesterone, and
progesterone were all identified as labelled
transformation products, each containing
less than 3% of the original radioactivity.
When 7-3H cholesterol was incubated with
adrenocortical tissue it was converted to
labelled pregnenolone (0.43%), progesterone
(0.091%), 17a-OH progesterone (0.023%),
cortisol (0.061%), cortisone (0.004%), corticosterone (0.001%), and 11-deoxycortisol
(0.047%). There was no evidence for the
occurrence of either 11-deoxycorticosterone
or aldosterone.
Only one holostean fish, A. calva, has
been examined for the presence of adrenocorticosteroids. Idler et al. (1971) reported
that a pooled plasma sample from four male
bowfins contained 0.76 jug cortisol/100 ml,
and 0.11 fx.g of corticosterone/100 ml; the
identification of the latter compound being
tentative only.
TABLE 1. Adrenocortical steroids in fish blood.
Corticosteroids, /ig/100 ml
Species
Experimental conditions
E
B
DOC 18-OH-B Aldo la-OH-B
Class Agnatha (jawless fishes)
ityxine
Maintained iii running sea wa- 0.022
0.013
glutinosa
ter. Blood collected by 3 dif- (tentative identification)
ferent methods—no anesthetic
0.368 N.D.
Myxine
Fish in captivity for 3 yr. 50 0.093 1.38
glutinosa
IXJ porcine ACTH/fish injected every 2nd day for 1 week.
MS222 was used before ACTH
(injected subcutaneously near
region of portal vein and
branchial heart). Blood collected from caudal sinus. Sea
water temp 0.9 to 1.9 C
Control fish injected with 0.1 nil 0.074 0.031 0.023 N.D.
of 0.9% saline on same days
as ACTH injection
Intaet hagfish acclimated 3 days 0.123 0.986 0.023 0.270
in sea water (5.5 C-8.0 C) 20
IU ACTH/fish injected on 1st,
4th, and 7th day. Bled on 10th
day. Values are for serum.
Petromyaon
Caught during May and June 0.005 0.002 0.002
marinus
during spawning migration, (tentative identification)
maintained in fresh water for
1 week before blood collection.
MS222 used; tail cut to obtain blood.
Class Cliondriehthyes (carilaginous fishes)
Torpedo
Female fish blood collected
marvwrata
from ventral aorta.
Raja radiata
Fluids collected immediately after anaesthetizing with MS222.
A
B
C
D
55.70 17.50 21.80
53.90 11.40 42.70
37.30 10.90 58.40
40.03 4.90 1.30
mature
S
A
Author(s)
Criteria for
identification
Weisbart and DIDA, C.I.E.
Idler (1970)
0.339
Idler et al.
(1971)
DIDDA including
crystallization
to C.I.R.
r
O
O
O
*1
Weisbart and
Idler (1970)
DIDA with crystallization to
C.I.E.
Lupo di Prisco identification by
et al. (1967) T.L.C. followed by
G.L.C. (quantitation by G.L.C. and
TJ.V. absorption)
5.80
1.77
0.15
0.38
P 2
PV16
PC 10
C 2
Idler and
Truscott
(1968)
isolated by T.L.C.
and quantified by
acid fluorescence
oo
C
TABLE
1 (continued)
Corticosteroids, /ig/100 ml
Species
Experimental conditions
Class Chondriehthyes (carilaginous fishes)
Peripheral blood collected by
Saja radiata
cannulation of conus arteriosus. Cranial, pericardia!, and
perivisceral fluids removed by
aspiration.
In the lab, fish were held in
Eaja radiata
tanks of running water. Blood
collected by cannulation of
conus arteriosus
Raja oeellata
Snja laevis
Squalus
acanthias
Prionaoe
glauoa
Imrus
oxyrinolms
Torpedo
marmorata
Caught in nets, transported
alive to lab, and bled immediately by cardiac puncture
Caught and bled on board ship
by cardiac puncture
Frozen plasma shipped on dry
ice by air to the laboratory.
E
B
DOC
18-OH-B
Aldo lo-OH-B
S
A
Author (s)
Criteria for
identification
mature
(P = Plasma; PV = Perivisceral fluid; PC = Pericardial fluid;
C = Cranial fluid)
immature, 12 N.M.
days postinterrenalectomy
0.021
immature
N.M.
immature
N.M.
mature
0.018
mature
0.16
mature
N.M.
mature
immature
0.57
2.5
immature
immature
0.033
unknown
N.M.
(serum)
0.052
immature
0.066
0.047
0.14
0.063
0.42
0.043
N.M.
0.70
0.60
0.29
0.57
0.94
0.90
0.016
0.077
2.8
0.92
0.82
N.M.
N.M.
N.M.
N.M.
0.026
0.029
0.054
0.16
0.034
N.M.
_
5.3
N.M.
0.082
N.M.
—
—
—
N.M. —
1.8
Truscott and
Idler (1972)
DIDDA with purification to C.I.R.
(no crystallization)
o
o
w
>>
i
so
(F and E were below detectable levels in all plasma samples)
Class Actinopterygi (higher bony fishes)
Aoipenser
Sexually immature acclimated 4
o&yrhynehus weeks in running sea water.
Blood obtained by cutting
tail. (No anaesthesia)
Mature held for 5 months in
lab. Frozen plasma analyzed.
Amia caXva
F
Experimental details not given.
Values are for serum.
0.181 0.016
0.007 0.008?
.007?
0.0499 —
N.D. 0.003?
N.D.
Sangalang
etal. (1971)
DIDDA involving
crystallization
to C.I.B.
Idler et al.
(1971)
DIDDA involving
crystallization
to C.I.R.
( ? = not crystallized to C.I.R.)
0.76
0.11
TABUS
1 (continued)
Corticoateroida, jig/100 ml
Species
Experimental conditions
F
E
Class Actinopterygi (higher bony fishes)
Fvndulus
+ weighing 7-9 g acclimated 10.7 to N.M.
heteroolitus
1 week after capture to 18 C. 11.9
MS222; cardiac puncture.
B
DOC 18-OH-B Aldo lo-OH-B
S
1.4
Eippoglossus
Pish caught by hook and line;
5.3 to 0.28 to
hippoglossus
maintained from 1 week to 46.9
3.2
several months in miming sea
water 33% salinity. Fishes removed from sea water and
quickly immobilized by stunning with several blows to the
head. Caudal peduncle was cut
and blood collected.
Carassius
auratus
Clupea
harengus
Clupea
harengus
Bemitripterus
americanus
Lepidosiren
paradoxa
7.2
0.8
22 and 23 held at 25 C for 3-4 44.0
4.3
0.11
days after capture. Blood obtained by terminal cardiac
puncture of restrained living
fish. Values are for serum.
0.008 &
2 pooled plasma samples coll. N.M. N.M. N.M. N.M.
N.M.
0.065
July.
N.D.
Pooled plasma from 100 fish— 75.0
7.0
0.06
no sampling methods given.
Adult fish, netted or taken by 7.2
1.1
0.2*
otter trawl, were held in large ±1.0 ±0.2
tanks for at least a fortnight
before use. Blood from 11 2
* Constant isotope ratios in crystals not obtained
was obtained from caudal vessel by severing the tail.
0.03
4 9 lungfish sampled 3-37 days 0.60
N.D. 0.16
0.58
after arrival; MS222, then
cannulation.
A
Author (s)
Criteria for
identification
Laversage
et al. (1971)
DIDDAwith chn>matographic purification to C.I.B.
quantification also by CPB and
fluorimetry
Weisbart and DIDDA involving
Idler (1971)
crystallization
to C.I.B.
Chavin and
Singley
(1972)
quantification by
DIDDA involving
crystallization
to C.I.B.
Truseott and
Idler (1969)
Sangalang
et al. (1972)
Owen and
Idler (1972)
DIDA—crystallization to C.I.B.
DIDA—no exptl.
details
DIDA—crystallization to C.I.B.
Idler et al.
(1972)
DIDA—crystallization to C.I.B.
Purification of a doubly labelled unknown compound to constant isotope ratios (C.I.B.); double isotope derivative assay (DIDA); double isotope dihition derivative assay (DIDDA); common names for steroids are abbreviated as follows: cortisol (F), cortisone (E), corticosterone (B), 11-deoxycorticosterono (DOC), la-hydroxycorticosterone (la-OH-B), 18-hydroxyeorticoeterone (18-OH-B), aldosterone (aldo), 11-deoxycortisol (S), 11-dehydrocorticosterone (A); not detected (N.D.), not measured (N.M.), competitive pTotein binding assay (CPB).
o
o
o
848
DAVID GORDON BUTLER
male and female killifish contained from
10.7 to 11.9 jug/100 ml of cortisol. Corticosterone levels ranged from 0.50 to 1.5 jug/100
ml. These authors made no attempt to measure cortisone.
Truscott and Idler (1969) were the first
to identify aldosterone in the blood of a
teleost fish, based on adequate chemical criteria for a positive identification. Two
pooled plasma samples (July) from Atlantic
herring (Clupea harengus harengus) contained 8 and 65 ng of aldosterone per 100
ml respectively. Two additional pooled samples collected from sexually mature herring
in mid-August of the same year each contained less than 1 ng of aldosterone per 100
ml. Only in the first two samples was the
identification of aldosterone based on purification to constant isotope ratios. Sangalang et al. (1972) re-examined herring
plasma, but were unable to detect aldosterone. Plasma was collected from 100 fish
a few hours after capture, pooled, and extracted. The following adrenocortical steroids were detected using the DIDA (jug/100
ml plasma): cortisol, 75; cortisone, 7; and
corticosterone, 0.06; there being no evidence
for either aldosterone or 18-OH corticosterone. Chavin and Singley (1972) measured
adrenocortical steroid levels in goldfish
(Carassius auratus) serum and found cortisol, 44.0 ju.g/100 ml; corticosterone 7.2 jug/
100 ml; cortisone 4.3jug/100 ml; 11-deoxycorticosterone, 0.8 ^g/100 ml; and aldosterone 0.11 jug/100 ml. Oxidation of aldosterone diacetate and re-crystallization of the
11, 18-lactone-21-acetate to constant isotope
ratios might have strengthened the case for
pldosterone.
In a relatively large number of experiments, adrenocortical steroid levels have
been measured with relatively non-specific
assays, and this in itself does not prove that
the steroids were actually present in the
blood. These include fluorimetric assays
(e.fr., Donaldson and Fagerlund, 1968; Donaldson et al., 1968; Donaldson and McBride,
1967; Butler et al., 1969; Bradshaw and
Fontaine-Bertrand, 1968) and the competitive protein binding assay (CPB) (e.g., Fageilund, 1070; Bradshaw and Fontaine-Bertrand, 1968; Freeman and Idler, 1971;
Hawkins et al., 1971; Ball et al., 1971).
