Download Phylogenetic Distribution and Function of the Hypophysiotropic

AMER. ZOOL., 18:385-399 (1978).
Phylogenetic Distribution and Function of the Hypophysiotropic Hormones
of the Hypothalamus
IVOR M. D.JACKSON
Division of Endocrinology, Department of Medicine,
New England Medical Center Hospital, Tufts University School of Medicine,
Boston, Massachusetts 02111
SYNOPSIS. Following the isolation, synthesis and subsequent development of specific and
sensitive radioimmunoassays for the hypothalamic hormones thyrotropin-releasing hormone (TRH), luteinizing hormone-releasing hormone (LH-RH) and growth hormone
release-inhibiting hormone (somatostatin), it was recognized that these peptides were not
localized solely in the hypothalamus, but were widely distributed throughout the mammalian nervous system. Somatostatin occurs outside the nervous system altogether, being
located in the gastrointestinal tract of vertebrates where it may have a physiologic role in
the secretion of gastrointestinal hormones. TRH, also, has been located outside the
nervous system, occurring in large quantities in the skin ofRana species where it may be of
physiologic importance in skin function. This tripeptide is found throughout the nervous
system of vertebrate and invertebrate species in situations where it has no pituitary-thyroid
function.
These peptides are present in brain synaptosomes and enzymatic degrading systems
have been recognized for each in brain tissue. For TRH, specific receptors and synthesizing activity have been detected outside the hypothalamic-pituitary system. The anatomic
location, phylogenetic distribution, neurophysiologic and behavioral effects strongly
support a role for these substances in neuronal regulation, apart from control of pituitary
secretion. Evolutionary studies, especially of TRH, suggest that their primary function may
be as neurotransmitters.
INTRODUCTION
It is generally accepted that the hormones secreted by the mammalian anterior pituitary are regulated by factors
synthesized and secreted by peptidergic
neurons in the hypothalamus (Reichlin,
1973; Blackwell and Guillemin, 1973). The
isolation and synthesis of three of these
hypothalamic hypophysiotropic factors or
hormones, thyrotropin-releasing hormone
(TRH), luteinizing hormone-releasing
hormone (LH-RH) and growth hormone
release inhibiting hormone (somatostatin)
have provided powerful tools for the investigation of pituitary function and have
permitted the development of specific
radioimmunoassays for the measurement
of these substances at exceedingly low concentrations (Reichlin et al., 1976). A wholly
The original research reported herein was supported by a grant from the National Institutes of
Health, AM 16684.
unanticipated outcome was the finding
that most of neural TRH and somatostatin
are located outside the hypothalamus
(Reichlin et al., 1976; Jackson, 1977;
Jackson, 1978 for review). Further, these
hormones are present in inframammalian
species, though the functional significance
of these substances in such animals essentially remains to be determined. Even
more surprising is the revelation that
somatostatin (Arimura et al., 1975) and
TRH (Jackson and Reichlin, 1977a) exist
in tissues other than the nervous system.
It is believed that the hypothalamic peptidergic neurons are in turn regulated by
neurotransmitters
largely of the
monoaminergic variety, and that the peptidergic neuron acts as a "neuroendocrine
transducer" converting neural information
from the brain into chemical information
(Wurtman, 1971). More recently it has
been recognized that the chemical products of the peptidergic neurons may
themselves act as neurotransmitters (Martin et al., 1975; Jackson, 1977; Jackson,
385
386
IVOR M. D.JACKSON
1978 for review). Still to be determined is
the question whether the same kind of
hormonal feed-back and/or neurotransmitter control characteristic of the hypothalamus is also operative at extrahypothalamic sites.
T h e finding of extrahypothalamic
sources of hypophysiotrophic hormones
provides some support for the view (ependymal tanycyte theory) that a portion of
the releasing hormones reach the primary
portal plexus by trans-median eminence
transport, it being postulated that the releasing hormones are secreted into the
ventricular system, taken up by the lumenal processes of the tanycytes of the
median eminence, and then actively transported for release at the capillary end of
the cell (Knigge and Silverman, 1972).
In this report I will review the more
recent findings concerning the anatomic
and phylogenetic distribution of TRH,
LH-RH and somatostatin and discuss their
extrapituitary functional significance.
