General and Comparative Endocrinology 146 (2006) 62–68
0016-6480/$ - see front matter 2005 Elsevier Inc. All rights reserved.
Role of corticotropin-releasing hormone as a thyrotropin-releasing
factor in non-mammalian vertebrates
Bert De Groef, Serge Van der Geyten, Veerle M. Darras, Eduard R. Kühn¤
Laboratory of Comparative Endocrinology, K.U. Leuven, B3000 Leuven, Belgium
Received 5 July 2005; revised 23 September 2005; accepted 21 October 2005
Available online 9 December 2005
The Wnding that thyrotropin-releasing hormone does not always act as a thyrotropin (TSH)-releasing factor in non-mammalian verte-
brates has led researchers to believe that another hypothalamic factor may exhibit this function. In representatives of all non-mammalian
vertebrate classes, corticotropin-releasing hormone (CRH) appears to be a potent stimulator of hypophyseal TSH secretion, and might
therefore function as a common regulator of both the thyroidal and adrenal/interrenal axes. CRH exerts its dual hypophysiotropic action
through two diVerent types of CRH receptors. Thyrotropes express type 2 CRH receptors, while CRH-induced corticotropin (ACTH)
secretion is mediated by type 1 CRH receptors on the corticotropic pituitary cells. The stimulating eVect of CRH on both TSH and
ACTH release has profound consequences for the peripheral action of both hormonal axes. The simultaneous stimulation of the thyroi-
dal and adrenal/interrenal axes by CRH, possibly Wne-tuned by diVerential regulation of the expression of the diVerent CRH receptor iso-
forms, provides a potential mechanism for developmental plasticity.
2005 Elsevier Inc. All rights reserved.
Keywords: CRH; TRH; TSH; Pituitary; Thyrotrope
In 1955, SaVran and Schally published a landmark paper
with the Wrst experimental proof of the existence of hypo-
thalamic hormones regulating anterior pituitary function,
as postulated by Harris (1948). They showed the presence
of a substance in hypothalamic and neurohypophyseal tis-
sue that stimulates the anterior pituitary cells to release cor-
ticotropin (ACTH). Despite major eVorts, only more than
25 years later, ovine corticotropin-releasing hormone
(oCRH) was isolated and sequenced by Vale and colleagues
(1981). Soon it became clear that CRH was the major regu-
lator of the hypothalamo–pituitary–adrenal (interrenal)
axis leading to the release of hypophyseal pro-opiomelano-
cortin (POMC)-derived peptides in all vertebrates tested. In
addition, several studies have shown that CRH, and the
family of CRH-related peptides, are the main integrators
of the stress response in vertebrates. Less than 10 years
after its characterization by Vale and co-workers, compara-
tive endocrinological research
important function of CRH: the control of thyrotropin
2. TRH—a TSH-releasing factor, but not always
Thyrotropin-releasing hormone (TRH) was the Wrst
hypothalamic hormone to be isolated and characterized,
and this was done in 1969, simultaneously by two research
groups after a breathtaking scientiWc battle (Burgus et al.,
1969; Schally et al., 1969). The puriWed preparations from
numerous ovine and porcine hypothalamic fragments were
extremely potent in stimulating TSH release from the mam-
malian pituitary both in vivo and in vitro. Ever since, TRH
has been regarded as the main regulator of TSH secretion
in mammals. The TSH-releasing activity of the tripeptide
was soon conWrmed in other vertebrates.
*Corresponding author. Fax : +32 16 32 42 62.
E-mail address: email@example.com (E.R. Kühn).
B. De Groef et al. / General and Comparative Endocrinology 146 (2006) 62–68
Only a couple of years after its discovery in mammals,
TRH was demonstrated to have a TSH-releasing activity in
the chicken using bioassays (Bolton et al., 1974; Breneman
and Rathkamp, 1973; Scanes, 1974). Kühn and Nouwen
(1978) showed that both cold treatment and injection of
TRH increases plasma thyroid hormones (thyroxine, T4,
and 3,3?,5-triiodothyronine, T3) in 40-day-old cockerels;
Kamis and Robinson (1978) made the same observation
after TRH injection in mature Japanese quail Coturnix
coturnix. Later TRH injections were also shown to increase
plasma T4 concentrations in both chicken embryos and
growing chicks (for a review, see Kühn et al., 1993). Using a
subtractive radioimmunoassay strategy, Berghman et al.