Nevertheless, these assays are practical and
give some insight into changes in adrenocortical activity in relation to changes in the
physiology of the animal.
In much of the experimental work concerning the types and quantities of adrenocortical steroids in fish blood, there has
tended to be an inverse relationship between the sophistication of the assay methods and sound experimental design. Close
scrutiny shows that very little attention has
been paid to details of the methods of capture of fish, temperature of acclimation,
methods for blood sampling, anesthetics
employed, etc. Details are often lacking.
This means that, quite apart from the
strengths and weaknesses of the assay procedure, very little is known about the resting levels in healthy fish in an unstressed
condition.
Biosynthesis of adrenocortical steroids.
Another experimental approach in studying
the physiology of adrenocortical cells in
teleost fishes has been to investigate the
biosynthetic properties of these cells in vitro.
It has been necessary to incubate cardinal
veins and head kidney since the adrenocortical cells are scattered therein; tissue from
the posterior (functional) kidney is often
used as a control.
Earlier research (Phillips and Mulrow,
\959a; Nandi and Bern, 1959, 1960, 1965;
Butler, 1965; Leloup-Hatey, 1966) showed
that, with or without added substrate,
adrenocortical tissue synthesized corticosteroids such as cortisol, cortisone, and corticosterone in vitro. Identification of these
steroids was only preliminary.
Sandor et al. (1966) studied the biogenesis of adrenocortical steroids in European
eel (A. anguilla L.) head kidney (containing
adrenocortical cells) in vitro. When head
kidney was incubated with 16-3H pregnenolone and 4-"C progesterone as substrates,
"double-labelled" transformation products
included progesterone, 17a OH-progesterone, cortisone, and cortisol. A fairly exhaustive search for aldosterone was unfruitful,
even when 4-"C corticosterone was added
as substrate. Nonetheless, these experiments
provided fairly definitive proof, involving
ADRENAL GLAND OF FISHES
849
purification of doubly labelled compounds significance in the intact fish. Radioactive
to constant isotope ratios during successive progesterone should be added as a substrate
re-crystallizations, that eel adrenocortical to test the hypothesis that progesterone is
tissue had transformed labelled substrate transformed directly to 11-deoxycorticoto adrenocortical steroids. Homogenates of sterone.
A. anguilla cardinal veins when incubated
Sandor et al. (1970) further strengthened
(30 C) with 7-3H cholesterol yielded la- the argument for the presence of an 18belled pregnenolone and cortisol as trans- hydroxylating enzyme in teleost adrenocorformation products. The substrates preg- tical tissue (see Arai and Tamaoki, 1967).
nenolone 16-3H and 4-14C progesterone Head kidney homogenates from three spewere transformed to double-labelled pro- cies of teleosts, the cod (Gadus morhna), the
gesterone 11-deoxycortisol, and cortisol. haddock (Melanogrammus aeglefinus), and
There was no indication that the labelled the eel (A. anguilla) all, to one degree or
substrates had been converted to corticos- another, transformed 4-14C corticosterone
terone, aldosterone, or 18-OH corticoster- to small amounts of 18-OH corticosterone,
one in vitro. Earlier, Arai and Tamaoki yet there was no evidence for aldosterone.
(1967) had reported that rainbow trout One questions the physiological significance
(Salmo gairnerii) head kidney had recast of this experiment since corticosterone has
4-14C deoxycorticostcrone into labelled cor- not been isolated from eel incubates (see
ticosterone and 18-OH, 11-deoxycorticos- Sandor et al., 1966). Only cod and haddock
terone in vitro. Truscott and Idler (1968£>) adrenocortical homogenates transformed
incubated 1.04 g of head kidney plus post- 4-14C corticosterone to labelled cortisol.
cardinal vein excised from five Atlantic
Experiments with Atlantic herring adreherring (C. harengus harengus) with 25.2 nocortical incubates were repeated and exixg (1,2-3H) corticosterone (spec. act. 6 fxgj panded (Sangalang et al., 1972), but these
me) and were able to isolate radioactive authors were unable to confirm the earlier
corticosterone and aldosterone as transfor- finding that labelled corticosterone is transmation products. However, the following formed to 18-OH corticosterone and aldopoints should be considered: (1) the per- sterone in vitro. There are, however, some
centage conversion of corticosterone to al- noteworthy differences between the protocol
dosterone was extremely small; (2) no in the earlier experiments (Truscott and
chemical derivatives of aldosterone were Idler, 19686) and those which followed
made at any stage of the chromatographic (Sangalang et al., 1972). For example, in the
purification of the unknown; and (3) only earlier experiment, 25.2 /nCi of 1,2,-3H cor6.5% of radioactivity that was located in ticosterone was added to 1.04 g of head kidthe aldosterone area after the first chroma- ney, whereas presently, 0.2 fj.d of 4-14C
tographic separation was present at the final corticosterone plus 0.5 of yuCi of a second
stage of purification.
labelled substrate (either 11 or 21-deoxyA year later, Arai et al. (1969) published cortisol) was added to a total of 500 mg of
further accounts of their studies on the bio- head kidney tissue. For both herring and
synthesis of adrenocortical steroids by rain- Atlantic salmon (Salmo salar L.) head kidbow trout adrenocortical tissue in vitro. ney incubates, cortisol was the preferred
3
Head kidney incubated (37C) transformed transformation product when 16- H preglabelled pregnenolone to 17a-OH pregnen- nenolene was added as substrate. However,
the added substrate was changed to
olone, U-deoxycortisol, and cortisol; how- when
14
ever, in none of the incubates was there any 4- C progesterone, cortisol remained the
evidence for the labelled biosynthetic inter- major transformation product in the salmediates 11-deoxycorticosterone or cortico- mon, but in herring the major transformasterone. For this reason, the conversion by tion product changed to corticosterone. The
the incubates of 11-deoxycorticosterone to latter finding lends greater significance to
labelled corticosterone and 18-OH, 11-deoxy- the earlier experiments in which corticocorticosterone may be of little physiological sterone was used as a substrate. Colombo
850
DAVID GORDON BUTLER
et al. (1972) reported that in Tilapia mossambica exogeneous progesterone was metabolized by head kidney tissue (adrenocortical
cells) via three possible routes, a major
route involving the 17-hydroxycorticosteroids leading to cortisol and cortisone, a
second route via 17-deoxycorticosteroids
leading to costicosterone and 11-dehydrocorticosterone, and a third including ll/?-hydroxyprogesterone and 21-deoxycortisol.
There was no evidence for the formation of
labelled aldosterone when 4-14C progesterone or 4-14C corticosterone were added as
substrates.
Chavin and Singley (1972) have employed
the DIDA to identify and quantify corticosteroids which were extracted from lyophylized head kidney from goldfish (C. au-
ratus). Compounds identified on the basis of
purification to constant isotope ratios included cortisol, cortisone, corticosterone, 11deoxycorticosterone, aldosterone, 17a-OH
progesterone, and progesterone. These authors thought that extracts of lyophilized
anterior head kidney would provide more
meaningful data about the biosynthetic processes than would studies on in vitro biosynthesis of adrenocortical steroids. The high
steroid concentrations are noteworthy, e.g.,
routes for steroid biosynthesis in teleost fish
13.5 /xg cortisol per g lyophilized head kidney tissue, when one considers that a very
small fraction of this tissue would comprise
adrenocortical cells together with the fact
that the adrenal is an organ of biosynthesis,
not storage. Figure 1 shows the proposed
FIG. 1. Proposed routes for the biosynthesis of
adrenocortical steroids in teleost fishes, a—Anguilla
anguilla (Sandor et al., 1966, 1967, 1970); b—Salmo
gairdneri (Arai and Tamaoki, 1967; Arai et al., 1969);
c—Melanogrammus aeglefmus (Sandor et al., 1970);
d—Gadus morhua (Sandor et al., 1970); e—Clupea
harengus harengus (Truscott and Idler, 1968; Sangalang et al., 1972); f—Salmo salar (Sangalang et al.,
1972); g—Tilapia mossambica (Colombo et al., 1972).
*taoii«fo'>«
TABLE 2. Biosynthesis of corticosteroids by the adrenal gland in fishes.
Species
Tissue
Temp
Substrate
Class Agnatha (jawless fishes)
Myxine glutinosa
posterior cardinal veins 25 C 4-"C-B
and supraintestinal vein
heart tissue incubated
4Cu-17a-OH-prog.
as a control
Petromyzon marinus
posterior cardinal vein
Class Chondriehyes (cartilaginous fishes)
Raja radiata
sectioned interrenal
R. ocellata
glands
Sphyrna lewini*
interrenal tissue sectioned
Prionace glauca*
Dasyatis violaoea*
Squalus acanthius
Carcharhinus obscurus*
C. falciformis*
Isurus oxyrinchus*
R. erinacea
Prionace glauca*
S. lewini*
R. radiata
7-3H-17o-OH-prog.
25 C 4-"C-prog.
26 C 4-"C-B
Corticosteroids in
medium
"C-la-OH-B doubtful
F,S; (DIDA failed to
provide adequate proof
for the presence of
these steroids)
F & S (doubtful)
"C-17a-OH-prog.
no other steroids
4-14C-lo-OH-B
26 C 4-»C-B
»C-la-OH-B •
"C-A
26 C 4-»C-B
26 C 4-"C-B
"C-A
"C-lo-OH-B
AuthoT(s)
Criteria for
identification
Weisbart and Idler
(1970)
DIDA involving crystallization to constant isotope
ratios
Idler and Truscott
(1967)
infra-red and ultra-violet
spectra, melting points
and derivatives
crystallization of doubly
labelled derivatives to
C.I.B.
Truscott and Idler
(1968)
purification of derivatives
to C.I.E.
O
z
r
•z
3
l
26 C 7-'H-prog.
26 C
Isurus oxyrinchus*
"
R. clavata
R. laevis
*—Tissue frozen, then thawed
S. acanthius
interrenal, frozen —28 C, 20 C
»H-la-OH-B
isopolarity with authentic la-OH-B
purification of "C-aeetylated products mixed with
authentic 7 3H-OH-B acetate to C.I.R.
B
"
"
la-OH-B
4-"C pregn.
Simpson and Wright derivatives chromatogprog.; DOC; B ;
raphy
(1970)
(labelled)
prog.; DOC;B; lo-OHB (labelled)
recrystallized to C.I.R.
labelled F but no la- Idler et al. (1969)
OH-B
<?. malastonxus
thawed, homogenized
S. canicula
fresh homogenized
20 C 4-"C pregn.
H. colliei
interrenals on ice 24 hr,
then sliced
26 C 4-"C-B
TABLE 2 (continued)
Tissue
Species
Class Actinopterygii (higher bony fishes)
Acipenser oxyyellow bodies (minced)
rhynchus
25 C 16-3H-pregn.
+4-"C-prog.