THYROTROPIN-RELEASING HORMONE
Thyrotropin-releasing hormone (TRH) radioimmunoassay and mammalian hypothalamic
TRH
Since the early studies of Greer (1951),
and Harris and Jacobsohn (1952), it has
been known that the hypothalamus exerts
an important influence on the regulation
of the pituitary-thyroid axis. Demonstration of the existence of a thyrotropic releasing factor (TRF), as well as its purification from hypothalamic extracts, was provided by Guillemin (1964), and subsequent
reports indicated that TRH activity was
present in whole hypothalamic extracts
and stalk median eminences (SME) of the
sheep and rat (Guillemin et al., 1965; Averill and Kennedy, 1967). Physiologic data
based on electrical stimulation of different
hypothalamic areas (D'Angelo and Snyder,
1963; Martin and Reichlin, 1972) and the
placement of intrahypothalamic pituitary
grafts (Flament-Durand, 1965) suggested
that TRH synthesis might occur diffusely
throughout the mammalian hypothalamus. In 1969 the chemical structure
of TRH was elucidated in the laboratories of Guillemin and Schally and
shown to be a tripeptide amide (P gluHis-Pro NH2), molecular weight 362, following rigorous chemical analysis of large
numbers of ovine and porcine hypothalamic extracts (B0ler et al., 1969;
Burgus et al., 1969). The availability of
chemically pure synthetic TRH led to the
development of radioimmunoassays for
TRH by several groups including those of
Utiger (Bassiri and Utiger, 1972), Wilber
(Montoya et al., 1973), Porter (Oliver et al.,
1973) and myself (Jackson and Reichlin,
1973). The conjugation of TRH to a large
carrier weight protein such as bovine
serum albumin (Bassiri and Utiger, 1972)
or bovine thyroglobulin (Tg) (Jackson and
Reichlin, 1974a) permits the generation of
antibody to TRH in rabbits with a high
degree of sensitivity and specificity. I
utilized Tg as the carrier protein following
reports that this substance augmented the
immunogenicity of small peptides. The
histidine of TRH is readily iodinated by
the Greenwood-Hunter
procedure
(Chloramine T, sodium metabisulfite), and
the labelled hormone is separated from
iodide with gel filtration on Sephadex
G-10. This label is very stable, and I have
used it for immunoassay purposes for
periods of up to 4 months following iodination, with little damage on storage. Delayed addition of I25 I-TRH in my hands
appears to increase sensitivity and charcoal
(0.1%) separation of "bound from "free"
hormone is a simple procedure. The double antibody technique (Bassiri and Utiger,
1972), and polyethylene glycol after short
incubation (Montoya rt al., 1973) have
also been reported to produce satisfactory separation. The levels of immunoreactive (IR)-TRH in the rat
hypothalamus reported from different
laboratories have given values of 2.7-15.7
ng (Bassiri and Utiger, 1974; Jackson and
Reichlin, 1974a; Oliver, et al., 1974). I have
found considerable variation in the
amount of IR-TRH in different groups of
rat hypothalami in different experiments.
These %'ariations may relate to the size of
tissue block in different experiments but
might also reflect seasonal or other differ-
387
DISTRIBUTION OF HYPOTHALAMIC HORMONES
ences. Bassiri and Utiger (1974) have also
reported variations in the levels of
IR-TRH in the hypothalamus when experiments were performed at different time
intervals apart. I have found immunoreactive TRH readily detectable in porcine
hypothalmi (500 pg/mg tissue wet weight),
hamster hypothalami (480 pg/mg tissue
wet weight), and in human stalk median
eminence with values up to 300 pg/mg
tissue (Jackson and Reichlin, 1974). Recent
studies by Okon and Koch (1976), Guansing and Murk (1976) and Kubek et al.
(1977) have demonstrated substantial
quantities of TRH throughout the
hypothalamus and SME of humans.
Abalation of the "thyrotrophic" area of
the hypothalamus induces hypothyroidism
in the rat, but the TRH levels in the
hypothalamus of such lesioned animals
were as much as 35% of the values found
in the controls (Jackson and Reichlin,
19776). The persistence of significant TRH
levels in the hypothalamus following lesion
provides an explanation for the fact that
depression of baseline thyroid function
after such a procedure is never as severe as
that occurring after hypophysectomy.
Studies by Brownstein et al. (1974) utilizing
a technique which allows discrete nuclei to
be dissected from the brain of the rat,
showed that TRH though present in highest concentrations within nuclei of the
"thyrotrophic area" were also found in the
hypothalamus outside this region. Their
data is in keeping with our findings in
lesioned animals, as well as our report
(Jackson and Reichlin, 19746) of a gradient of TRH from dorsal hypothalamus
(49 pg/mg tissue) to SME (3570 pg/mg
tissue) (Table 1). TRH has been reported
to show immunofluorescent staining of
nerve terminals in the medial part of the
external layer of the median emminence
(Hokfelt et al., 1975a) although no immunopositive TRH perikarya were observed.
The estimate of total rat hypothalamic
TRH by radioimmunoassay has given
levels 10-20 times those previously reported by in vivo bioassay (Reichlin et al.,
1972). The discrepancy is explicable on the
basis of the presence of somatostatin which
inhibits TRH induced TSH rise (Vale et al.,
1974a).
Extrahypothalamic distribution of TRH
mammalian species
The first reports that IR-TRH was present in the brain outside the confines of the
hypothalamus were provided by my group
(Jackson and Reichlin, 1973) and that of
Oliver (Oliver et al, 1973). Significant concentrations of TRH are found in the rat
extrahypothalamic brain (Jackson and
Reichlin, 19746) (Table 1). Although such
concentrations are small when compared
with the levels in the hypothalamus, quantitatively over 70% of total brain TRH is
found outside this region (Oliver et al.,
TABLE 1. TRH distribution in rat brain.