(1993) demonstrated the involvement of TSH in this inter-
action; a 3-fold increase of calculated TSH index upon
TRH challenge was recorded from chicken pituitaries in a
perifusion experiment. TRH was also found to upregulate
TSH-? mRNA expression in pituitary cultures of the Mus-
covy duck (Hsieh et al., 2000), but no eVect was seen on
TSH-? mRNA expression in chicken pituitary cell culture
after a 20–24h TRH treatment (Gregory and Porter, 1997).
By means of a combined in situ hybridization and immuno-
cytochemical staining, we localized TRH receptor mRNA
in chicken thyrotropes (De Groef et al., 2003a). This Wnding
suggests that the TSH-releasing ability of TRH in the
chicken is likely to be the result of a direct neuroendocrine
eVect on the thyrotropes. However, in several of the above-
mentioned studies, TRH injection was found to increase
preferentially T3 instead of T4, the rise of T3 being mediated
through the growth hormone (GH)-releasing action of
TRH in birds and the subsequent inhibiting eVect of GH on
T3 breakdown (Darras et al., 1992). Moreover, in adult
chickens, TRH treatment caused no stimulation of T4
secretion whatsoever; only a signiWcant increase in plasma
T3 and GH was noted (Kühn et al., 1988, 1991). These
observations might indicate that the primary action of
TRH in birds is a somatotropic one, rather than a thyrotro-
pic one. An autoradiographic study of the pituitary of chick
embryos and adult chickens reveals a preferential binding
of TRH to the somatotropes, located in the caudal lobe of
the anterior pituitary, compared to the binding to the thy-
rotropes, located in the cephalic lobe. Moreover, these lat-
ter binding places are much more pronounced in embryos
(Kühn et al., 1993).
In 1987, Preece and Licht found a signiWcant 2- to 6-fold
increase in TSH secretion by hemipituitaries of adult turtles
(Chrysemys picta) when stimulated in vitro with synthetic
TRH. Although previous in vivo studies had suggested that
TSH secretion is not aVected by TRH in turtles, these data
indicate that the dose sensitivity of the chelonian pituitary
is comparable with that of mammalian and avian glands.
Also in the lizard Anolis carolinensis, TRH was found to
stimulate TSH release by the pituitary, both in vivo and
in vitro (Licht and Denver, 1988). Like in birds, these exper-
iments indicate that functional TRH receptors are
expressed by the reptilian thyrotropes, but evidence for the
role of TRH in endogenous TSH regulation is still lacking.
For a long time, TRH has been considered to exhibit no
TSH-releasing activity in amphibians, as several attempts
to induce metamorphosis with TRH were unsuccessful
(Norris and Dent, 1983). In 1982, however, Darras and
Kühn found that an intravenous injection of TRH is able
to increase plasma T4 and T3 levels in adult Rana ridibunda
frogs for several hours. Moreover, since no eVect was dem-
onstrated in frogs with their pars distalis removed, it is
highly probable that TRH exerted this action through a
stimulatory eVect on the release of TSH from the pituitary
gland. Denver (1988) showed indeed that pituitaries from
adult Rana pipiens, Hyla regilla, and Xenopus laevis,
exposed to TRH, increase their TSH secretion in a dose-
dependent manner, as judged by the thyrotropic activity of
the culture medium. Similarly, TRH is able to induce a sig-
niWcant increase in plasma T4 levels in metamorphosed
axolotls, but not in neotenic ones (Jacobs et al., 1988). A
stimulation of Rana perezi thyrotropes by TRH, measured
by ultrastructural morphometry of the cellular biosynthetic
machinery, was demonstrated by Castaño and co-workers
(1992), although the response of the thyrotropes towards
TRH is more delayed than that of prolactin cells. Together
with the above-mentioned results in reptiles, these Wndings
argue against the hypothesis that the TSH-stimulating
activity of TRH evolved relatively recently in association
with endothermy. Furthermore, it can be concluded that
TRH only has a TSH-releasing action in metamorphosed
but not in larval amphibians. Further investigation con-
Wrmed this conclusion. Denver and Licht (1989b) showed
that TRH stimulates the release of thyrotropic bioactivity
from adult bullfrog (Rana catesbeiana) pituitaries but not
from larval ones. Recently, this Wnding was conWrmed using
a homologous bullfrog TSH radioimmunoassay (Okada
et al., 2004).
