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AMER. ZOOL., 18:401-410 (1978).
Hypophysiotropic Centers in the Brain of Amphibians and Fish
H . J . T H . GOOS
Zoological Laboratory, Section for Comparative Endocrinology,
State University, Padualaan 8, Utrecht, The Netherlands
SYNOPSIS The subject is the localization of three different hypophysiotropic centers in the
brain of amphibians and fish.
The thyrotropic hormone-releasing hormone (TRH) in Xenapus may originate from the
dorsal magno-cellular neurons of the preoptic nucleus. This hypothesis is based on
correlative changes between these cells and alterations in thyroid activity during
metamorphosis. Experimental data are in support of a functional relationship between
certain preoptic neurons and the thyrotropic activity of the pituitary.
The MSH inhibiting activity of the hypothalamus is effected by means of an aminergic
innervation of the pars intermedia in amphibians, teleosts and elasmobranchs. In amphibians the aminergic fibers originate from the caudal part of the paraventricular organ
(PVO); in elasmobranchs probably from the nucleus medius hypothalamicus (NMI); in teleosts
the origin still has to be investigated.
Two centers producing gonadotropic hormone-releasing hormone (GRH) have been
demonstrated. Lesion experiments lead to the hypothesis that GRH is produced in the
caudal hypothalamus, i.e., in the nucleus infundibularis ventralts of amphibians and in the
nucleus lateralis tuberis of fishes. ImmunoHuorescence studies indicate in both groups the
presence of neurons, in front of the preoptic area in the telencephalon, and these neurons
are immuno-ieactive with anti-mammalian LH-RH.
quired. These are generally based upon
the histochemical properties of particular
Many experiments have been carried cellular substances, e.g., hormones and
out to establish the relation between carrier proteins in the neurosecretory cell.
hypothalamic neurosecretory processes Bargmann (1949) selectively stained such
and certain pituitary functions. Some of neurons by applying Gomori's haematoxythese experiments were carried out to de- lin method in the diencephalon of mamtermine the location of certain mals. The cells form two paired nuclei: the
hypophysiotropic centers, others to study nucleus supra-opticus and the nucleus
the functional interrelationship between paraventricularis. Both are connected morthe centers and the pituitary functions, or phologically with the pituitary and have a
to ascertain the chemical nature of the functional relation to this organ (for review see Bargmann, 1954). The rostral
neurosecretory products.
This paper contains our findings, and hypothalamus of birds and reptiles condata from the literature on the localization tains two Gomori-positive nuclei also. The
of the thyrotropic center in Xenopus laevis, diencephalon of amphibians and fish conthe melanotropin inhibiting center in tains only one paired Gomori-positive nuXenopus laevis and elasmobranchs, and the cleus preopticus, which is homologous with
gonadotropic center in some amphibian both the nucleus supra-opticus and nucleus
and teleost species. A short introduction paraventricularis. Biochemical and imdeals with definitions and terminology of munohistochemical studies prove that
these neurosecretory cells synthesize large
different types of neurosecretion.
carrier proteins, the neurophysins, and
biologically active peptides. Electron microPeptidergic neurosecretion
scopically, the neurosecretory cells show
To demonstrate the various types of the characteristics of glandular cells. Seneurosecretory cells with the light micro- cretory material is synthesized in the rough
endoplasmatic reticulum, transported to
scope, specific staining methods are reINTRODUCTION
401
402
H.J. TH. Goos
the Golgi system, and stored in the socalled elementary granules with a diameter
of 1000 to 3000 A (Bargmann, 1966;
Sloper, 1966). On the basis of the peptidergic nature of the biologically active
substances in the Gomori-positive neurons,
these are classified as peptidergic
neurosecretory cells. Since this type of
neurosecretory cell was the first to be
described, it is often referred to as "the
classical neurosecretory system." Not all
neurons described as peptidergic
neurosecretory cells are Gomori-positive
and contain large elementary granules. In
teleosts the cells of the nucleus lateralk
tuberis (see below) are an example of such
neurons. They are referred to as Gomorinegative peptidergic neurosecretory cells
and many contain granules of 800-1000 A.