Extrahypothalamic
brain
Hypothalamic
pituitary
complex
a
b
c
Brain
stem
5b
(4-5)<
Dorsal
hypothalamus
49
(41-61)
Cerebellum
2
(1-3)
Ventral
hypothalamus
64
(23-106)
Diencephalon
6
(3-12)
Stalk
median
eminence
3570
(920-7600)
TRH (pg/mg tissue)
Jackson and Reichlin, 19746.
Mean concentration.
Range of values.
in
Olfactory
lobe
6
(5-8)
Cerebral
cortex
2
(1-3)
Posterior
pituitary
Anterior
pituitary
10
(8-11)
155
(150-160)
388
IVOR M. D.JACKSON
1974; Winokur and Utiger, 1974). In an
attempt to determine the source of extrahypothalamic TRH, we have studied
the effects of classical thyrotrophic area
lesions which bring about a reduction in
hypothalamic TRH by two-thirds (Table
2). The extrahypothalamic brain TRH
content was unaffected in rats so treated
providing support for the intriguing
hypothesis that synthesis occurs in situ
(Jackson and Reichlin, 1977*). Studies
using hypothalamic deafferentation, complementary to these experiments (Brownstein, et al., 1975a) demonstrated that such
procedures not only leave the levels of
TRH in the extrahypothalamic brain unaltered, but cause a marked reduction in
hypothalamic content, suggesting that
much of hypothalamic TRH may be synthesized by cells outside this area.
It is of note that quantitatively the
amount of TRH in the posterior pituitary
is much greater than that in the anterior
pituitary (Table 2). The high concentration of TRH in the posterior pituitary
relative to the anterior pituitary in normal
rats (15 times as shown in the study reported in Table 1) has also been observed by
others (Oliver^ al., 1974). The depletion
of TRH from the posterior pituitary in the
lesioned animals (Table 2) supports the
concept of a third neurosecretory hypothalamo-hypophysial system extending
into the neurohypophysis, as suggested by
Hokfelt and his colleagues {I975a,b) on the
basis of immunohistochemical staining of
TRH and somatostatin positive fibers
reaching into the posterior pituitary from
the median eminence. Whether TRH has
a role in the posterior pituitary function is
uncertain, but it may be of relevance that
TRH is found in extraordinarily high concentration in the pituitary complex of
lower vertebrates (Jackson and Reichlin,
1974a), and there is evidence (reviewed by
Sawyer, 1964) that in the bony fish the
neurohypophysis may be a homologue of
the median eminence in higher animals.
Substantial quantities of TRH are found in
the spinal cord (Jackson, unpublished;
WilberetaL, 1976; KardonetaL, 1977)and
immunohistochemical examination has
localized TRH around the motoneurons of
the spinal cord (Hokfelt et al., 1975a).
Networks of TRH-positive nerve terminals
have also been found in many cranial
nerve nuclei (Hokfelt et al., 1975a).
As in the fetal rat (Eskay et al., 1974);
significant concentrations of TRH are
present in the extrahypothalamic brain of
the human fetus (Winters et al., 1974),
TRH being detected in the cerebellum as
early as 9 weeks. Interestingly, the cerebellum of an anencephalic infant contained a relatively high concentration of
TRH (Winters et al., 1974). It should be
noted however that the area cerebrovasculosa—an area lacking in nerve cells—taken
from an anencephalic fetus has been reported to synthesize a TSH releasing
substance in vitro (Ishikawa et al., 1976).
Extrahypothalamic brain tissue from
normal human adults (killed in traffic
accidents) contains significant concentrations of immunoassayable TRH in
the thalamus and cerebral cortex (Okon
and Koch, 1976). Guansing and Murk
(1976) and Kubek et al. (1977) have also
identified
IR-TRH
in
the
extrahypothalamic brain tissue of humans.
TABLE 2. Effect of a lesion of the "thyrotrophic area" of the hypothalamus on brain distribution of TRH in the rat.'
Hypothalamus
Anterior pituitary
Posterior pituitary
Extrahypothalamic brain
Lesionb
3,625±249°
15±8
2
17,040±925
"Jackson and Reichlin, 19776.
Six lesioned and seven control animals were studied.
Results (mean ± s.e.m.) are given as pg per tissue.
b
c
Control"
9,357+449
64±5
157±32
18,929±737
Significance
P < 0.001
P < 0.001
P < 0.001
P > 0.1
DISTRIBUTION OF HYPOTHALAMIC HORMONES
Physiologic role of TRH in the regulation of
thyroid function in inframammalian species
Aves. The importance of TRH in the
regulation of pituitary-thyroid function in
inframammalian species is uncertain. In
the chick, Ochi et al. (1972) reported that
the pituitary thyroid axis was unresponsive
to TRH stimulation whereas Breneman
and Rathkamp (1973), Newcomer and
Huang (1974) and Scanes (1974) provide
evidence that thyroid function can be
stimulated by exogenous TRH. I
examined the hypothalamus of adult
chicks for TRH and found levels slightly
less than that present in rat hypothalamus
(Fig. 1). Although not readily measurable
in mammalian blood, TRH can be measured in the circulation of the chicken and
incubation1 of plasma at 37°C does not degrade the endogenous IR-TRH (Jackson,
unpublished).