7-sH-cholesterol
Anguilla anguilla
Anguilla anguilla
Gadus morhua
Melanogrammus
acglcfinus
Anguilla anguilla
Salmo gairdneri
Salmo gairdneri
anterior region of the 30 C 4-"C-prog.
posterior cardinal vein
4-"C-prog.
+ head kidney
+ 16-3H-pregn.
37 C 4-"C-prog.
+ 16-3H-pregn.
-j-Angiotensin I I
posterior cardinal veins 30 C 7-3H-cholesterol
+ head kidney minced
and homogenized
6-3H-pregn.
+4-"C-prog.
2-3H-pregn.
+4-"C-17tt-OH-prog.
30 C 3H-B
homogonized anterior
head kidney
B
homogenized head kid-
30 C
Corticosteroida in
medium
Substrate
Temp
3
H-B
B
posterior cardinal vein 35 C 4-"C-B
and head kidney ("mitochondria")
head kidney
37 C 4-"C-DOC
ney
head kidney;—body kid- 37 C 4-"C-pregn.
ney used as control
4-"C-17a-OH-prog.
4-"C-DOC
Author (s)
Criteria for
identification
3
H-F;
Idler and Sangalang crystallization to C.I.B.
Double labelled E ; 3 S; (1970)
B; 17a-OH prog. + Hprog. in low yields
3
it
H-pregn.
3
H-prog.
3
H-17a-OH-prog.
3
H-F, 3H-E, 3H-B,
+ 3H-S
"C-E + "C-F
Sandor et al. (1966) doubly labelled comE + F (doubly labelled)
pounds crystallized
to C.T.R.
»
E + F (doubly labelled)
no aldosterone
3
3
3
3
3
3
3
H-F
H-Preg.
H, "C-F
H, "C-S
H, "C-F
H, "C-S
H-18-OH-B
Sandor et al. (1967)
tt
Sandor et al. (1970)
H-18-OH-B
"
serial derivative formation and purification to
C.I.R.
»
F
"C-18-OH-B
"C-B (major product)
"C-18-OH-DOC
"C-17a-OH-pregn;
"C-S; "C-F
"C-S; "C-F
"C-B; "C-18-OH-DOC
Arai and Tamaoki
(1967)
Arai et al. (1969)
D
O
fc
G
•*
F
3
doubly Labelled compounds crystallized
to C.I.B.
>
<
3
O
crystallization to constant
specific activity
crystallization to constant
specific activity
M
7
TABLE 2 (continued)
Species
Salmo gairdneri
Clupea harengus
harengus
Clupea harengus
harengus
Salmo salar
Poecilia latipinna
Hippoglossus
hippoglossus
Tilapia mossambica
Substrate
Corticosteroids in
medium
minced head kidney in 10 C 16-aH-pregn.
dialysis bag
+4-"C-prog.
F (doubly labelled)
Tissue
Temp
non-dialysod homoge25 C
nized head kidney
head kidney with an- 26 C
terior section of posterior cardinal vein.
head kidney contain- 24-25 C
ing "interrenal"
tissue (minced)
head kidney contain- 24^25 C
ing "interrenal"
tissue (minced)
[l,2-aH]-D0C
+4-"C-B
[1,2-3H]-B
16-3H-preg.
+4-"C-prog.
16-sH-pregn.
[1,2-3H]-DOC
+4-"C-B
minced head kidney in 27 C 16-31<
H-pregn.
dialysis bag
+4- C-prog.
head kidney (minced)
trunk kidney used as a
control
CUUMU1
head kidney (minced)
F (possibly)
no 18-OH-DOC
3
H-aldo
•'H-18-OH-B
head kidney (homogenized)
26 C 4-"C-prog.
head kidney (minced)
26 C 4-"C-B
4-"C-17-OH-prog.
derivative formation an
purification to consta
specific activity (no cry
tallization)
Truscott and Idler
(1968)
re-crystallization to C.I.B
(derivatives of aldo we
not made)
purification followed b
derivative formation an
crystallization to C.I.B.
Weisbart and Idler
(1971)
17a-OH-prog.; S ; F ; E ; Colombo et al.
DOC; B ; A; 11/3-OH- (1972)
prog.; 21-dO-F (labelled
•vvitli 14C)
17a-OH-prog.;S;F;B;
11/3-OH-prog. (labelled
ivith 14C)
"C-S; "C-F;
"C-E; 3 H-B;
+ [l,2-3H]-D0C
S
l-"C-acetate
"C-prog.
H-A
Criteria for
identification
Hargreaves et al.
(1970)
Prog.; DOC; B ; 17a- Sangalang et al.
(1972)
OH-prog.
S; F (doubly labeUed)
Prog.; DOC; B ; 11,3OH-prog.; 17a-0Hprog.; S; 21-dO-F; F.
(doubly labelled)
B ; S; F (doubly
labelled)
F (doubly labelled)
Hargreaves et al.
(1970)
25 C no exogenous
substrate
26 C 4-"C-prog.
Author (s)
derivative formation an
purification to consta
specific activity (no cry
tallization)
D.I.A. involving crysta
lization of derivatives t
C.I.B.
crystallization to C.I.B.
854
DAVID GORDON BUTLER
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routes for steroid biosynthesis in teleost fish
adrenocortical tissue in vitro. Table 2 provides details of substrates and their transformation products.
Corpuscles of Stannius as a possible
source of adrenocortical steroids in teleost
fishes. In spite of important differences in
embryological origins, histochemistry, and
fine structure between the Stannius corpuscles and adrenocortical cells in teleost
fishes, there have been repeated attempts to
implicate these structures in the biosynthesis of adrenocortical steroids. Corpuscular tissue, when incubated with labelled
substrates, has consistently failed to produce
adrenocortical steroids as transformation
products (Ford, 1959; Phillips and Mulrow,
19596; Chester Jones et al., 1965). In contrast, Idler and Freeman (1966) were able
to demonstrate a slight conversion of 4-14C
pregnenolone to labelled progesterone and
of 4-14C progesterone to 11-deoxycorticosterone (0.032% of the original radioactivity)
by sliced cod (G. morhua) corpuscles. When
homogenates of rainbow trout corpuscles
were incubated with labelled progesterone
or 11-deoxycorticosterone, there was no evidence that corticosteroids were transformation products (Arai et al., 1969).
The findings of Colombo et al, (1972)
demonstrated the importance of incubating
"control" tissue concurrently with head kidney (containing adrenocortical cells) or Stannius corpuscles, a factor that has often been
overlooked in the earlier experiments. This
is particularly important when the percentage transformation of substrate is low.
Minced corpuscles of Stannius from rainbow trout, incubated at the fishes acclimation temperature of 11.5 C, converted
4-14C progesterone to 11-deoxycorticosterone. However, body kidney incubated introduced greater percentage conversion of
labelled substrate to 11-deoxycorticosterone than did the corpuscles. It was concluded that no important role in corticosteroid biosynthesis is played by Stannius
corpuscles in rainbow trout.
Even though there have been reports that
Stannius corpuscles in Atlantic salmon contain cortisol and cortisone (Fontaine and
Leloup-Hatey, 1959; Cedard and Fontaine,
ADRENAL GLAND OF FISHES
1963) and that goldfish corpuscles contain
11-deoxycorticosterone (Ogawa, 1963), the
consensus, based on the physiological data,
is that Stannius corpuscles are not organs
of steroidogenesis.
Subclass Sarcopterygii
(Order Dipnoi—hingfishes)
Sarcopterygian fishes have received scant
attention as regards adrenocortical function.
Of the two orders within this subclass, only
the dipnoans have been studied. Phillips
and Chester Jones (1957) reported that the
plasma of Protopterus annectens contained
15.0 mg cortisol/100 ml. Leloup-Hatey (1964)
extracted plasma from the same species and
reported that it contained 12.0 ^,g of 17hydroxy steroids per 100 ml, but the author
did not exclude the possible occurrence of
corticosterone. In both of these papers the
methods imply that identifications were
tentative. Janssens et al. (1965) incubated
the presumptive adrenocortical tissue (cardinal veins) from the African lungfish (Protopterus sp.) with 4-14C progesterone and
reported that 0.05% of the original radioactivity was incorporated into labelled corticosterone. Under these conditions cortisol
was not a transformation product.
Recently, Idler et al. (1972) collected,
pooled, and extracted the plasma from four
South American lungfish (Lepidosiren paradoxn). They identified cortisol (0.60 ^tg/100
ml plasma) and aldosterone (0.58 ^g/100 ml
plasma) together with smaller amounts of
II-deoxycortisol and corticosterone. The
occurrence of aldosterone in this Sarcopterygian fish is particularly exciting because
the lungfish is a forerunner of amphibians
in which aldosterone is not only widely distributed but also of demonstrated physiological importance.
THE PITUITARY-ADRENOCORTICAL AXIS IN FISH
Adrenocorticotrophic hormone (ACTH)
when released by the anterior pituitary
gland of higher vertebrates increases steroidogenesis and the release of adrenocorticosteroids into the circulation. Normally,
if the corticosteroid levels increase beyond
a certain point, the rate of ACTH secretion
855
is lowered and the corticosteroid levels decrease. Superimposed on this negative-feedback system is the ability of most animals
to drastically increase ACTH secretion and
the release of corticosteroids from the
adrenal cortex (adrenocortical cells) in response to stress or other stimuli. This functional relationship between the pituitary
gland and the adrenal cortex is known as
the pituitary-adrenocortical axis. Strahan
(1959) reported that adrenocortical hypertrophy in mice followed injections of Lampetra pituitary extracts. Olsson et al. (1965)
found that lamprey erythrosinophils increased in number following the injection
of metopirone, presumably due to the blockage of 11-hydroxylase and subsequent release of ACTH (see Ball and Olivereau,
1966). Idler et al. (1971) reported that
plasma cortisol and cortisone concentrations increased in the Atlantic hagfish following the injection of 60 IU of mammalian
ACTH administered during a period of 10
days. Studies based on the assumption that
jawless fishes possess a pituitary-adrenocortical axis are premature until it has been
shown whether or not the presumptive
adrenocortical tissue actually produces corticosteroids (see Weisbart and Idler, 1970).
Even the histological data are equivocal.
Dittus (1937, 1941) studied the pituitaryinterrenal axis in elasmobranchs. Hypophysectomy of Torpedo was followed by
degenerative changes in the interrenal (adrenocortical) cells. These degenerative changes
could be prevented by the injection of mammalian ACTH. Dodd (1961), however, did
not observe any significant changes in the
cytology of the interrenal gland of the spotted dogfish (S. canicula) one year after
hvnophysectomy.