Although TRH was recently found to increase in vitro
hypophyseal TSH-? mRNA expression in bighead carp
(Aristichthys nobilis) and in Japanese eel (Anguilla japonica)
(Chatterjee et al., 2001; Han et al., 2004), apart from some
positive reports (e.g., Eales and Himick, 1988), TRH treat-
ment did not stimulate TSH release in teleosts, lungWsh or
hagWsh (for an overview, see Larsen et al., 1998). In the sea
bass Dicentrarchus labrax (Perciformes), TRH Wbers were
found to be numerous in the posterior neurohypophysis,
forming varicosities between groups of melanotropes in the
pars intermedia, but no clear relationship was seen between
TRH Wbers and the thyrotropes, or any other cell type of
B. De Groef et al. / General and Comparative Endocrinology 146 (2006) 62–68
the pars distalis (Batten et al., 1990). A similar observation
was made in the carp Cyprinus carpio (Cypriniformes)
(Hamano et al., 1990). It appears that TRH is not a major
TSH-releasing factor in Wsh, but the in vivo eVects of TRH
on TSH release in Wsh need further investigation.
3. CRH as common neuroregulator of the thyroidal and
In search of hypothalamic TSH-releasing factors in
amphibians, Denver (1988) discovered that ovine CRH sig-
niWcantly stimulates TSH release by adult male R. pipiens
pituitaries in vitro, as quantiWed by the T4-releasing eVect
of the medium on cultured frog thyroid glands. Moreover,
CRH produced a much greater TSH output than TRH or
mammalian gonadotropin-releasing hormone did. This
important observation suggests that CRH controls both
ACTH and TSH release in amphibians. On a cellular level,
this can be seen by the activation of the secretory pathway
in corticotropes and thyrotropes in CRH-treated pituitaries
from adult R. perezi (Malagon et al., 1991), and by the
decrease in TSH and ACTH immunoreactivity in pituitar-
ies from CRH-treated Bufo arenarum larvae (Miranda
et al., 2000). Human and ovine CRH increase T4 levels in R.
perezi larvae (Gancedo et al., 1992), and homologous Xeno-
pus CRH (xCRH) increases whole body amounts of both
corticosterone and T4 in prometamorphic larvae (Boorse
and Denver, 2004). In vitro treatment of pituitaries from
Xenopus tadpoles with xCRH caused an increase of TSH
immunoreactivity in the culture medium (Boorse and Den-
ver, 2004), and homologous CRH stimulated the in vitro
secretion of authentic TSH in adult bullfrogs (Ito et al.,
In 1989, results in chickens pointed towards a similar
interaction of CRH with the thyroidal axis in birds. When
injected in 18-day-old chicken embryos, ovine CRH
caused not only a rise in plasma corticosterone levels, but
also a marked increase in plasma T4 and somewhat later
T3 (Meeuwis et al., 1989). The involvement of TSH in this
eVect was later conWrmed in vivo and in vitro (Geris et al.,
1996, 1999). These experiments indicated that CRH is a
potent TSH-releasing factor in the chicken, and that it
exerts this function directly at the level of the pituitary.
On an equimolar base, ovine CRH appeared twice as
potent as TRH in inducing TSH release by pituitaries
from one-day-old chicks in vitro (De Groef et al., 2005).