Amphibia. The significance of the
hypothalamus in the regulation of amphibian thyroid function has not been determined for sure (Hanke, 1976 for review).
However, there is evidence for hypothalamic control over amphibian metamorphosis, since lesions of the hypothalamus
(Voitkevich, 1962; Hanaoka, 1967) impair
metamorphosis. Thyroid hormone injected directly into the hypothalamus of
the neotenic tiger salamander induces
metamorphosis (Norris and Gern, 1976),
findings that are in keeping with a critical
TA0?OLE
SALWOM
FIG. 1. Concentration of TRH (pg per mg of tissue)
(mean ±s.e.m.) in the olfactory lobe (telencephalon)
of a number of different vertebrates. The mean TRH
concentration in the hypothalamus is given for comparison (from Jackson, 1977).
389
role for TRH in tadpole metamorphosis as
postulated by Etkin (1963). He suggested
that development of the tadpole
hypothalamus is under positive feedback
control by thyroid hormone and that a
gradually rising level of circulating thyroid
hormone during prometamorphosis induces maturation of the hypothalamic tissue concerned with the synthesis of TRH.
This positive feedback system results in the
stimulation of thyroid activity required for
metamorphosis. However Gona and Gona
(1974) failed to induce metamorphosis in
the tadpole with TRH administration.
TRH has also been given to the neotenic
Mexican axolotl (salamander), a species
whose plasma does not degrade TRH in
vitro, and in spite of achieving high circulating levels of the exogenous material in
the blood, as measured by radioimmunoassay, metamorphosis was not induced (Taurog^a/., 1974).
These findings raise the possibility that
in amphibia the mammalian TRH is not a
physiologic TSH releasing factor in these
species. Alternative explanations are possible. The TRH may induce simultaneous
release of prolacin, which in the tadpole, at
least, blocks metamorphosis (Etkin and
Gona, 1967). Indeed antiserum to bullfrog
prolactin injected into prometamorphic
tadpoles of R. catesbeiana will accelerate
metamorphic climax (demons and Nicoll,
1977). However, TRH given to the red eft,
a species that undergoes water drive or
second metamorphosis, which is known to
be specially controlled by prolactin (Grant
and Grant, 1958), had no effect on
metamorphosis (Gona and Gona, 1974).
demons et al., (1976) have reported that
TRH induces prolactin release in the
bullfrog and have suggested that TRH
may have functioned as a prolactin releasing factor before it became a stimulator for
TSH release. TRH has also been shown to
release MSH from the pituitary of R. esculenta (Vaudry et al., 1977), but whether
this has any relation to thyroid function is
unclear at this time.
Pisces. In the lung fish, TRH in high
doses was not found to have any effect in
stimulating thyroid function (Gorbman
and Hyder, 1973). However, evidence for
390
IVOR M. D. JACKSON
a thyrotropin inhibitory factor from the
hypothalamus (TIF) has been provided by
Peter and McKeown (1975) for the
goldfish and other teleost fishes, and, it
should be mentioned, by Rosenkilde
(1972) for some species of amphibia also.
Bromage (1975) has raised the interesting
possibility that TRH may function as a TIF
in teleost fishes.
The overall evidence suggests that the
hypothalamus is of importance in the regulation of thyroid function in lower animals
but that the degree of autonomy of the
pituitary-thyroid axis is much greater than
in mamilian species. In amphibia there is
evidence for a physiologic hypothalamic
thyrotropin releasing factor, but this might
be different from, or in addition to, the
tripeptide amide, TRH.
TABLE 3. Levels of TRH in the brain and pituitary complex
of the ammocetes larva of lamprey and of amphioxus.a
Species
Lamprey (4)b
(ammocetes)
(Petromyzon marinus)
Amphioxus (4)
Whole
brain0
Pituitary
complex'
38
145
(25-60)"
2e
Not examined
(Branchiostoma
lanceolatum)
"Jackson and Reichlin, 1974a.
Number of animals examined.
c
Pg/mg tissue wet weight.
" Range of values.
e
Pg/whole undissected head.
' Pg/whole pituitary.
b
mones. In a sense, the pituitary has "coopted" TRH as a regulatory hormone.
TRH distribution in submammalian chordates
In view of the reported absence of a role
for TRH in the regulation of thyroid function in inframammalian species, I examined
the hypothalami of a number of vertebrates including snake, frog and salmon for
TRH content, and found high concentrations (Jackson and Reichlin, 1974a), with
values up to 10 times that found in rat
hypothalamus (Figure 1). Elevated TRH
levels in amphibian hypothalamus have
also been reported by Taurog^ al. (1974).
Further high concentrations of TRH were
also found in the extrahypothalamic brain
of these vertebrates (Figure 1) and evidence for its authenticity was shown by the
ability of a frog brain extract to release rat
TSH in vivo (Jackson and Reichlin, 1974a).
We have also shown TRH to be present
in the whole brain of the larval lamprey,
in the head end of the amphioxus (Jackson
and Reichlin, 1974a; Table 3) and in the
circumesophageal ganglia of the invertebrate snail (Grimm-Jorgensen et al., 1975).