Aminergic neurosecretion
Another type of specialized Gomorinegative neurosecretory cell in the brain
was described in the early sixties after the
introduction of a histochemical method for
demonstrating catecholamines and indolamines (Falck et al., 1962). Cells containing these substances are present in the
caudal hypothalamus of mammals, i.e., in
the nucleus arcuatus (Fuxe, 1964), and also
in the hypothalamus of birds (Sharp and
Follett, 1968; Oehmke, 1969), reptiles
(Baumgarten and Braak, 1968), amphibians (Goos and van Halewijn, 1968; Braak,
1970; Bartels, 1971) and fish (Baumgarten
and Braak, 1967). The cells are commonly
referred to as "Falck-positive" cells. Electron microscopically, they can be seen to
contain many dense-core vesicles with a
diameter of 700-1000 A throughout the
cytoplasm.
Neurosecretory processes of all the mentioned types were considered to have a
relation to certain hypophysial functions.
The present concept is that neurosecretory
cells in the brain produce hormones that
either stimulate or inhibit the production
and/or the release of hypophysial hormones. These neurosecretory products are
the so-called releasing or inhibiting hormones. In addition, peptidergic neurosecretory cells produce the octapeptides
oxytocin and vasopressin or related peptides, which are released into the general
circulation in the pars nervosa of the
hypophysis.
LOCALIZATION OF TRH PRODUCING NEURONS IN
XENOPUS l^AEVIS TADPOLES
In anuran metamorphosis, three periods
can be destinguished (Etkin, 1964): premetamorphosis, marked by rapid growth
and few morphological changes; prometamorphosis, with a differential growth of
the legs; metamorphic climax, during
which drastic changes take place, including
changes of the feeding system and the
reduction of the tail. For a detailed description of changes during metamorphosis, see Dent (1968). The thyroid activity increases during metamorphosis and
reaches a maximum at metamorphic
climax. Then, the activity decreases again
to a relatively low level that is maintained
during adult life (Saxen et al., 1957 a, b;
Leloup et al., 1960). Etkin demonstrated
already in 1935 that the sequence of
metamorphic events in thyroidectomized
tadpoles depends on the amount of
exogenous thyroxine.
The question arises as to the cause of the
change of thyroid activity during
metamorphosis. The work of D'Angelo et
al., (1941), Gordon et al., (1945) and Etkin
(1966) indicates that at all stages the
thyroid gland may react to hypophysial
thyroid stimulating hormone (TSH). In
other words, if there is no change in the
sensitivity of the thyroid for TSH, there
may be a change in the extrusion of TSH
from the adenohypophysis that primarily
determines the variations in thyroid activity during metamorphosis. Etkin (1964)
and Hanaoka (1967) succeeded in inducing thyroid hypertrophy with goitrogens
during premetamorphic stages in Rana pipiens. This means that even during early
stages of metamorphosis the thyrotropic
capacity of the pituitary is adequate to
induce a high thyroid activity. Consequently, it may be concluded that in the
absence of goitrogens during these early
stages the pituitan is not stimulated to a
high thyrotropic activity, and it must be
HYPOPHYSIOTROPIC CENTERS IN AMPHIBIANS AND FISH
some factor outside the pituitary-thyroid
axis that causes the gradual increase in
TSH output during the later stages of
metamorphosis.
According to the current concept, the
TSH cells in the adenohypophysis are
stimulated by a thyrotropin-releasing
hormone (TRH) that originates from
neurosecretory cells in the hypothalamus
and reaches the adenohypophysis via
hypothalamo-hypophysial track, median
eminence and portal vessels. Even before
the functions of the neurosecretory cells
were known, the relation of the
hypothalamo-hypophysial tract, median
tigated in studies on the regulation of
amphibian metamorphosis. Etkin (1938)
transplanted the pituitary to various sites,
e.g., to the eye muscle; the histological
picture of the autotransplant remained
nearly normal but metamorphosis was arrested or retarded. These results were
confirmed for many other amphibian
species. Etkin (1964) explained the retardation of metamorphosis that follows
hetertopic transplantation by assuming
that the pituitary gland cannot be reached
by TRH.
To answer the question of where TRH is
produced, amphibian larvae proved to be
excellent experimental animals. The
reason is obvious: During metamorphosis
the pituitary-thyroid axis is subjected to
drastic changes, and if these depend on
TRH secretion by one or more
hypothalamic centers, metamorphic processes must be accompanied by changes in
the TRH-producing neurosecretory cells.