CRH shows an in vitro TSH-releasing action both before
and after hatch, and the magnitude of the hypophyseal
response increases with age (Geris et al., 2003). However,
the relative increase in TSH secretion induced by CRH is
much lower in adult birds due to a markedly higher basal
TSH secretion. The recent cloning of chicken proCRH
cDNA showed that the chicken CRH peptide is identical
to human and rat CRH (Vandenborne et al., 2005). Pre-
liminary data indicate that this peptide, like ovine CRH, is
able to stimulate TSH release in young chicks (B. De
Groef, unpublished results).
Although by now the TSH-releasing capacity of CRH
has been conWrmed by direct homologous TSH assays in
several non-mammalian vertebrates, this phenomenon was
demonstrated unequivocally for the Wrst time in reptiles.
Ovine CRH was shown to possess a TSH-releasing activity
as it stimulated TSH secretion by in vitro cultured pituitary
cells from both hatchling and adult turtles (Denver and
Licht, 1989a, 1990, 1991). Larsen et al. (1998) used the same
technique to examine the hypothalamic control of TSH
release in the coho salmon Oncorhynchus kisutch. Whereas
TRH and gonadotropin-releasing hormone did not stimu-
late TSH secretion, both oCRH and the CRH-related pep-
tide sauvagine caused a signiWcant and dose-dependent
increase in TSH secretion. Another CRH-related peptide,
carp urotensin I, was also found to be highly stimulatory.
Pretreatment with the non-speciWc CRH receptor blocker
?-helical CRH(9–41) suppressed the TSH-releasing activity
of oCRH, suggesting the involvement of a functional CRH
4. Molecular mechanism of the dual hypophysiotropic action
As CRH was shown to exert its TSH-releasing eVect at the
level of the pituitary, we studied the expression of CRH
receptors (CRH-Rs) in the chicken pituitary (De Groef et al.,
2003a,b). Type 1 CRH receptor (CRH-R1) expression is con-
Wned to corticotropic cells, whereas thyrotropes express only
type 2 CRH receptors (CRH-R2). The involvement of CRH-
R2 in the transduction of the CRH stimulus for TSH release
was further conWrmed as the selective CRH-R2 agonist uro-
cortin III also stimulated TSH release, and the selective
CRH-R2 blocker antisauvagine-30 completely abolished the
TSH-releasing action of CRH (De Groef et al., 2003b). The
negative feedback of thyroid hormones on CRH-induced
TSH release (Geris et al., 1999) might be mediated through a
down-regulating eVect on CRH-R2 expression (De Groef
et al., 2005). Corticosterone on the other hand, is not able to
diminish the TSH-releasing action of CRH (Geris et al.,
1999), suggesting that CRH-R2 expression on the thyro-
tropes is not inXuenced by corticoids.
Recent experiments using bullfrog anterior pituitary cell
cultures indicate that CRH-induced TSH release is also
mediated by CRH-R2 in amphibians. In bullfrog, the
CRH-R2-speciWc ligands urocortin II and III markedly
stimulated TSH secretion, whereas CRH-induced TSH
release was totally blocked by the CRH-R2-speciWc antago-
nist antisauvagine-30. The CRH-R1-speciWc blocker anta-
larmin showed no eVect on the secretion of TSH (R. Okada,
personal communication). These experiments indicate that
the CRH-R expression pattern in the chicken might be rep-
resentative for all non-mammalian vertebrates: CRH
induces TSH release through activation of CRH-R2 on the
thyrotropes, and stimulates ACTH secretion by binding
CRH-R1 expressed by the corticotropes. Both CRH recep-
tors are likely to be diVerentially regulated by thyroid hor-
mones, corticoids, and other factors.
B. De Groef et al. / General and Comparative Endocrinology 146 (2006) 62–68
5. Roles for the dual hypophysiotropic action of CRH
The thyroidal and adrenal/interrenal axes are not only
closely interwoven at the level of the hypothalamus in non-
mammalian vertebrates. The dual hypophysiotropic action of
CRH seems to have profound eVects on the peripheral hor-
mone function of both axes as well. The same suggestion can
be made for the dual releasing capacity of TRH in the
chicken, as—like corticosteroids—GH has profound periphe-
ral eVects on the control of deiodination of thyroid hormones.