Extracts of elasmobranch pituitaries contain material that accelerated steroid biosvn thesis in chicken adrenocortical tissue
in vitro (de Roos and de Roos, 1967). In
two species of elasmobranchs, S. acanthius
and R. rhina and one holocephalian, H.
colliei most of the corticotrophic activity
was found in the pars distalis. Other authors
have used the opposite approach, that of
testing the activity of ACTH from a higher
vertebrate on elasmobranch interrenal tis-
856
DAVID GORDON BUTLER
sue. Macchi and Rizzo (1962) found that
mammalian ACTH stimulated the production of UV absorbing (240 m^) blue tetrazolium positive materials, presumably corticosteroids, by interrenals in vitro. However,
Bern et al. (1962) failed to observe the
increased production of corticosteroids when
mammalian ACTH was added to interrenal
incubates from S. acanthius, R. rhina, and
H. colliei. Ideally, elasmobranch pituitary
extracts (corticotropin) should be tested on
elasmobranch interrenal tissue. Experimental design should take into account the fact
that there is a lack of definite proof that
17-hydroxylated steroids are present in the
blood of elasmobranchs and that the major
circulating steroid is probably la-OH corticosterone.
Fish pituitary extracts and mammalian
ACTH preparations with various degrees
of chemical purity have produced hypertrophy of the adrenocortical tissue in a relatively large number of intact teleost fishes
(Burden, 1956; Chavin, 1956; Chavin and
Kovacevic, 1961; Pickford and Atz, 1957;
Rasquin, 1951; Hanke and Chester Jones,
1966; Hanke et al., 1967; Mahon et al.,
1962; Fleming et al., 1971). Conversely,
ablation of the pituitary gland was followed,
in most cases, by adrenocortical atrophy
which is characterized by a reduction in
nuclear diameter and cell size (Chavin,
1956; Fontaine and Hatey, 1953; Olivereau
and Fromentin, 1954; Burden, 1956; Ball
et al., 1965; Donaldson and McBride, 1967).
Adrenocortical activity was restored in
hypophysectomized fish that were injected
with fish pituitary brei (Burden, 1956; van
Overbeeke and Ahsan, 1966) and mammalian ACTH preparations (Fontaine and
Hatey, 1953; Chavin, 1956; Leloup-Hatey,
1964; Basu et al., 1965; van Overbeeke and
Ahsan, 1966). Restoration of adrenocortical
function has been observed in hypophysectomized teleosts with transplanted pituitary
glands (Chavin, 1956; Ball et al., 1965).
Metopirone (SU 4885, Ciba, metyrapone)
is a potent chemical blocker of Ilj8-hydroxylase activity in the mammalian adrenal cortex with the result that circulating levels
of 11-oxosteroids are reduced. If a feedback
system is operating, the rate of ACTH se-
cretion increased in an attempt to elevate
the circulating levels of corticosteroids. Injections of this drug to intact A. anguilla
and Poecilia latipinna activated the Epsilon
(corticotropin-secreting) cells in the pituitary gland and caused a hypertrophy of the
adrenocortical cells. Stimulation of the
adrenocortical tissue by metopirone could
be fully blocked in Poecilia and partially
blocked in A. anguilla by hypophysectomy
(Olivereau and Ball, 1963; Olivereau, 1965;
Ball and Olivereau, 1966). These findings
support the case for the existence of a pituitary-adrenocortical axis in teleost fishes.
It has been suggested that the reduction
in size and nuclear diameter of teleost
adrenocortical cells which followed the injection of corticosteroids (Krauter, 1958;
Robertson et al., 1963; Basu et al., 1965;
Hanke et al., 1967; Olivereau, 1966) was due
to a negative feedback rather than to a direct suppressive action on the adrenocortical cells. Hanke (1967) and Olivereau and
Olivereau (1968), using a more direct
method of testing the negative feedback
theory, have reported that the partial adrenalectomy of A. Anguilla is accompanied by
hypertrophy of the corticotrophic cells in
the pituitary gland.
Donaldson and McBride (1967) were the
first to provide physiological evidence for
a pituitary-adrenocortical feedback mechanism in fish. They observed that hypophysectomy or the injection of dexamethasone (16a-methyl-9-a-fluoroprednisolone) to
sham-operated controls was followed by a
very significant reduction in plasma cortisol levels. In 1966, Donaldson (personal
comm.) was able to increase plasma cortisol in dexamethasone-inhibited fish by injecting ACTH, thereby excluding the possibility that dexamethasone had acted directly on the adrenocortical cells. Dexamethasone also decreased cortisol levels in
European eels (Bradshaw and FontaineBertrand, 1968) and in North American
eels (Butler et al., 1969). In both species,
as well as in the Japanese eel (Hirano,
1969), plasma cortisol concentrations decreased following hypophysectomy and increased when operated fish were injected
with mammalian ACTH. Hawkins et al.
ADRENAL GLAND OF FISHES
(1971) have shown that cortisol and
ACTH are in a negative feedback relationship in intact P. latipinna. All of these
workers have employed either fluorimetric
or competitive protein binding (CPB) assays which to some degree may lack specificity.
STEROID DYNAMICS IN FISH
Binding of adrenocorticosteroids
The binding potential of fish blood is not
achieved through strong binding of the
transcortin type, as in mammals, but rather
by employing a system with a low or medium binding affinity together with a reasonably large total binding capacity. Association constants for blood proteins were
determined for adrenocorticosteroids in the
thorny skate (R. radiata), Atlantic cod (G.
morhua), and Atlantic salmon (S. salar)
using the equilibrium dialysis-Scatchard
plot methods (Idler and Freeman, 1968;
Freeman and Idler, 1971). The skate was
reported to possess a single low affinity
(kj = 0.9 X 104) binding system with a total
capacity of from 18-25% of the circulating
la-OH corticosterone. Both the cod and
salmon were found to have two binding systems, the first with an "intermediate" binding affinity but with a relatively low capacity and the second with a lower affinity but
larger capacity. In salmon, for example, the
high affinity-low capacity system (lq — 1.7
X 105) can bind as much as 20-40 fig cortisol/100 ml plasma which is greather than
the total binding capacity of human transcortin (20^g/100 ml) where the association
constant is 3 X 107. In salmon, the binding
affinity of the low affinity-high capacity system (2.8 X 104) approximates that of 0.5 —
7.0 X 104 f° r human serum albumen. Together, the two binding systems in Atlantic
salmon bind from 30-55% of the endogenous cortisol at 4 C, whereas in man (37 C)
the transcortin and albumin systems together bind as much as 90% of circulating
cortisol (Daughaday and Mariz, 1961; Freeman and Idler, 1971).
A good deal of interest has been given to
the possibility that adrenocortical steroids
are involved with the control of active so-
857
dium transport by fish gills. Attempts have
been made to show whether or not they are
bound to gill epithelial cells. Snart and
Dalton (1970) scraped epithelial cells from
the gill bars of European eels, suspended
them in Ringer's solution and measured
displacable binding of a number of tritiated
compounds including cortisol, corticosterone, aldosterone, and 11-deoxycorticosterone. There was no indication that any of
these steroids was specifically bound to gill
epithelial cells in vitro. Goodman and
Butler (1972) injected both 4-14C cortisol
and 4-14C progesterone into North American eels. Cortisol, but not progesterone, was
preferentially bound (localized) in the gill
epithelium which suggested that cortisol
acts directly on the cells which actively
transport Na+ (see Mayer et al., 1967).
Aldosterone was not tested since it has not
been identified as a transformation product
of eel interrenal tissue after repeated attempts to find it (Butler, 1965; LeloupHatey, 1966; Sandor et al., 1966, 1967, 1970).
Metabolic clearance rate for
a dren ocort icosteroids
The turnover rate or metabolic clearance
rate (MCR) has been defined by Tait (1963)
as "the volume of blood cleared completely
and irreversibly of steroid per unit time."
In the elasmobranch R. radiata, in which
the naturally occurring corticosteroid lahydroxycorticosterone is loosely bound to
plasma protein (Freeman and Idler, 1971),
the MCR is reasonably high, 92 ml/kg body
wt/hr. Owen and Idler (1972) have discussed the possibility that there is an inverse
relationship between MCR and the degree
of binding affinity for a given steroid. For
example, in 7?. radiata, testosterone, in contrast to la-hydroxycorticosterone, is relatively tightly bound (association constant
6 X 107 compared with 2 X 104 for lahydroxycorticosterone) and the MRC is 9
ml/kg body wt/hr. Recently, Goodman and
Butler (unpublished) measured the MCR
in freshwater A. roslrata after single injection of 4-14C cortisol. To rule out the possibility that serial sampling of fish had affected steroid dynamics, we killed and sam-
858
DAVID GORDON BUTLER
pled blood from six or seven eels at each of
a series of times after the injection of
steroid. Adequate methods of purification
preceded measurement of labelled cortisol.
We found that the MCR for these immature freshwater eels was 44.6 ml/kg body
wt/hr. Donaldson and Fagerlund (1970)
measured the MCR in sockeye salmon (0.
nerka) with a view to understanding the
relationship between cortisol dynamics and
maturation. They reported that the MCR
for sexually immature fish was 83 ml /kg
body wt/hr for males and 54 ml/kg body
wt/hr for females. At sexual maturity, the
MCR increased to 267 and 170 ml /kg body
wt/hr for males and females respectively.
It was shown by these authors that estrogens
(Donaldson and Fagerlund, 1969a,b) and
androgens (Fagerlund and Donaldson, 1969)
had brought about these increases in MCR
since they were not evident following gonadectomy. Recently, Owen and Idler (1972)
have studied the MCR for cortisol and cortisone in a relatively modern marine teleost,
the sea raven (H. americanus). The mean
MCR for cortisone in 6 of these fish was
449 ± 48 ml/kg body wt/hr.
Secretion rates for adrenocortical steroids
Secretion rates have been calculated as
S = MCR times plasma corticosteroid concentration, assuming the fish is in the steady
state. Donaldson and Fagerlund (1969a) reported that the cortisol secretion rate in
resting gonadectomized female O. nerka was
0.9 ± 0.5 ^g/kg body wt/hr, considerably
lower than in intact salmon where the secretion rate was 6.2 ± 3.2 /*g/kg body wt/hr
(Donaldson and Fagerlund, 1970). When
estradiol was injected into gonadectomized
females, the cortisol secretion rate increased
to 10.0 ± 4.4 /xg/kg body wt/hr. These experiments were expanded to show that there
is a progressive increase in the rates of cortisol (Donaldson and Fagerlund, 1970) and
cortisone (Fagerlund and Donaldson, 1970)
secretion as sockeye salmon approach sexual
maturity. For example, the cortisol secretion rate in male sockeye salmon increased
from 6.2 ± 3.2 to 13.9 ± 3.6 ^g/kg body
wt/hr. These elevated secretion rates for
both cortisol and cortisone in mature salmon could be prevented by hypophysectomy.
The experiments showed that there is a
direct correlation between cortisol secretion
rates and circulating levels of estrogens
(Donaldson and Fagerlund, 1969a, 1970;
Fagerlund and Donaldson, 1970). Recently,
Owen and Idler (1972) have reported that
cortisol and cortisone secretion rates in the
sea raven (H. americanus) were 9.0 /xg/kg
body wt/hr and 4.9 /ag/kg body wt/hr,
respectively. Calculations were based on
mean values for MCR and mean plasma
steroid levels in different fish but from the
same experimental stock.