Of the amphibian larvae those of Xenopus
laevis are suitable to study various aspects
of the endocrine regulation of metamorphosis; the larvae can be raised in the
laboratory at all times of the year and
survive surgical and chemical treatment if
handled with sufficient care. For these
reasons tadpoles of Xenopus laevis have
been selected as experimental animals for
determining the TRH center in the
hypothalamus of amphibians. In presenting the results, it should be mentioned that
these apply only to the Gomori-positive
peptidergic center, i.e., the paired preoptic
nucleus, and its tract towards the median
403
eminence. The Gomori-negative centers
were not included, and questions about the
hypothalamic regulation of TSH secretion
in amphibians cannot be conclusively
answered.
With a variant of Gomori's hematoxylin
technique, i.e., Schiebler's (1958/59)
pseudoisocyanine (PlC)-method, preoptic
neurosecretory cells can be observed from
the stage of very early premetamorphosis.
Their appearance coincides with the differentiation of the first thyroid follicles.
During pre- and prometamorphosis the
number of preoptic cells gradually increases and the nucleus reaches its full size
at metamorphic climax (Goos et al., 1967).
After thyroidectomy, this differentiation
of neurosecretory cells has not been observed, but increasing amounts of exogenous thyroxine caused a return to normal
development. It is concluded that during
larval life, thyroxine has a positive effect
on the hypothalamo-hypophysial system.
According to Etkin (1963, 1965) this effect
applies to the development of the system as
well as to its secretory activity. The latter
proved to be wrong. Treatment of Xenopus
tadpoles with the goitrogenic agent propylthiouracil (PTU) caused a hypertrophy of
the thyroids, a degranulation and hypertrophy of the TSH-cells, and a degranulation and hyperactivity of the dorsal, magnocellular part of the preoptic nucleus
(Goos et al., 1967). Exogenous thyroxine or
a restoration of hormone synthesis by the
thyroid caused a regranulation of the
TSH-cells and the above-mentioned
neurosecretory cells (Goos et al., 1968;
Goos, 1968). This experiment allows one
conclusion and one hypothesis. The conclusion is that thyroxine at all metamorphic stages causes a negative feed-back
action in the secretory activity of the
hypothalamo-hypophysial system, at least
for the production of TRH and TSH. The
hypothesis is that the dorsal, magnocellular part of the preoptic nucleus is involved
in TRH production. This is based on correlative changes between the amount of
PIC-positive material in the neurosecretory cells, the TSH-cell activity and the
thyroid activity. Such correlations should
be interpreted with the utmost care, as .
404
H.J.
T H . GOOS
they do not include a functional interrelationship.
A relationship can be proved if extirpation of the cells of the dorsal part of the
preoptic nucleus leads to retardation of
metamorphic processes and to the impossibility of activating the thyroid gland with
goitrogens. This experiment was carried
out (Goos, 1969/;) by extirpating partly or
completely the preoptic nucleus in a large
number of Xenopus tadpoles. Some of the
operated animals were treated with PTU
and their metamorphosis, as well as their
thyroids and pituitaries, was compared
with that in unoperated and operated
non-PTU-treated animals. In tadpoles
without the dorsal part of the preoptic
nucleus, metamorphosis was not completed and the thyroid could not be stimulated with PTU to the same extent as in
control animals. This means that the dorsal, magnocellular part of the preoptic
nucleus is an essential link in the
hypothalamo-hypophysial-thyroid axis in
Xenopus tadpoles, and that these neurosecretory cells might produce TRH. A direct
method for demonstrating TRH by means
of histo-immunological methods may provide additional evidence for the presented
hypothesis.
LOCALIZATION OF AN MSH-INHIBITING CENTER
Many poikiloterms are able to change
their skin color. This is based on the presence of integumentary pigment cells or
chromatophores. One of the types of
chromatophores is the melanophore. In
reaction to an appropriate stimulus, melanin granules migrate into or away from
static processes of the cells; these movements are called dispersion and aggregation respectively. Pigment migration may
be a response to a variety of environmental stimuli, which usually do not have a
direct effect on the melanophores. The
direct stimulus may be neural or hormonal.
In teleosts the chromatophores are controlled by nerve fibers (Jacobowitz and
Laties, 1968), but for some species a hormonal control of melanophores cannot be
excluded. In amphibians the regulation is
primarily hormonal, as demonstrated in
early experiments by Allen (1916) and
Smith (1916). They observed that
hypophysectomy of young tadpoles gave
rise to "albino" larvae. Hogben and Winton (1923) were the first to show the importance of a principle in the pars intermedia (PI) of the pituitary. This principle
was named intermedin, melanophore
stimulating hormone (MSH) or melanotropin.