As the lamprey lacks TSH, and the amphioxus and snail lack a pituitary I propose
that the TSH-regulating function of TRH
may be a late evolutionary development
representing an example of an organism
acquiring a new function for a pre-existing
chemical substance or hormone, analogous
to the evolution of neurohypophysial hor-
Pineal
I also found TRH to be present in the
frog (Rana pipiens) pineal in high concentrations which are influenced by the degree
of photoillumination; changing seasons
are associated with swings in pineal TRH
concentrations as much as 10-20 fold
(Jackson et al., 1977).
Comparable findings have also been reported by Kiihn and Engelen (1976) who,
with an in vivo bioassay in the rat, have
demonstrated a seasonal variation in the
PRL and TSH-releasing activity in the
hypothalamus of the frog. The function of
TRH in the frog pineal is unknown, but
the circannual rhythm and effect of illumination bespeak for a role in neurotransmission and this is supported by evidence that TRH has an excitatory action
on frog motoneurones (Nicoll, 1977).
Blood
Unlike TRH in mammals (Jackson and
Reichlin, 19746), TRH circulates in the
blood of Rana pipiens in high concentration
(22-132 ng/ml) and shows rapid degradation in vitro with a tl/2 of 1.8 min at 26°C
and 0.95 min at 37°C (Jackson and Reichlin, 1977c). An extract of frog blood containing 100 ng IR-TRH produced a TSH
DISTRIBUTION OF HYPOTHALAMIC HORMONES
rise in the rat in vivo comparable to synthetic TRH, 100 ng, while a sample of the
same frog blood, allowed first to incubate
at 37°C, contained only 12 pg IR-TRH in
the extract and caused no elevation in rat
TSH (Jackson and Reichlin, 1977c). These
studies support the authenticity of frog
blood IR-TRH. Since frog brain weighs
only 100-150 mg, and contains approximately 100 ng TRH, it seems unlikely that
brain TRH can account for the bulk of
blood TRH.
Skin
Examination of organ distribution
showed huge quantities of immunoreactive
and bio-active TRH in the skin (mean 48
ng/mg protein) (Fig. 2), concentrations 3-4
times that of the hypothalamus; the retina
contained lesser quantities of TRH, while
thoracic or gastrointestinal organs contained no significant levels of TRH (Table
4). Since frog skin is approximately 10 g in
weight I estimate frog skin to contain over
EXTRACT OF
FROG SKIN
Time (minutes)
FIG. 2. Effect of an extract of skin from the frog
(Rana pipiens) on the release of TSH in the ratm vivo.
The skin was extracted in 90% methanol and the dried
supernatant, reconstituted in buffer, was assayed for
IR-TRH content. Skin extract containing 100 ng
IR-TRH, made up to 1 ml with saline, was injected IV
into each of 5 Sprague-Dawley male rats, under
nembutal anesthesia, and blood sampled at 2 and 5
min for TSH measurement. Each of the 5 control rats
received saline alone. Results show mean ±S.E.M. rise
in serum TSH. The skin extract exhibited biologic
potency appropriate to its content of IR-TRH. Saline
treated controls showed no TSH rise. (From Jackson
and Reichlin, 1977a)
391
TABLE 4. TRH concentration in various frog (Rana
pipiens) tissues removed from a group of four animals.3
TRH
Organ
Hypothalamus
Extrahypothalamic
brain
Spinal cord
Splanchnic nerve
Skin" 0
Retina
Heart
Lung
Tongue
Stomach
Intestine
Liver
Spleen
Kidney
Gonad
Muscle
Blood
(pig per g of protein)
14.9 ± 1.0
7.7 ± 1.2
2.3 ± 1.1
0.072 ± 0.03
26.1 ±15.4
3.3 ± 0.4
0.057± 0.009
0.058± 0.018
0.28 ± 0.13
0.041± 0.007
0.023± 0.004
0.019± 0.009
0.025± 0.009
0.019± 0.007
0.015± 0.006
0.021 ± 0.010
0.045+ 0.007
/xg/ml
whole blood
a
The blood TRH level is given for comparison. Results shown mean ± S.E.M. (from Jackson and Reichlin, 1977a).
b
Protein content of skin is 15.7 ± 0.4% wet weight
(n = 6). The mean skin: blood concentration gradient
ofTRHis91:l.
c
Tissue obtained from a separate group of 6 frogs.
50 fig TRH. In the frog the skin is an
important organ in salt and water balance
and excretion and generally in maintaining homeostasis. The specific function of
skin TRH is unknown but the massive
quantities of TRH present in the integument of this species suggests a role for this
peptide in skin function. It appears that
blood TRH is derived from the skin in this
species and that frog skin is an active
peptide secreting organ. This study provides evidence that the physiological role
of TRH, like that of other neural peptides
such as somatostatin (Arimura et al., 1975),
is not restricted to the CNS (Jackson and
Reichlin, 1977a). Support for the authenticity of the TRH present in the frog skin is
further provided by the work of Yasuhara
and Nakajima (1975) who described the
occurrence of a tripeptide chemically
characterized as pyroglutamylhistidyl prolineamide, in an extract of skin from the
Korean frog, Bombina orientalis.