Half life of adrenocortical steroids in fish
Idler and Truscott (1963), using calculations that were based on a single-pool system, reported that the half-lives for cortisol
and cortisone in sockeye salmon (O. nerka)
were 15 hr and 3.4 hr, respectively. If 4-14C
cortisol was injected into healthy (as opposed to moribund) Atlantic cod (G. morhua), the half life for total radioactivity in
plasma ranged from 5.4 to 4.7 hr (Idler and
Freeman, 1965). If, following the injection
of radioactive cortisol, the clearance of labelled cortisol is measured rather than total
radioactivity, the half life is considerably
reduced. For example, maturing and mature sockeye salmon were injected with
4-14C cortisol and the half lives for total
radioactivity were reported as 1.5 hr and
2.2 hr. In a second experiment, when the
rate of clearance was measured for labelled
cortisol only, the half life was reduced to
35.8 ± 10.4 min in immature male salmon
and 32.6 ± 6.0 min in mature male salmon
(Donaldson and Fagerlund, 1968, 1970).
Donaldson and Fagerlund (19696) reported
that gonadectomy decreased the cortisol
secretion rate in female sockeye salmon and
that the secretion rate returned to normal
following the injection of estradiol. The
half life for cortisol did not change in parallel with secretion rates, the T i/o being 46.2
± 10.0 min in gonadectomized fish compared with 50.8 ± 5.6 min following treatment with estradiol. The metabolic fate of
4-14C cortisone was also studied (Fagerlund
ADRENAL GLAND OF FISHES
and Donaldson, 1970). The half life for cortisone was relatively low in female salmon
(9.3 ± 2.7 min) and it remained low at maturity (13.2 ± 0.6 min) and after gonadectomy (9.9 ± 5.2 min). However the half life
of cortisone in males is higher in immature
salmon (49.1 ± 13.7 min) than mature salmon (116.9 ± 15.9 min). Goodman and
Butler (unpublished), using a single injection technique followed by the sampling of
separate groups of A. rostrata seriatum after
injection, have found that the half life for
4-14C cortisol to be 15.1 min, a value within
the range of those for mature salmon (Donaldson and Fagerlund, 1968).
PHYSIOLOGY OF ADRENOCORTICAL STEROIDS
IN FISH
Role of adrenocorticosteroids in
ionoregulation
The ionic composition of the cells of the
hagfish M. glutinosa was found to be very
similar to the cells of marine invertebrates
with the exception of the cellular potassium
concentration which was not unlike that of
higher vertebrates (Bellamy and Chester
Jones, 1961). Serum electrolyte concentrations including Na+, K+, C l - and Ca2+
were not significantly different from the
external concentrations when hagfish were
in either full or 60% sea water. A number
of exogenous steroids, including aldosterone, deoxycortkosterone acetate, and cortisone were injected to hagfish that had
been acclimated to 60% sea water or full
sea water, but there were no statistically
significant changes in serum or muscle
electrolyte concentrations (Chester Jones
et al., 1962). The freshwater lamprey
(Lampetra fluviatilis), however, is not an
"adjuster" and must regulate the ionic
composition of the body fluids. The continuous renal (Hardisty, 1956) branchial
and integumental loss of electrolytes would
be counterbalanced by dietary sources of
salt or the active uptake of ions through the
gills (see Wikgren, 1953). Bentley and Follet (1962) found that the rate of total sodium excretion from freshwater lampreys
(L. fluviatilis) decreased after the injection
of aldosterone. It was suggested that aldo-
859
sterone acted by reducing the renal excretion
of sodium and that an accelerated rate of
sodium excretion, following injections of
SCI 1927, was due to the blocking of aldosterone. ACTH reduced the rate of sodium
loss, but cortisol had no measurable effect.
The results for these experiments could be
explained by way of the effects that test
compounds may have had on renal hemodynamics. Other explanations are possible.
In a second series of experiments, urine was
collected and its flow rate measured. Urinary sodium and potassium concentrations
were also measured (Bentley and Follet,
1963). Neither aldosterone nor cortisol injections were followed by significant changes
in the urinary concentrations of either Na+
or K+. Urine flow rates decreased after
treatment with aldosterone, but the reduction was not great enough to lower significantly the rate of sodium excretion. The
filtered load of sodium was not measured so
there is no way of knowing whether or not
aldosterone had affected the transtubular
flux of Na+. Cortisol had no measurable
effect on urine flow rates. To date there is
no conclusive evidence that adrenocortical
steroids are present in cyclostome blood (see
Weisbart and Idler, 1970). Even if they prove
to be (see Table 1), there is no evidence that
endogenous steroids affect ionoregulation
in jawless fishes.
Chondricthyes. Elasmobranch fishes are
able to keep their extracellular fluids hypertonic, relative to the external environment,
by the retention of urea. The kidney can
actively conserve the nitrogenous compounds such as urea and trimethylamine
oxide and the gills are largely impermeable
to nitrogenous end-products. Such an adaptation for existence in a hypertonic environment is shared by the sharks, rays, and
skates. The osmotic concentration of the
blood is such that water is drawn across the
gills and integument and into the fish without the expenditure of free energy. Urine
flow rates are comparable with those in
dium excretion from freshwater lampreys
freshwater teleosts; for example, in the
dogfish (S. acanthius) the urine flow rate
was reported to be 27.60 ml/kg body wt/
day and the glomerular filtration rate was
84.0 ml/kg body wt/day (Hickman and
860
DAVID GORDON BUTLER
Trump, 1969). Sodium and chloride appear
as the dominant solutes in dogfish urine,
but ions can be selectively reabsorbed. To
date no work has been published on the
adrenocortical control of transtubular electrolyte fluxes; this would be a profitable
area for investigation.
The elasmobranch rectal gland secretes a
fluid with greater Na+ and Cl~ concentration than in the plasma, thereby providing
an extra-renal route for the excretion of
Na+ and Cl~. Chan et al. (19676) found
that the rectal gland of the lip shark
Hemiscyllium plagiosum (Bennett) was influenced by two test steroids, neither of
which may be present in the blood of this
species. Cortisol and 11-deoxycorticosterone both decreased the rate of fluid secretion by the rectal gland but neither compound changed the NaCl concentration in
rectal fluid. These changes may have been
generated as the result of a change in
blood flow to the gland.
Hartman et al. (1944) attempted to show
whether or not the interrenal secretions are
important for ionoregulation by interrenalectomizing R. erinacea. In general, there
was no conclusive evidence that the interrenal of this species secreted corticosteroids
that were essential for maintaining plasma
electrolyte concentrations. Idler and Szeplaki (1968) have repeated the interrenalectomy of R. erinacea, but the findings were
essentially the same. As long as 44 days after
the operation there were no measurable
changes in plasma Na+, K+, Cl~, Mg2+,
or osmotic concentrations. Plasma Ca2+
concentrations increased by about 10%.
Grimm et al. (1969) have reported that
the major circulating adrenocortical steroid
in elasmobranch. fishes, la-OH corticosterone, stimulated active transport of sodium
by toad bladder in vitro. However, this compound was not tested in R. erinacea because
interrenalectomy (Hartman et al., 1944;
Idler and Szeplaki, 1968) had not affected
tissue electrolyte levels.
Prior to the development of techniques
for surgical adrenalectomy of teleost fishes,
two experimental approaches were used to
study the effects of corticosteroids, or their
deficiency, on ionoregulation. Intact fish
were injected with a variety of exogenous
steroids, often in non-physiological closes,
in an attempt to alter the branchial and/or
renal fluxes of electrolytes (Sexton, 1955;
Smith, 1956; Spalding, 1956; Holmes, 1959;
Edelman, et al., 1960; Holmes and Butler,
1963; Henderson and Chester Jones, 1967).
Taken together, these findings showed that
some corticosteroids will change the rates of
sodium transport through the gills and/or
the renal handling of sodium.
The second experimental approach was
to attempt to block adrenocortical function
with chemical inhibitors such as metyrapone
(Ciba, SU 4885) and betamethasone (Betnesol, Glaxo), singly or in combination,
(Chan et al., 19676) and to observe the pattern of changes or lack thereof in tissue
electrolytes. The findings are difficult to
assess because of the possible "side effects"
of these compounds in the doses administered, together with inadquate evidence that
the test animals were actually in a state of
adrenocortical insufficiency, e.g., plasma
levels of corticosteroids. In a number of experiments it has been assumed that aldosterone is present in fish, and accordingly,
several anti-aldosterone compounds including aldactone (SC9420, Searle Ltd.), SC
11927 and SC 14266, (G. D. Searle Ltd.) have
been injected into eels (Olivereau and Chartier-Baraduc, 1965; Chan et al., 1967a). A
number of minor changes in plasma and
muscle electrolyte concentrations were observed, but they may be of pharmacological
significance only since repeated attempts to
identify aldosterone in eels have been unsuccessful (Butler, 1965; Leloup-Hatey, 1966;
Sandor et al, 1966, 1967, 1970).
Chester Jones et al. (1964) first developed
a method for the partial surgical adrenalectomy of eels, although the operation had
been successfully performed on goldfish a
year earlier by Etoh and Egami (1963). In
eels, the bulk of the adrenocortical tissue
was removed by excising segments of the
postcardinals together with the surrounding
anterior head kidney. The anterior cardinals
often contain islets of adrenocortical tissue
so the degree of completeness of adrenalectomy was improved later by removing segments of these veins and more of the an-
861
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terior head kidney (Butler and Langford,
1967; Chan et al, 1967a; Butler et al., 1969).
Partial surgical adrenalectomy of freshwater European eels was followed, in three
weeks, by a decreased concentration of
serum Na+ and Mg++ but there were no
statisically significant changes in K+ or
Ca++ concentrations. Parietal and tongue
muscle Na+ and K+ concentrations decreased but Ca2+ and Mg2+ were unchanged. The percentage fat-free muscle
water and intracellular water content
(muscle) both increased (Chan et al.,
1967a). Unfortunately, these observations
are largely invalidated because comparisons were made with intact control eels.
Adrenalectomy was achieved by way of
entry through a long, mid-ventral incision
through the body wall which was subsequently stitched shut. Any influx of water
or outflux of ions through the incision
would drastically alter the picture. Two additional uncontrolled factors were postoperative stress and the effect on renal
hemodynamics of removal of segments of
the cardinal veins. Changes in the pattern
of renal function would soon alter tissue
electrolyte and water concentrations.
Butler and Langford (1967) used the
methods described by Chester Jones et al.