Amphibians
According to Etkin, MSH secretion in
amphibians is inhibitively controlled by the
central nervous system. After transplanting a single pituitary in hypophysectomized axolotls and various American
Ranidae, he noticed an excessive pigmentation and a loss of background color response. Histological examination of the
grafts revealed a striking hyperplasia and
cellular hypertrophy of the PI. Destruction
of the infundibulum also caused hyperpigmentation. The inhibiting principle was
called melanotropin inhibiting factor
(MIF). The control may be hormonal or
nervous, i.e., via nerve fibers from or passing through the infundibular area. A hormonal control seems unlikely since restoration of background adaptation after
pituitary stalk sectioning takes much
longer than portal vessel regeneration (Etkin, 1962; J0rgensen and Larsen, 1963).
If a nervous control is accepted, the
question arises whether the neurons involved are peptidergic or aminergic. Some
Gomori-positive peptidergic fibers have
been observed in the pars intermedia; it
seems unlikely, however, that these play an
important role in MSH-regulation, for extirpation of the preoptic nucleus in Rana
temporaria was not followed by loss of
background adaptation (Dierickx, 1967).
A great number of fluorescence and
electron microscopical studies (review in
Terlou et al., 1974) indicated that these
fibers originate in the post-optic region. In
Xenopus laevis this area contains many
aminergic neurons (Goos and van
Halewijn, 1968), which according to Terlou and Ploemacher (1973) are concentrated in the paraventricular organ (PVO)
HYPOPHYSIOTROPIC CENTERS IN AMPHIBIANS AND FISH
fe
and the nucleus infundibularis dorsalis (NID).
Moreover, a tract of monoaminecontaining fibers, originating from these
nuclei, was found to run via the median
eminence into the pars intermedia where
the fibers terminate on the glandular cells
(Goos et al., 1972; Terlou and Ploemacher,
1973). Apart from these more descriptive
studies, experimental work showed the
importance of aminergic innervation for
the inhibition of MSH-release. Reserpine,
which causes depletion of monoamines,
induces an uncontrolled MSH-release in
Bufo arenarum (Iturriza, 1966) and Xenopus
laevis (Goos, 1969«)- Likewise, the injection
near the PI of chlorpromazine (an adrenergic receptor blocking agent) disturbs
the inhibition of MSH-release in Rana pipiens (Dierst-Davies, et al., 1966).
Another argument in support of an adrenergic control of MSH-secretion was
presented in developmental studies by
Terlou and Van Straaten (1973). A dispersion of the melanophores, irrespective of
background color, was observed in Xenopus
tadpoles up to stage 39 (Nieuwkoop and
Fabers normal table). From stage 39-41 on,
the animals are able to inhibit MSHsecretion when placed on a white
background. In the PVO and NID, the
first monoamine-producing neurons can
be demonstrated in these stages. This
confirms earlier results of Nyholm (1972),
who observed the appearance of aminergic
fibers in the median eminence and nerve
endings in the PI at the moment when the
animals acquire the capacity of
background adaptation. All these in vivo
experiments indicate that in amphibians
the MIF is a bioamine. This idea was
supported by in vitro studies by Jenks
(1977); when PI tissue was incubated in
the presence of bioamines, the production
of MSH was suppressed.
In the amphibian brain, bioamineproducing neurons are restricted to the
PVO and NID. The MIF therefore must
have its origins in these nuclei. Lesion
experiments were carried out to establish
the source of MIF more precisely (Terlou
et al., 1975). Lesioning of the NID or the
rostral half of the PVO did not affect MSH
secretion, but destruction of the caudal
405
part of the PVO caused an uninhibited
MSH secretion. This leads to the conclusion that in Xenopus laevis the MIF is produced in the caudal part of the PVO.
Biochemical studies by Goos et al. (1972)
demonstrated that mainly dopamine is
present in the hypothalamus of the Xenopus
tadpole. This was confirmed by microspectrofluorometric identification (Terlou and
van Kooten, 1974).
Teleosts
With regard to the regulation of
background adaptation in teleosts, it was
generally assumed that fish melanophores
are neurally rather than hormonally controlled. This probably explains why only a
few studies were made on MSH, its origin,
effects and its control of release in fish.