392
IVOR M. D. JACKSON
LUTEINIZING HORMONE-RELEASING HORMONE
Distribution of LH-RH in mammalian species
septo-pre-optic region up to the retromammillary area, that give rise to two main
tracts ending in the infundibulum and the
lamina terminalis. The highest concentrations of LH-RH occur in the preoptic
region and in the pituitary stalk, the
proximal portion of which is the homologue of the rat median, eminence (Okon
and Koch, 1976).
Initial reports of large quantities of
LH-RH in extracts of rat extrahypothalamic brain (White et al., 1974)
have not been confirmed by us. Rather, we
have found that LH-RH in extrahypothalamic brain of the rat amounted
to only 17% of total brain LH-RH
(Jackson, in preparation). We have also
reported negligible levels of LH-RH in the
extrahypothalamic brain of the mouse
(Lechan et al., 1976). The absence of
LH-RH in brain tissues outside the
hypothalamus (in marked contrast to
TRH) was also reported in humans (Okon
and Koch, 1976).
It seems clear that the LH-RH decapeptide isolated and synthesized in Schally's
laboratory is a physiologic releasing hormone for both LH and FSH from the
mammalian pituitary (Schally et al., 1973,
for review). In the rat, immunoassayable
and bioassayable LH-RH are present in the
medial basal hypothalamus—especially in
the arcuate nucleus (ARC), and in the
preoptic suprachiasmatic tissue (Wheaton
et al., 1975). As for TRH and somatostatin,
deafferentation of the rat hypothalamus
causes a marked reduction in the LH-RH
content of the medial basal hypothalamus
(MBH) suggesting that such LH-RH arises
from, or is controlled by, cells elsewhere in
the brain (Brownstein et al., 1976). This
view is supported by work from our group.
Mice given monosodium glutamate show
degeneration of > 80% of the cell bodies in
the ARC, but the LH-RH content, and
intensity of immunohistochemical staining
is not affected. This suggests that LH-RH
may not be synthesized there, but transported to the ME by axons passing Distribution of LH-RH in lower vertebrates
through the ARC (Lechan et al., 1976).
LH-RH is detectable in the chicken and
The data are consistent with the hypothesis shows immunochemical and chromatoof a dual central influence on pituitary graphic similarity with the mammalian degonadotropin secretion — the LH-RH capeptide (Jeffcoatee<a/., 1974). However,
passing through the MBH controlling the previous studies by G. L. Jackson (1971)
tonic, and the LH-RH from the pre-optic suggested that chicken LH-RH was not the
suprachiasmatic nuclei the cyclic, secretion same as mammalian LH-RH on the basis of
of LH. However the LH-RH in the ARC of differences in the chromatographic propthe rat (and mouse) involved in the tonic erties of biologically active fractions on
discharge of LH from the anterior pitui- ion-exchange columns. Immunohistary may in fact be synthesized in the tochemical staining for LH-RH was repreoptic area (Kalra, 1976). It seems likely ported by deReviers and Dubois (1974) in
that species differences exist, for surgical the median eminence of the cockerel, and
isolation of the MBH of the guinea pig by McNeill et al. (1976) in the duck where
causes only a slight reduction in LH-RH nerve fibers were observed to be projected
content of the ME (Silverman, 1976). This to the portal systems of both cephalic and
data implies an LH-RH synthesizing locus caudal parts of the anterior pituitary
intrinsic to the MBH and in this regard the where it probably regulates both LH and
guinea pig resembles the monkey (Krey et FSH secretion. Exogenous LH-RH given
al., 1975) rather than the rat.
to the cockerel induces a rapid rise in LH
LH-RH has been detected in extracts of (Turret al.\ 1973).
fetal human brain as early as 4 1/2 weeks
In amphibia, LH-RH is effective in
(Winters et al., 1974). In the adult human stimulating gonadal function (Mazzi et al.,
brain, Barry (1977) has observed LH-RH 1974; Thornton and Geschwind, 1974).
positive perikarya, scattered from the Studies by ourselves (Alpert et al., 1976a)
m
DISTRIBUTION OF HYPOTHALAMIC HORMONES
*
393
and others (Deery, 1974; Doer-Schott and hypothalamus behind the optic chiasm (a
Dubois, 1976; Goos et al., 1976) have dem- procedure which severs the LH-RH cononstrated the presence of immuno-reactive taining fibers as well as the pre-optico
LH-RH in amphibian hypothalamus. In hypophysial pathway,) prevents ovulation
frogs (Rana pipiens and Rana catesbeiana) in amphibia. The existence of such a
immunoreactive LH-RH was found within "higher" center regulating cyclical
neuronal perikarya in the diagonal band of gonadotropin activity located outside the
Broca and in the median septal nucleus hypothalamus has been postulated by
intermingled with non-immunoreactive Dierickx (1967) in the frog. The latter
neurons (Figure 3).
investigator also postulated the presence of
These findings are comparable to those a tonic gonadotropin regulating center in
of Goos et al. (1976) who report IR-LH-RH the ventral hypothalamus, but neither ourin perikarya in front of the preoptic recess selves nor Goos et al. (1976) found LH-RH
and giving rise to axons radiating to the positive cells in such an area.