(1964) to partially adrenalectomize some
immature freshwater North American eels
(A. rostrata). Three weeks after the operation, there were no significant changes in
tissue electrolyte concentrations compared
with sham-operated controls provided the
mid-ventral incision had been closed with
stainless steel wire. On the other hand, if
surgical silk was used, the wound was inflamed, and the tissue around the sutures
had begun to rot. In this group of adrenalectomized eels, tissue electrolyte concentrations were significantly lower than in
sham-adrenalectomized eels (steel sutures).
Two years later we repeated the experiments using a slightly improved method for
adrenalectomy in which segments of the
anterior cardinal veins were excised together with larger segments of the postcardinals and anterior head kidney (Butler
et al., 1969). Three groups of freshwater
eels were adrenalectomized, but in only one
I- O
48
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TABLE 4. Plasma cortisol, Hood glucose, and liver and muscle glycogen concentrations in fresh-water yellow and silver North American eels three weeks
after adrenalectomy.
Liver glycogen Muscle glycogen
Cortisol
(mg/100 mg
(/ig/100
(mg/100 mg
n
wet tissue)
Group
Treatment
ml plasma)
wet tissue)
(g)
Intact controls
6.9644
1301.9
0.2240
3.29
10
±88.9
±0.14
±0.5136
±0.0177
±7.7
1. Fresh-water silver eels
1036.4
156.2
5.5971
0.2752
7
Sham-adrenalectomy
2.43
±0.88
18 C—March 1968
±130.9
±0.2945
±0.0257
±16.4
3 weeks
133.7
Adrenalectomy
5.7128
8
0.1950t
0.58*t
1265.0
±0.5327
±0.0207
±0.24
±75.6
±12.3
3 weeks
10
Intact controls
5.2880
977.1
166.9
0.2646
2.08
±0.1180
±0.0178
±0.09
±93.0
±13.8
2. Fresh-water yellow eels
11
232.9*
Sham-adrenalectomy
4.6094
1170.6
0.2774
1.87
±0.3280
±131.2
±17.7
±0.0170
10 C—June 1968
±0.23
3 weeks
9
Adrenalectomy
1075.0
0.1940t
0.99*t
105.9t
3.1440* t
±120.7
±0.4417
±0.0166
±0.15
±21.2
3 weeks
Intact controls
1148.2
0.2932
3.93
7
56.63
5.6205
±102.6
±0.56
±0.3441
±0.0153
±12.70
3. Fresh-water yellow eels
3.02*
Sham-adrenalectomy
1383.8
4.2691
0.2487
10
83.45
±107.8
±0.3872
10 C—July 1968
±0.33
±0.0181
±9.46
3 weeks
11
Adrenalectomy
1055.5
0.2350
1.05*t
56.75
2.2683*t
±184.0
±9.71
±0.0192
±0.11
±0.2108
3 weeks
Values are means ± SEM. Multiple comparisons of means in each experimental group were made using Tukey's w-proeedure (Tukey, 1953).
* P < 0.05 compared with intact controls and t P < 0.05 compared with sham-aclrenalectomized controls (From Butler ct al., 1969a).
Body weight
Blood glucose
(mg%)
159.5
u
>
o
o
o
UTLE
w
ADRENAL GLAND OF FISHES
863
doses of cortisol (10 yug per eel body wt range
500-800 g) injected to adrenalectomized eels
gave "normal" Na+ gains (Henderson and
Chester Jones, 1967). Unfortunately, these
data do not show whether the rates of active
uptake or passive outward diffusion of Na+
had been affected. Observed changes in net
flux could have been due entirely to changes
in passive permeability of the gill membranes to Na+. However, this is unlikely
since Chan (1968, unpublished observations
cited by Chester Jones et al., 1969) and
Maetz (1969) both reported that adrenalectomy of fresh-water A. anguilla was followed
by a reduction in active Na+ uptake by the
gills but no change in the rate of passive
outflux. This suggested that accelerated net
influx of Na+ in adrenalectomized. and intact fresh-water eels injected with different
doses of cortisol ranging from 100 /»g/kg
body wt/day to 2 mg/kg body wt/day for
Role of adrenocortical steroids in regulating 3 or 4 days, was due to a stimulation of active Na+ transport (Chan et al., 1969).
ion fluxes through the gills
Adrenalectomized freshwater A. anguilla
Holmes (1959) observed that cortisol and failed to excrete Na+ at the normal rate
deoxycorticosterone acetate increased the after transfer to sea water; the exchange rate
rate of Na24 excretion through the gills of being about i/s of that in sham-adrenalecsalt-loaded rainbow trout (S. gairdneri) ac- tomized controls. After the adrenalectoclimated to fresh water. Deoxycorticosterone mized eels were injected with cortisol hemiacetate impaired the rate of uptake of Na22 succinate (500 /*g/kg body wt), rates of
from fresh water by salt-loaded trout. These sodium excretion were accelerated and the
findings suggested that corticosteroids might Na+ turnover rate returned to normal.
normally influence ion transport in trout
Cortisol may change the rate of active
during acclimation to sea water. Henderson Na+ transport in freshwater and seawater
and Chester Jones (1967) transferred fresh- eels in an indirect fashion, e.g., through its
water A. anguilla to distilled water for 6-8 effects on intermediary metabolism or by
weeks. After they were transferred back to changing the rate and direction of blood
fresh water (tap water), the net rate of gain flow to the gills. On the other hand, cortisol
of Na+ by the gills of intact eels increased could act directly on the gill epithelium. Eel
to 46.1 ± 4.3 juM/kg body wt/hr, whereas gills seem to have the capacity to selectively
in adrenalectomized eels the rate was low- bind cortisol (Goodman and Butler, 1972),
ered to 4.5 ± 6.4 //.M/kg body wt/hr. When an association which could result in a change
adrenalectomized eels were injected with a in gill permeability to ions or activation of
large, non-physiological dose of 250 jug aldo- the sodium pump. Sodium pump activity
sterone (Aldocorten) per eel the net rate of may be regulated, in part, by the formation
gain of Na+ increased to 82.1 ± 7.9 /ttM/kg of new enzyme(s) or changes in the activity
body wt/hr. Aldactone (SC 9420, Searle) in- of pre-existing enzyme(s). With this in mind
jected to intact eels in doses ranging from we studied Na+/K+-ATPase activity in
5 to 90 mg per fish lowered the rate of gain the gills of North American eels (A. rostrata)
of Na+. Even though the daily injection of in relation to environmental salinity and
10 mg of cortisol per eel for 4 days was fol- adrenocortical function (Butler and Carlowed by a net branchial outflux of Na+ michael, 1972).
(-54.8 ± 27.0 /xM/kg body wt/hr), smaller
Epstein et ai. (1967) found that Na+/K+-
group was there a clear-cut response to removal of the adrenocortical tissue (see Table
3). In this group, plasma Na+, Ca++, and
osmotic concentrations were lower than in
sham-operated controls, but there was no
significant change in plasma K+ or Cl~.
Epaxial muscle K concentrations and content decreased significantly but there was no
change in the concentrations of other ions
nor in muscle water. Table 4 shows that, in
each of the three experimental groups,
adrenalectomy was followed by a significant
reduction in plasma cortisol. Blood glucose
concentrations were not affected, but in the
second and third groups of adrenalectomized eels, liver glycogen concentrations decreased significantly. This may mean that,
in starved eels, gluconeogenesis was impaired
as a result of decreased circulating levels of
cortisol.
864
DAVID GORDON BUTLER
ATPase activity in the gills of F. heleroclitus, acclimated to sea water, decreased after
hypophysectomy. A similar response to
hypophysectomy was observed in seawater
A. anguilla (Milne et al., 1971). Na+/K+ATPase activity fell from 3.70 ± 0.18 to
1.56 ± 0.27 /*m Pi/mg protein/hr within
10-14 days after removal of the pituitary,
and increased to 3.48 ±0.17 ^m Pi/mg protein/hr if the hypophysectomized eels were
injected with mammalian ACTH (5 1U per
eel/day) for the last 3 days before sampling.
These authors suggested that enzyme activity was restored by way of the "ACTH-corticosteroid" axis. In these experiments,
enzyme activity was extremely low in comparison with earlier values for Na+/K+ATPase activity in the gills of the same
species (Motais, 1970; Langford, 1971) and
in A. rostrata (Carmichael, 1968; Epstein et
al., 1971) and A. japonica (Kamiya and
Utida, 1968; Utida et al., 1971). This may
have been largely due to the use of relatively
crude enzyme preparations; comparisons are
difficult with a very low activity.
It seemed possible that normal rates of
sodium excretion by the gills of euryhaline
fishes acclimated to sea water was linked to
Na+/K+-ATPase activity which in turn was
controlled through the pituitary-adrenocortisol axis. To indirectly test this hypothesis,
cortisol hemisuccinate was injected into
hypophysectomized Fundulus that had been
acclimated to sea water (Pickford et al.,
1970). At the lower dose, a total of four injections of 2.5 mg cortisol hemisuccinate/kg
body wt ip on alternate days produced no
change in enzyme activity. However, when
hypophysectomized fish were given a total
of 15 daily injections of 2.5 mg of cortisol
hemisuccinate, Na+/K+-ATPase activity increased by 31% in males and 24% in females.
Epstein et al. (1971) found that 7-14 daily
injections of 4 mg cortisol/kg body wt ip
into intact freshwater A. rostrata was followed by a threefold increase in Na+/K+ATPase activity. We have extended these
observations by measuring Na+ /K+-ATPase
activity in hypophysectomized (4 days) freshwater eels and found that enzyme activity
was reduced after removal of the pituitary
and that it increased following injection of
cortisol to hypophysectomized eels (Table
5). If hypophysectomized eels transferred to
50% sea water for 2 days, Na+/K+-ATPase
activity was lower than in sham-hypophysectomized controls but higher than in intact
freshwater eels. This demonstrated that
hypophysectomy would impair enzyme activity, but would not hold it below the
freshwater control level. Adrenalectomy of
freshwater eels had no measurable effect on
gill enzyme activity. These data can be interpreted in a number of ways, for example:
(1) Hypophysectomy may have decreased
gill Na+/K+-ATPase activity due to the
absence of a pituitary factor other than
ACTH; increased enzyme activity being
caused by a pharmacological effect at these
doses of cortisal; (2) the pituitary-adrenocortical axis may regulate ion transport by
virtue of its influence on intermediary metabolism; or (3) cortisol may directly control
the activity and/or induction of Na+/K+ATPase within the epithelial transport cells.
If this last alternative is correct, then some
adrenocortisol tissue may have remained in
situ, thereby secreting enough cortisol to
maintain normal enzyme activity.