There is no doubt, however, that MSH has
a certain effect on fish melanophores.
Hypophysectomy causes aggregation of
melanin granules; injection with MSH or
pituitary extracts causes a dispersion (for
review, see Pickford and Atz, 1957).
Moreover, in the pituitary certain cells
have been identified as the source of MSH
(Olivereau and Ball, 1966).
Most information about the regulation
of the MSH-release in teleosts supports an
inhibitory control by the central nervous
system, as in amphibians. In organ cultures
of pituitaries from Poecilia latipinna, Carassius auratus, and Anguilla anguilla, the MSH
cells hypertrophied and the MSHsecretion increased (Ball et al., 1972)
Heterotopic autotransplantation of the
pituitary of Gillichthys mirabilis causes a
hyperactivity of the MSH-cells (Nishioka et
al., 1973).
The nature of this control seems to be
aminergic, since reserpine caused depletion of granules from the MSH-cells
and a dispersion of the melanophores
(Olivereau, 1972). Treating Gillichthys
mirabilis with 6-OH-dopamine, a false
neurotransmitter, caused also a degranulation of the MSH-cells and an increasing
amount of rough endoplasmic reticulum
(Nishioka et al., 1973). Moreover, aminergic nerve fibers are known to be in contact
with the MSH-cells (Bage etal, 1975).
406
H.J. TH. Goos
Although a number of amine-containing cells, testosterone treatment an inactivastructures have been demonstrated in the tion. Thus is was suggested that the
hypothalamus of teleosts, information on gonadotropin releasing hormone (GRH)
the origin of the fibers innervating the has its origin in the NIV.
MSH-cells is not available.
No definite conclusion can be drawn
Surgical (Meurling and Bjorklund, about the cellular source of hypothalamic
1970) and pharmacological experiments hormones until these have been dem(Wilson and Dodd, 1973) proved that in onstrated directly in their respective
elasmobranchs also, certain amines, prob- neurons, in the perikarya as well as in the
ably dopamine, are involved in the inhibi- axons ending in the neurohypophysis. For
tory control of the MSH-secretion. Two a direct intracellular demonstration of
amine-containing nuclei were described peptide hormones, immunohistochemical
for these animals: the nuclei lobi inferiores methods can be applied, provided that
(NLI) and the nucleus medius hypothalamicuspure antigens are available. Isolated and
(NMI) (Wilson et al., 1974). Lesion exper- purified amphibian GRH is not yet availaiments revealed that from these two only ble; there are some indications of biologithe NMI may play a role in the color- cal activity and immunological crossreactivity of mammalian luteinizing
changing mechanism.
hormone-releasing hormone (LH-RH) in
amphibians. Thornton and Geschwind
LOCALIZATION OF GRH-PRODUCING NEURONS
(1974) found that mammalian LH-RH enIt is generally accepted that the central hances GTH-secretion in Rana pipiens, and
nervous system regulates the gonadotropic Deery (1974) observed immunological
activity of the pituitary and causes periodic binding ofXenopus laevi% hypothalamus exchanges in GTH-release. One of the well- tract to anti-mammalian LH-RH, when
known examples is the hypothalamic in- tested in radio-immunoassay. This means
duction of an LH surge in mammals and that amphibian GRH is physiologically and
birds prior to ovulation. A similar concept chemically related to mammalian LH-RH;
was postulated for amphibians by van when anti-LH-RH is used, the immunohisOordt (1960) and for teleosts by Lam et al. tochemical technique may provide information on the cellular source of GRH in
(1976).
the amphibian brain.
Applying the double antibody technique
to brain tissue, we found perikarya reactIn a series of surgical studies Dierickx ing with anti-mammalian LH-RH in an
(1974) demonstrated that the ventral tuber unpaired nucleus, situated in the ventral
cinereum of Rana temporaria is involved in part of the area where the telencephalon
the regulation of the gonadotropic activity merges into the diencephalon, imrequired for gametogenesis and produc- mediately in front of the optic recess (Goos
tion of gonadal hormones. Dierickx et al. el al., 1976). A paired nerve tract, contain(1972) provided ultrastructural evidence ing immunoreactive material, can be
for the presence of several neurosecretory traced towards the median eminence (ME).