outer zone of the ME in R. esculenta. The
The only published data providing meapresence of distinct bundles of LH-RH surements of the total amount of LH-RH
containing fibers extending from the vicin- in the amphibian hypothalamus is that of
ity of the neuronal cell bodies to the ME is ourselves (Alpert et al. 1976a), who found
evidence for the existence of an LH-RH 3.3 ng/hypothalamus in the frog, Rana
peptidergic septo-infundibular-axonal pipiens, and of Deery (1974), who reported
pathway in frogs (Alpert et al., 1976a). This 0.9 ng in the toad, Xenopus laevis. Our
pathway probably functions in the control studies demonstrated that 16% of total
of gonadal activity since transection of the brain LH-RH was located outside the
hypothalamus (a value similar to that obtained in mammals) within the
telencephalon-septum-optic chiasm regions. Since we extracted these tissues with
90% methanol the absolute levels need to
be re-examined after acetic acid extraction.
Carp or trout hypothalamic extract
stimulates LH secretion from ovine
pituitaries in vitro (Breton et al., 1972).
Exogenous mammalian LH-RH stimulates
gonadotropin secretion in the fish, but
' /? «
extracts from teleost hypothalamus produce
different
profiles of teleost gonadotropin
' . . * . . „ •
• *
secretion than the LH-RH decapeptide
(Breton and Weil, 1973). However Deery
(1974) was unable to find IR-LH-RH in the
extracts of hypothalamus from dogfish or
goldfish. Since the sensitivity of his RIA
was 80 pg, it is possible that significant
levels were undetected. Recently, in the
1 t
trout fish, Goos and Murathanoglu (1977),
by immunohistochemical staining, have
localized LH-RH to perikarya in the forebrain and their axons. However no
fluorescence was observed in the nucleus
preopticus or nuclear lateralis tuberis areas
that previously have been correlated with
FIG. 3. Immunohistochemical staining for luteinizing reproductive activity in the fish (Peter,
hormone-releasing hormone (LH-RH) in the median
1973). It still remains to be settled whether
septal region of R. pipiens. The immunopositive another gonadotropin releasing hormone
.1 •• ">* ,;f
K
neuronal perikarya appear dark. (Alpert etal., 1976a)
394
IVOR M. D. JACKSON
is present in the fish (or indeed in other felt et al., 19756) and in nerves in different layers of the small and large intestine
vertebrates).
(Hokfelt et al., 19756). The distribution of
somatostatin in the gastrointestinal tract
SOMATOSTATIN
corresponds with its site of action in inhibDistribution of somatostatin in mammalian iting glucagon, insulin, gastrin and HC1
secretions (Gomez-Pan and Hall, 1977). It
tissues
seems likely that somatostatin is formed in
Nervous system. Somatostatin, a tet- situ in the gastrointestinal tract and may
radecapeptide isolated from ovine have a physiologic role in the regulation of
hypothalamus (Brazeau et al., 1973), ini- many gut hormones.
tially shown to inhibit growth hormone
secretion from the anterior pituitary, was Somatostatin in submairunalian species.
subsequently shown to inhibit TSH secretion as well as gastrointestinal hormone,
A survey of the phylogenetic distribupancreatic endocrine and exocrine secre- tion of somatostatin in a number of differtion (Vale et al., 1974a; Gomez-Pan and ent vertebrates has been reported by Vale
Hall, 1977; for review). Both by bioassay et al. (1976). The highest concentration of
(Vale et al., 19746) and radioimmunoassay somatostatin in the rat was found in the
(Brownstein et al., 19756) somatostatin has hypothalamus although the gastrointestibeen found widely distributed throughout nal tract contains the greatest amount of
the mammalian extrahypothalamic brain, somatostatin. Somatostatin was found in
including the pineal gland. Using an im- the brain and pancreas of the frog, catfish,
munoperoxidase technique, somatostatin torpedo and hagfish. In studies performed
has been localized in the circumventricular in this laboratory we found somatostatin to
organs, in addition to the external zone of be present in the skin of the frog (Rana
the median eminence (ME) (Pelletier et al., pipiens) (Jackson, unpublished). The sig1975). Somatostatinergic neurons have nificance and function of somatostatin in
been detected immunohistochemically in chordates requires further study.
the anterior periventricular hypothalamus
Recently a gene for somatostatin was
and in the pre-optic area, in part outside
chemically
synthesized and fused to plasthe confines of the hypothalamus (Alpert^
mid
elements
in E. Coli. These organisms
al., 19766). It is likely that these neurons
were
subsequently
able to synthesize the
inhibit the release of GH, and probably
also TSH, from the anterior pituitary. polypeptide in vitro (Itakurae/ al., 1977).