Adrenocorticosteroids and renal fimction
Holmes and McBean (1963) studied the
changes in glomerular nitration rate (GFR)
in freshwater rainbow trout (S. gairdneri)
following the injection of steroids. A single
injection of 25 /xg of d-aldosterone had no
measurable effect but 5.0 mg of corticosterone decreased the glomerular filtration rate
from 168.9 ml/kg body wt/day to 126.0
ml/kg body wt/day. It was implied that a
reduction in GFR. might normally be
achieved in sea-run trout by increased secretion of an endogenous corticosteroid,
assuming that urine flow rates were proportional to glomerular nitration rates
(see also Holmes and Stainer, 1966).
Chan et al. (1969) studied the patterns of
renal function in freshwater A. anguilla
after surgical adrenalectomy. This operation entails removal of most of the length
of the postcaidinal veins together with adjacent anterior head kidney; part of the
865
ADRENAL GLAND OF FISHES
TABLE 5. Na*-K*-activated ATPase in eel gills in relation to environmental salinity and the pituitaryadrenocortical axis.
Plasma
Experimental
group
Intact controls
Sham hypox 4 days
Hyp ox 4 days
Hypox 2 days—saline i.p.
4 days
Hypox 2 days—400 /ig cortisol/100 g bw/day i.p.
4 days
Sham hypox 4 days FW—•
2 days 50% SW
Hypox 4 days FW—2 days
50% SW
Sham ADX 4 days
ADX 4 days
K+
n
(meq/1)
3.15 ±
3.3
3.69 ±
4.7
3.56 ±
1.7
4.1
3.51 ±
0.04
0.32
0.25
0.32
Gill (Na*-K+)-ATPase
(,um Pi/mg protein/hr)
35.70 ± 1.72
30.87 ± 1.94
16.64 ± 1.93ab
23.73 ± 1.24
6
155.0 ±
150.3 ±
158.4 ±
140.8 ±
6
139.7 ± 3.0
3.67 ± 0.12
40.53 ± 3.18"
7
152.7 ± 2.7
3.67 ± 0.16
40.61 ± 3.47
6
150.7 ± 4.4
3.87 ± 0.26
26.60 ± 2.06"
6
146.8 ± 3.3
144.5 ± 5.2
3.71 ± 0.20
3.63 ± 0.16
34.50 ± 3.42
38.74 ± 3.49
6
6
7
All eels were acclimated to fresh water during the course of experiments unless otherwise shown. Values are means ± SE. Duncan's multiple range test was used for a comparison of the means, a = P
< 0.05 compared with intact controls, b = P < 0.05 compared with sham operated control. For experimental details see Butler and Carmichael (1972).
anterior cardinals are also excised. Venous
return from the kidney to the heart is impaired. Following this operation there was a
significant reduction in GFR (36.1 ± 4.4 to
12.4 ± 1 . 8 ml/kg body wt/day) and urine
flow rate (26.4 ± 3.5 to 12.9 ± 2.6 ml/kg
body wt/day). However, this comparison is
dubious because intact eels were used as controls (see Rankin, 1967). Confusion arises
since the same control eels were later reported to have been injected with saline
(Chan et al., 1969) and finally as being shamoperated controls (Chester Jones et al.,
1969).
If adrenalectomized freshwater silver eels
were maintained on daily im injections of
cortisol (200 /ig/kg body wt), the GFR and
urine flow rate remained within the normal
range for intact controls, as did urinary Na+
and K+ concentrations (Chan et al., 1969).
In a second experiment, cortisol, 11-deoxycorti coster one, 11-deoxycortisol, or aldosterone (200 ug/kg body wt/day) were injected
into different groups of adrenalectomized
eels for 7 or 8 days after the operation. Only
cortisol seemed to prevent the changes in
renal function that usually followed surgical
adrenalectomy, i.e., an increase in urinary
Na+ concentrations and a gradual decrease
in urine flow rate. An increased urinary K+
concentration was the only measurable
change that followed the injection of 11deoxycorticosterone or aldosterone to adrenalectomized eels. These experiments were
adequately "controlled" since the comparisons were made between non-treated adrenalectomized eels and adrenalectomized eels
that were given a daily injection of the particular test steroid. The method for shamadrenalectomy as used in these experiments
has never been described in the literature.
That is important since the conclusion that
adrenalectomy, and not the surgical procedure, had produced the observed changes
in renal function was the basis for further
comparisons with the test steroids.
A few investigators have tried to circumvent the technical difficulties with surgical
adrenalectomy of teleost fishes by removing
the pituitary gland and then studying renal
function. This experimental approach rests
on the assumption that a pituitary-adrenocortical axis is present in the species being
studied; problems arise because changes
may reflect the absence of any number of
the pituitary hormones. Butler (1966) reported that urinary Na+ concentrations
increased and urine flow rates decreased 3
weeks after hypophysectomy of freshwater
A. anguilla. However, the lack of informa-
866
DAVID GORDON BUTLER
tion about GFR and plasma electrolyte
concentrations precluded the calculation of
percentage tubular reabsorption of electrolytes or water. Changes in other species were
similar. Urine flow rates decreased and urinary Na+ concentrations increased after
hypophysectomy of Fundulus kansae (Stanley and Fleming, 1966) and C. auratus (Lahlou and Sawyer, 1969). In the latter experiment, hypophysectomized goldfish were
injected with ACTH (1.0 IU/per fish/day;
body wt 40-120 g), but this treatment failed
to re-establish the normal pattern of renal
function, possibly because the dose was too
high and the adrenocortical tissue became
exhausted. The argument that adrenocortical secretions influence renal function in
some teleosts is supported by the finding
that elevated urinary sodium concentrations
in hypophysectomized goldfish were lowered
to normal by the injection of cortisol (Lahlou and Giordan, 1970).
I have recently studied kidney function in
hypophysectomized freshwater eels (A. rostrata). Three weeks after removal of the
pituitary gland, both the GFR and urine
flow rates were lower than in sham-hypophysectomized eels. There was, however, no
change in the relative free-water clearance
which meant that hypophysectomy had not
produced a water diuresis (Table 6). There
were significant increases in the fraction of
filtered Na+ (174%), K+ (68%), and Cl
(267%) and total osmolytes (130%) excreted
in the urine of hypophysectomized eels
(Table 7). Hypophysectomy was confirmed
histologically and with measurements of
plasma cortisol concentrations which decreased from 2.78 ± 0.32 to 1.00 ± 0.40 (SE)
Mg/100 ml (Butler, 1973). These changes in
renal function may be due to inactivity of
the adrenocortical tissue and impaired secretion of a mineralocorticoid, possibly cortisol.
are absorbed through the wall of the intestine, and ions are subsequently excreted
by the gills and, to a lesser extent, by the
kidney (see Sharratt et al., 1964; House and
Green, 1965; Potts and Evans, 1967; Maetz,
1970; Parry, 1966). The transport of water
across the intestine involves a solute-solvent
interaction. Water can flow along an osmotic gradient or it can be absorbed against
a gradient (House and Green, 1965; Maetz,
1970). When the drinking rate increases,
there is a concommitant increase in the
active transport of salt, which, because of
the solute-solvent interaction, results in an
increased uptake of water. Both Maetz and
Skadhauge (1968) and Utida et al. (1967)
have shown that, in the eel, the permeability of the gut to water as well as the ionic
absorptive capacity of the gut increases as
a function of an increased external salinity.
The specific activity of alkaline phosphatase was three times as great in the intestines of sea water-adapted Japanese eels as
in those of fresh water-adapted eels, the increase being correlated with an increased
intestinal absorption of water (Oide and
Utida, 1967, 1968; Oide, 1970). However, it
is too early to implicate alkaline phosphatase activity in the transport mechanism
since there is no sound evidence that the
enzyme is linked to intestinal transport in
other vertebrate animals. The pituitaryadrenocortical axis may be related to alkaline phosphatase activity since hypophysectomy of eels was followed by a decrease in
intestinal activity of Cl~ activated alkaline
phosphatase (Utida et al., 1966). Adrenalectomy of the rat is followed by a decrease
in intestinal alkaline phosphatase activity
(Rodgers et al., 1967; Watson et al., 1967).
Hypophysectomy prevented the accelerated uptake of salt and water by the intestine of Japanese eels that would normally
occur when the fish were transferred from
fresh water to sea water (Hirano, 1967);
Intestinal transport of water and ions; pos- Hirano et al., 1967). If intact A. japonica
sible involvement of adrenocortical steroids were injected with ACTH, water uptake by
the intestine increased within 24 hours, the
Smith (1930) observed that sea water- response being dose-dependent (Hirano and
acclimated eels counterbalance the loss of Utida, 1968). Other hypophysial hormones
water through gill membranes and kidney including prolactin, TSH, growth hormone,
by drinking the medium. Water and ions oxytocin, lysine vasopressin, and vasotocin
867
ADRENAL GLAND OF FISHES
were ineffective. Only cortisol or cortisol
acetate augmented the rate of uptake of
water by intestinal segments in vitro (Hirano and Utida, 1968, 1971). A number of
other steroids including aldosterone, deoxycorticosterone acetate, corticosterone, and
cortisone acetate had no demonstrable effect
(Hirano and Utida, 1968).
Since water flow is solute-linked and since
the active transport of ions through the intestine is energy dependent, it is possible
that Na+/K+-ATPase activity is indirectly
involved in the regulation of water uptake.
Oide (1967) reported that intestinal Na+/
K+-ATPase activity doubled when eels were
transferred to sea water. Taking this hypothesis one step further, it is possible that Na+ /
K+-ATPase activity and/or induction is
regulated, in part, by the pituitary-adrenocortical axis. For example, intestinal Na+/
K+-ATPase activity decreased by hypophysectomy and increased when F. heteroclitus
were injected with cortisol (Pickford et al.,
1970). Cortisol injections (4 mg/kg body
wt/day for 7-10 days) also increased intestinal Na+/K+-ATPase activity in freshwater North American eels (A. rostrata)
(Epstein et al., 1971). Gaitskell and Chester
Jones (1970) reported that adrenalectomy
impaired the transport of water by eel intestine and that cortisol injections re-established rates of uptake, i.e., they were not
significantly different from those in shamadrenalectomized controls. These experiments might have been strengthened by
measurements of the rate of water uptake
by the intestine in vitro following the addition of the test compound cortisol to the
incubation medium (see Hirano and Utida,
1968). Adrenalectomy, which involves removal of extensive sections of the postcardinals together with head kidney or injections of cortisol could both modify intestinal
blood flow. Metabolic processes in the transport cells would be affected and these results might have little to do with the endocrine control of intestinal water-flow.
2-2
co t -
IS
t- '
I o
' +1
©
-*
i
" '+1 S
q
rA
H
J.I
j
id L-;
i^ xt
'I
id •
Hi •
ri
«
id
c>
ss.
d
O O
O O
Cl i—t
O O
between
adrenocortical
i
CO rH
rid
in
o
U3 l ^
Adrenocortical steroids and intermediary
metabolism in fishes
Relationships
o
I
868
DAVID GORDON BUTLER
TABLE 7. Fractions of filtered solutes excreted by freshwater eels three weelcs after hypophysectomy.