cell types in this area of the brain. Similar Before entering the ME, the two tracts
neurosecretory cells were found by Peute join, and in the ME the)' split up into
and Mey (1973) in electron microscopical numerous fibers, apparently ending on the
studies of the caudal hypothalamus of portal vessels. Essentially the same results
Rana esculenta. The cells are the principal were obtained in other amphibians by Alneurosecretory elements of the Gomori- pert et al. (1976) and Doerr-Schott and
negative, peptidergic nucleus infundibularis Dubois(1976).
ventralu (NIV). One of these cell types
Just as other immunohistochemical data,
showed changes that can be correlated these results have to be considered with
with the amount of circulating androgen: caution. For the time being it may be
Castration caused an activation of these concluded that the amphibian brain conAmphibians
HYPOPHYSIOTROPIC CENTERS IN AMPHIBIANS AND FISH
407
tains at least two different centers for and in hypophysectomized animals with
regulating the gonadotropic activity of the homotransplanted hypophyses (Ball et al.,
pituitary: one in front of the preoptic 1965). Consequently, gonadotropinreleasing hormone must be present in
recess, the other in the NIV.
In attempting to formulate a hypothesis teleosts.
for the functional significance of two difFor the origin of this hormone several
ferent GRH centers, it can be argued that opinions have been expressed; most of
neurons in the ventral tuber cinereum them give special attention to the nucleus
hypothalami might affect the activity of lateralis tuberis (NLT). Cytological signs of
GRH-axons originating in the prechias- secretory activity in the NLT were corrematic region, or the reverse might be the lated with reproduction in a number of
case. These possibilities, however, seem teleost species (for lit., see Peter, 1970).
highly unlikely; Dierickx (1974) did not More direct evidence for the NLT being
find impairment of gametogenesis in Rana involved in gonadal activity was provided
temporaria after completely isolating the by Peter (1970). Lesions in certain and
ventral tuber cinereum hypothalami to- distinct parts of this nucleus caused a lesser
gether with the pituitary from more rostral gonadal activity. These correlative and exand dorsal parts of the brain. More likely, perimental studies, however, are not the
both centers act independently. The one in only indications of the NLT being the
the caudal hypothalamus seems to be in- source of GRH. Several ultrastructural
volved in the seasonal production of ga- studies suggest that fibers innervating the
metes and gonadal hormones, and the gonadotropic cells originate in the NLT
GRH center in front of the preoptic recess (for review, see Peute et al., 1976). With the
might well bring about a GTH surge re- immunofluorescence technique, it has
quired for ovulation. The results of lesion been attempted to demonstrate GRH diexperiments by Dierickx (1974) support rectly in teleosts, just as in Rana esculenta.
this idea. Whatever the ultimate sig- The problems remained: pure teleost
nificance may be of the two centers in GRH is not available, and again antiregulating GTH-secretion, these centers mammalian-LH-RH had to be used. In
have a different location and a different several studies it was found that
hormone content. One center produces mammalian-LH-RH is biologically active in
and stores a substance immunochemically teleosts (Breton and Weil, 1973; Crim and
related to LH-RH; the other either pro- Cluett, 1974; Deery and Jones, 1975; Lam
duces a substance immunochemically dif- et al., 1976); although teleost GRH is not
ferent from LH-RH, or stores an LH-RH- identical to mammalian LH-RH (Breton
like principle in such a way or in such small and Weil, 1973; Deery, 1974), it seems
quantities that it cannot be demonstrated justified to apply anti-mammalian-LH-RH
with immunohistochemical techniques in for localizing teleost GRH.
which an anti-LH-RH is applied.
Applying the immunofluorescence
technique to the brain tissue of the rainbow trout (Salmo gairdneri), we realized that
Teleosts
Deery (1974) failed to demonstrate a
The situation in teleosts resembles that cross-reaction between mammalian LH-RH
in amphibians. There is conclusive evi- and fish GRH. It is clear that crossdence that in teleosts, as in all other verte- reactivities in radio-immunoassay and imbrates, normal functioning of the pituitary munohistochemistry cannot always be
depends on its stalk connection with the compared, for in the latter the immune
hypothalamus, and it is known that the reaction is carried out after fixation of the
hypothalamus stimulates the gonadotropic tissue. This proves to be a serious drawhormone secretion of the pituitary. For back of immunohistochemical techniques
example, gonadal atrophy was found in as far as specificity is concerned, and reteleosts with a heterotopically auto- sults of these techniques should be intertransplanted pituitary (Johanson, 1967), preted with proper restriction.
408
H.J. TH. Goos
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