Thyroid hormones play an important role in many cru-
cial developmental events in all vertebrate classes. In preco-
cial birds, high T3 levels at the end of incubation have been
showed to be essential for yolk sac retraction, functional
maturation of the lungs, and the initiation of pipping and
hatching (Decuypere et al., 1990). These high levels are
mainly obtained by increasing hepatic type I deiodinase
(D1) and decreasing the type III deiodinase (D3), responsi-
ble for T3 production and degradation, respectively. In
embryonic chickens, corticosterone and GH will inXuence
thyroid hormone metabolism by decreasing D3 (Darras
et al., 1992, 1996). The hepatic D1 activity, mRNA, and
protein levels, however, remain unaVected, following
administration of either corticoids or GH, whereas these
parameters decrease sharply for D3 (Van der Geyten et al.,
1999; Verhoelst et al., 2004a). An increased hepatic T4
uptake—and hence T4 to T3 conversion—can be excluded,
since T4 concentrations in the liver decrease after corticoid
treatment (Reyns et al., 2005). Although the very rapid
downregulation of hepatic D3 activity after corticoid and
GH treatment seems to favor the involvement of posttrans-
lational enzyme inactivation, a series of experiments includ-
ing blocking gene transcription via actinomycin D or
protein synthesis via cycloheximide showed that GH and
corticoids directly inhibit hepatic D3 gene transcription.
This downregulation by corticoids seems to be tissue-spe-
ciWc, since the in vitro activity of neither brain nor kidney
D3 is aVected (Van der Geyten et al., 1999, 2001; Verhoelst
et al., 2004a). Whereas brain D3 activity is unaVected by
corticosteroids, glucocorticoids (but not GH) stimulate the
overall cerebral type II deiodinase (D2) expression (locally
converting T4 into T3). Corticoids increase brain D2 at the
activity and protein level, as well as at the mRNA level
(Van der Geyten et al., 2001; Verhoelst et al., 2004b).
The same interaction exists in amphibians, as shown by
the signiWcant decrease in hepatic D3 activity and increase
in brain D2 in neotenic axolotls (Ambystoma mexicanum)
treated with corticoids (Darras et al., 2002; Kühn et al.,
2005), and probably also in reptiles as suggested by the
results of Shepherdley and colleagues (2002) using embry-
onic saltwater crocodiles (Crocodylus porosus). Corticoste-
roids are known to potentiate the actions of thyroid
hormone during amphibian metamorphosis. In premeta-
morphic Xenopus larvae, corticosterone administration
upregulates thyroid hormone receptor ? mRNA expression
in the intestine (Krain and Denver, 2004). The synergistic
action of thyroid hormones and corticoids was also demon-
strated in the axolotl. In this neotenic urodele species, low
submetamorphic doses of T4 or dexamethasone, a synthetic
corticoid, were ineVective to induce metmorphosis-like
morphological changes. However, when these submetamor-
phic doses were injected together, morphological changes
were observed within 1 week leading to complete metamor-
phosis (Kühn et al., 2004). Vice versa, thyroid hormones
could be important modulating factors of the activity of the
adrenal/interrenal axis. Carps injected with T4 indeed show
lower basal plasma cortisol levels, corresponding with an
elevated expression of CRH-binding protein in the nucleus
preopticus (P. Klaren, personal communication). No eVect
of T4 was found on TRH, CRH, or CRH-R1. In premeta-
morphic Xenopus larvae, glucocorticoid receptor mRNA is
upregulated by exogenous T3 in the tail but down-regulated
in the brain (Krain and Denver, 2004). Such complex inter-
actions between thyroid hormones and corticoids need a
carefully orchestrated control mechanism, which might be
provided by the common regulating action of CRH.