O solute /GFR X 100%
Controls
Sham hypox
Hypox
n
Na
K
11
11
11
9.94 ± 1.07
11.94 ± 1.23
32.76 ± 3.39»'b
15.77 ± 1.71
26.68 ± 5.75
44.79 ± 4.38a'b
Cl
urea
7.77 ± 1.45
48.01 ± 4.73
5.49 ± 0.78
59.10 ± 4.83
16.13 ± 3.18"'" 65.25 ± 10.44
niilliosmoles
14.41 ± 1.15
15.57 ± 0.93
35.84 ± 3.15"'*
Values are means ± SE. a z= P < 0.05 compared with intact control eels; b = P < 0.05 compared
with sham hypophysectomized eels using Duncan's multiple range test. (Prom Butler, 1973).
steroids and metabolic processes in higher
vertebrates, particularly the eutherian mammals, have received a good deal more study
than in fishes. Most of the fish work has been
based on measurement of changes in carbohydrate, protein, and fat composition in tissues following hypophysectomy, adrenalectomy, injection of test steroids, and the injection of chemical inhibitors of adrenocortical function.
As a rule, hypophysectomy is not followed
by a significant change in blood glucose
concentrations in cyclostomes, M. glutinose
(Falkmer and Matty, 1966b); elasmobranchs,
Mustelis canis (Orias, 1932); or teleosts, Cottus scorpio (Falkmer and Matty, 1966a),
Cyprinus carpio (Mazeaud, 1964), A. rostrata
(Butler, 1968). There are exceptions. Umminger (1971) has observed that serum glucose levels in F. heteroclitus decreased from
69.6 ± 3.2 to 55.6 ± 4.0 mg/100 ml 15 weeks
after hypophysectomy. These findings offer
no evidence that the pituitary-adrenocortical
axis controls blood glucose levels in fish. It
is possible, however, that hypoglycemia followed removal of the pituitary, but the
levels were quickly re-adjusted by the release of hyperglycemic factor such as glucagon or adrenalin, into the circulation.
Thorny skates (R. erinacea) have been
interrenalectomized (Hartman et al., 1944;
Idler et al., 1969). In both experiments, liver
glycogen concentrations tended to be lower
than in sham-interrenalectomized skates,
but were very similar to the intact controls.
Both groups of workers suggested that there
was no evidence for the interrenal control
of liver glycogen deposition, but this may
not be the case. Sham-operated controls are
used to account for surgical stress and other
variables, and the removal of the interrenal
did tend to decrease the liver glycogen con-
centration. Liver glycogen concentrations
are often reduced in teleosts after hypophysectomy or adrenalectomy (for eels see
Hatey, 1951; Butler, 1968).
Results following the injection of steroids
often seem contradictory. Both corticosterone and cortisol produced hyperglycemia
in intact dogfish, S. acanthius, but only corticosterone increased liver glycogen concentrations (1432 ± 458 cf. 534 ± 294 mg glucose/ 100 g wet weight liver) (Wright, 1961).
Perhaps the greater response to corticosterone than to cortisol occurred because laOH corticosterone is the major circulating
steroid in elasmobranch fishes, whereas 17OH steroids have not been identified, on the
basis of adequate chemical characterization,
in the blood of sharks, skates, or rays. However, in holocephalians, the major circulating interrenal steroid is cortisol. When cortisol or corticosterone were injected into a
holocephalian, H. colliei, neither blood
pressure nor liver glycogen concentrations
changed, there being only a trace amount of
glycogen in the liver. Wright (1961) also
injected cortisol into thorny skates, but
failed to observe any change in liver glycogen, a finding that was later confirmed
by Idler et al. (1969).
Following the injection of corticosteroids
blood glucose and liver and/or muscle glycogen concentrations increased in a number
of species of teleosts: Opsanus tau (Nace,
1955), S. gairdneri (Robertson et al., 1963;
Hill and Fromm, 1968), Nopterus nopterus
(Kumar et al., 1966), A. rostrata (Butler,
1968).
The argument that ACTH may normally
be a glycogenolytic hormone in Poecilia
latipinna and T. mossambica (see Chester
Tones et al., 1969) may not be entirely justified on the basis of some of the experimental
ADRENAL GLAND OF FISHES
findings (Ball et al., 1965, 1966; Swallow and
Fleming, 1966). In Poecilia, for example,
liver glycogen concentrations were elevated
in both starved and fed fish that had been
hypophysectomized, but since comparisons
were made with intact fish rather than shamoperated controls, one cannot be certain
that elevated glycogen concentrations were
not imposed by the stress of the operation.
Twenty-three days after hypophysectomy of
Poecilia, liver glycogen concentrations had
increased from 1.38 ± 0.28% (intact controls) to 7.39 ± 1.68%. A number of mammalian preparations including GH, prolactin, TSH, and ACTH were injected into
groups of hypophysectomized fish, but only
ACTH changed liver glycogen concentrations. Changes that were observed following injection of ACTH, i.e., the reduction
of liver glycogen concentration from 8.73 ±
0.58% to 6.70 ± 0.57% may have been due
to the exhaustion and failure of adrenocortical tissue, since the dose of 75 IU/100 g
body wt/day every second day for 2 weeks
was bound to have a dramatic effect on
adrenocortical function. If the pituitary
gland was transplanted to epaxial muscle,
liver glycogen concentrations were lower
than those observed in hypophysectomized
fish (7.92 ± 0.82% compared with 13.62 ±
1.72%). This finding shows that hypophysectomy lowered the liver glycogen content,
but it does not implicate ACTH in the control of glycogen metabolism. However, the
findings of Swallow and Flemming (1966)
show that much lower doses of ACTH (0.4
IU per fish) injected into T. mossambica
prevented the increased net synthesis of glycogen normally seen in intact fish following
the injection of glucose. The rate of incorporation of C14 glucose into glycogen, however, was unaffected, which may mean that
ACTH was glycogenolytic. If this assumption is correct, it is difficult to explain the
failure of the same dose of ACTH to decrease the glycogen concentrations in "well
fed" intact fish.
In N. nopterns (Kumar et al., 1966), injections of 15 IU ACTH/100 g body wt increased liver glycogen concentrations from
56.63 ± 5.50 to 87.66 ± 5.34 mg/g wet liver
during the first 90 m.in, then the concentra-
869
tion fell to 49.36 ± 3.36 at 180 min, the final
concentration being within the normal
range.
In mammals, the injection of corticosteroids is followed by a significant increase in
the activity of a number of enzymes, deposition of glycogen in the liver, and an increased renal excretion of nitrogen. Processes which accelerate gluconeogenesis are
associated with an increase in glutamicpyruvic transaminase activity (Beaton et al.,
1957; Rosen et al., 1959; Fajans, 1961; Bellamy and Leonard, 1964). Gluconeogenesis
in the goldfish (C. auratus), stimulated by
injections of cortisol, was also accompanied
by increased glutamic-pyruvic transaminase
activity together with an increased rate of
excretion of ammonia (Storer, 1967). Cortisol, in doses ranging from 1-21 mg/100 g
body wt, increased the rate of nitrogen elimination in Gobius cephalarges, G. melanostomum, and Trachurus and mediterraneus.
A similar response followed treatment with
ACTH. These authors suggested that corticosteroids had stimulated the catabolism of
protein and enhanced gluconeogenesis (Pora
and Precup, 1971). Gluconeogenesis has also
been observed in starved A. rostrata following daily im injections of cortisol (5 mg/kg
body wt) for three weeks; both liver and
muscle glycogen concentrations increased
significantly.
Only a slight increase in hepatic alanine
amino transferase was observed in goldfish
after injection of cortisol. Moreover, cortisol failed to increase hepatic tyrosine transaminase in the white bass (Roccus chrysops)
and the black crappie (Proxomis nigromaculatus) (Chan and Cohen, 1964). Janssens (1970) observed a similar response to
cortisol injections by starved Xenopus laevis
even though there was no change in the activity of either tyrosine or alanine amino
transferase. However, since cortisol treatment was followed by a significant increase
in liver glycogen in starved goldfish (Storer,
1967) and toads (Janssens, 1970), changes
in amino transferase activity may not have
been detectable under those experimental
conditions.
It is well established that salmon plasma
corticosteroid concentrations, increase dur-
870
DAVID GORDON BUTLER
ing the latter stages of the spawning migration (Hane and Robertson, 1959; Idler et
al., 1959; Robertson et al., 1961; Schmidt
and Idler, 1962) and that, correlated with
this increase in substantial conversion of
protein, particularly muscle protein, to carbohydrate (Miescher-Riisch, 1880; Pentegov
et al., 1928; Greene, 1919, 1921, 1926; Idler
and Clemens, 1959; Chang and Idler, 1960).
Fasting, starved salmon are, in this manner,
provided with energy for nervous and muscular activity, together with a carbon source
for the development of the maturing gonads
(Black, 1958).
Recently, we have investigated some of
the metabolic changes that occur in mature
A. rostrata after several months of starvation
at 15 C. One of the most significant changes
was that of lypolysis; plasma glycerol and
free fatty acid levels increased, a change that
also accompanied starvation in rainbow
trout (Bilinski and Gardner, 1968). Liver
glycogen was depleted, but there were no
significant differences in blood glucose concentrations between starved and fed eels.
Gluconeogenesis may have partially replaced carbohydrate reserves, but both
plasma protein concentrations and glutamic-oxalic transaminase activity did not
change in comparison with fed eels.
Mature female A. rostrata that had been
starved for 8 months and acclimated during
that time to 5 C were selected from stock,
assigned to two experimental groups, and
transferred to experimental tanks (10 C).
One group of eels was given daily injections
of saline, and the other were given daily
injections of cortisol (5 mg/kg body wt suspended in 1.15% saline) for 10 days. At the
end of this period, the eels were killed by
decapitation and the tissues were analyzed.
Results are given in Table 8.
Cortisol injections were followed by a
hyperglycemia in these starved freshwater
eels, but the liver glycogen concentration
was not significantly different from either
the saline-injected controls or from fed eels
that had been acclimated to 15 C (Mayerle
and Butler, 1971). In an earlier experiment
in which eels were starved for 2 months then
given 21 daily injections of cortisol (5 mg/
kg body wt), blood glucose also increased,
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ADRENAL GLAND OF FISHES
but in addition, there was a significant increase in liver glycogen concentrations (Butler, 1968).
In the present experiments (Table 8),
serum glutamic-oxalic transaminase activity
increased, suggesting that cortisol had increased protein catabolism. Carbon generated through the breakdown on protein and
deamination of amino acids, in addition to
maintaining liver glycogen levels in these
starving eels, may have been directed into
the synthesis of fatty acids. This would account for the increased fatty acid levels in
blood and increased concentration of lipid
in eel liver. A great deal more work must
be done on the endocrine control of intermediary metabolism in fishes before unifying principles will emerge.
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