Hypothalamic deafferentation caudal to
the optic chasm has been shown to mark- FUNCTION OF THE EXTRAHYPOTHALAMIC DISTRIBUTION OF THE RELEASING HORMONES
edly reduce the immunoassayable and
immunohistochemical content of somatoThe widespread distribution of TRH,
statin in the medial basal hypothalamus
LH-RH
and somatostatin in brain and/or
(Jackson, 1977) suggesting that, like TRH,
the
extrahypothalamic neural somatostatin is neural tissue remote from
hypothalamus suggests that these subsynthesized in situ.
stances could have a role in neuronal funcWith the indirect immunofluorescence tion apart from anterior pituitary regulatechnique somatostatin has been detected tion. The evidence supporting such a view
in some neuronal cell bodies in spinal is summarized as follows:
dorsal root ganglia, as well as in fibers in
the substantia gelatinosa of the spinal cord Anatomic and phylogenetic distribution
(Hokfelt et al, 19756).
Gastro-intestinal tract. Somatostatin is
The large quantities of T R H and
present in mammalian stomach and pan- somatostatin found in the mammalian excreas {A.r\muT3.etaL, 1975) where it is local- trahypothalamic brain are independent of
ized in the argyrophilic D (Aj) cells (Hok- hypothalarnic secretion as determined b)
DISTRIBUTION OF HYPOTHALAMIC HORMONES
395
studies utilizing surgical ablation or lesions ported an excitatory action of TRH on R.
isolating the hypothalamus from the rest pipiens spinal motoneurons in contrast to
of the brain (Jackson and Reichlin, 19776; its inhibitory action on supraspinal
Brovvnstein et al., 1975a). The location of neurons described above, suggesting that
TRH in several cranial nerve nuclei of the TRH might act as an excitatory transmitter
brain stem and motor nuclei of the spinal on one cell type and as an inhibitory
cord (Hokfelt et al., 1975a) and of somato- transmitter on another.
statin in dorsal root ganglia (Hokfelt et al.,
19756), as well as the subcellular location of Behavioral and CNS effects
these substances in synaptosomes (Bennett
etal., 1975; Styne etal., 1977) suggest these
The hypophysiotrophic hormones have
peptides might function as neurotransmit- marked central nervous system (CNS) efters. The presence of TRH in neuronal fects unrelated to their role in the regulatissues of vertebrate and invertebrate tion of pituitary function. Hypophysecspecies (Jackson and Reichlin, 1974a; tomized mice pretreated by pargyline, a
Taurog et al., 1974; Grimm-j0rgensen et monoamine oxidase inhibitor, show enal., 1975) in which TRH clearly has no role hancement of the motor activity induced
in the regulation of any pituitary-thyroid by L Dopa when TRH is concomitantly
axis that might be present, lends credence administered (Plotnikoff et al., 1972). The
to this hypothesis.
tripeptide enhances cerebral norepinephrine turnover (Keller et al., 1974) and has
profound effects on thermoregulation
Specific synthesizing and degrading systems and
(Metcalf, 1974). It is of note that somatoreceptors in brain tissue for hypothalamic pepstatin shows prolongation of barbiturate
tides
anesthesia and shortening of strychnine
Grimm-j0rgensen and McKelvy (1974) seizure activity in the rat, effects that are
have demonstrated the in vitro synthesis of opposite those produced by TRH (Brown
TRH by hypothalamic and forebrain and Vale, 1975). These behavioral effects
fragments from adult newts (Triturus vir- are consistant with their anatomic
idescens) and active enzyme degrading sys- location—TRH being immunohistochemtems for all three hypophysiotropic princi- ically detected around motor neurons and
ples have been demonstrated in ex- having predominantly motor activity—
trahypothalamic brain tissue of the rat whereas somatostatin present in sensory
(Griffiths, 1976). Further, high affinity neurons appears to act primarily as a senbinding sites for TRH in the synaptic sory depressant. LH-RH has been shown to
membrane fraction of rat extrahy- stimulate sexual activity in rats in which
pothalamic brain tissue have been demon- gonadal function is held constant (Moss
strated (Burt and Snyder, 1975). These and McCann, 1973).
workers have shown that such receptors
have properties similar to those of pituiCONCLUSIONS
tary membranes.
It is now clear that the regulation of
anterior
pituitary secretion is only one asNeurophysiologic studies
pect of the function of the hypophysioThe hypothalamic hormones have been tropic hormones. The anatomic, subcellushown to have a profound effect on the lar, and phylogenetic distribution of these
electrical activity of single neurons. Re- peptides, the presence of specific brain
naud et al. (1975) applied TRH, LH-RH receptors and active neuronal synthesizing
and somatostatin directly to central and degrading systems, along with
neurons microiontophoretically and re- neurophysiologic and behavior studies
ported a marked depressant action on the provide powe "ful support for the view that
activity of neurons at several levels of the these substances might operate as neurocentral nervous system. Nicoll (1977) re- transmitters. The evidence, at least for
396
IVOR M. D. JACKSON
secretion in vitro des hormones gonadotropes
C-HG et LH respectivement par des hypophyses de
Carpe et de Belier. C. R. Hebd. Seances Acad. Sci.
274:2530-2533.
Bromage, N. R. 1975. The effects of mammalian
thyrotropin-releasing hormone on the pituitarythyroid axis of teleost fish. General and Comparative Endocrinology. 25:292-297.
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