Amphibian metamorphosis is such a process, as several
studies have demonstrated the need of increasing both
plasma thyroid hormone and corticoid levels to accomplish
metamorphosis. Gancedo et al. (1992) found that certain
metamorphic changes, such as hind limb growth and tail
resorption, are signiWcantly enhanced when R. perezi larvae
are chronically treated with ovine or human CRH. Similar
observations were made by Denver (1993). Treatment with
ovine CRH or the CRH-related peptide sauvagine acceler-
ated metamorphosis in representatives of two diVerent
anuran families, namely R. catesbeiana (Ranidae) and Sca-
phiopus hammondii (Pelobatidae). Injection of antisera
against several CRH-like peptides resulted in a retardation
of the metamorphosis process. Both research groups found
rapidly increasing levels of thyroid hormones after CRH
treatment, suggesting that the acceleration of metamorpho-
sis occurs via stimulation of the thyroidal axis. Like in anu-
rans, metamorphosis is also accelerated by CRH in urodeles.
Daily injection of ovine CRH accelerated metamorphosis in
the tiger salamander Ambystoma tigrinum (Boorse and Den-
ver, 2002). This is not the case in the neotenic axolotl
Ambystoma mexicanum, however. In axolotl, metamorpho-
sis can only be induced when low submetamorphic doses of
T4 are added to the CRH treatment, suggesting that CRH-
induced TSH release is impaired in this neotenic species and
that the main eVect of CRH is to increase corticosteroid
production which might have synergized with the low dose
of administered T4 (Kühn et al., 2005).
In some amphibian species, the rate of metamorphosis is
dependent on environmental stress stimuli. For instance, S.
hammondii larvae live in ephemeral desert pools and accel-
erate metamorphosis when these pools are drying out. This
phenomenon was named adaptive phenotypic plasticity
(Denver, 1999). Denver (1997) mimicked the natural habi-
tat of these frogs in the laboratory by reducing the water
level of the aquaria and observed increased hypothalamic
CRH content in these animals. The elevation of CRH levels
coincided with increasing whole body content of thyroid
B. De Groef et al. / General and Comparative Endocrinology 146 (2006) 62–68
hormones and corticoids. Injection of the CRH receptor
antagonist ?-helical CRH(9–41) or passive immunization
using antisera against CRH-related peptides signiWcantly
reduced the response of the animals to decreasing water
levels. It is thought that environmental cues are translated
in the brain by the CRH signaling system, which in turn is
responsible for the combining activation of the thyroidal
and interrenal axes, leading to an accelerated metamorpho-
sis (Denver, 1997). Currently, no information is available
about a role for CRH-induced TSH secretion in develop-
mental plasticity in other non-mammalian vertebrates.
Nevertheless, a parallel between amphibian metamorphosis
and, for instance, the hatching process in birds is easily
drawn. Amphibian metamorphosis is generally associated
with the changes that prepare an aquatic organism for a
more terrestrial existence, while in hatching the bird
changes from a well-protected aquatic environment to a
more hazardous terrestrial life outside the egg. Both devel-
opmental processes are primarily dependent on marked
changes in thyroid function as greatly increasing thyroid
hormone levels characterize both events. Several environ-
mental parameters are known to inXuence hatching in
chickens, including clicking sounds (Decuypere et al., 1990).
It is possible that these environmental stimuli also exert
their eVect on the timing of hatching through the CRH sig-
naling system, but currently the involvement of endogenous
CRH in the hatching process is still under investigation.
Although the control of TSH secretion has been attrib-
uted mainly to TRH for the past decades, this does not
always seem to be case. The TSH-releasing function of
TRH is far from clear in Wsh, and does not occur in larval
amphibians. CRH appears to have a dual hypophysiotropic
role in non-mammalian vertebrates. Through its ACTH-
and TSH-releasing capacity it controls both the adrenal/
interrenal and the thyroidal axes. Experiments in chickens
and frogs suggest that the underlying receptor mechanism
might be similar in all non-mammalian vertebrates. The
closely interwoven functions of the thyroidal and adrenal/
interrenal axis in certain developmental processes such as
amphibian metamorphosis and hatching in birds, provide a
possible explanation for the common neuro-endocrine con-
trol by CRH. Whether CRH is also a TSH-releasing factor
in mammalian species remains to be demonstrated.
Bert De Groef and Serge Van der Geyten are Wnancially
supported by the Fund for ScientiWc Research—Flanders
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