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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 berg. 1975. The pituitary gland of the roach, After application of the double antibody Leuciscus rutilus. Ill The pars intermedia and its fluorescence technique, the perikarya were innervation. Acta Zool., Stockh. 56:43-60. found to react with the anti-LH-RH globu- Ball, J. N., M. Olivereau, A. M. Slider, and K. D. lin at both sides of the ventriculus communis Kallman. 1965. Functional capacity of ectopic in the area dorsalis partis medialis of the pituitary transplants in the teleost fish, Poeciha formosa, with a comparative discussion on the transplanted pituitary. Phil. Trans. Roy. B. 249:69-99. Ball, J. N., B. I. Baker, M. Olivereau, and R. E. Peter. 1972. Investigations on hypothalamic control of adenohypophysial functions in teleost fish. Gen. Comp. Endocr. Suppl. 3:1 1-21. Bargmann, W. 1949. Uber die neurosekretorische Verkniipfung von Hypothalamus und Neurohypophyse. Z. Zellforsch. 34:610-634. Bargmann, W. 1954. Das Zwischenhirn — Hypophysensystem. Springer-Verlag, Berlin, Gottingen, Heidelberg. Bargmann, W. 1966. Neurosecretion. Int. Rev. Cytol. 19:183-201. Bartels, W. 1971. Die Ontogenese der aminhaltigen Neurosysteme im Gehirn von Rana temporarw.. Z. Zellforsch. 116:94-118. Baumgarten, H. G. and H. Braak. 1967. Catecholamine im Hypothalamus vom Goldfisch (Carassius auratus). Z. Zellforsch. 80:246-263. Baumgarten, H. G. and H. Braak. 1968. Catecholamire im Gehirn der Eidechse (Lacerla virides und Lacerta muralis). Z. Zellforsch. 86:574602. Braak, H. 1970. Biogene Amine im Gehirn vom Frosch (Rana esculenta). Z. Zellforsch. 106:269-308. Breton, B. and C. Weil. 1973. Effects du LH/FSH-RH synthetique et d'extraits hypotholamiques de carpe sur la secretion d'hormone gonadotropin in vivo chez le carp (Cypnnus carpio L.). C. R. Acad. Sci. (Paris) Ser. D. 277:2061-2064. Crim, L. W. and D. J. Cluett. 1974. Elevation of plasma gonadotropin concentration in response to mammalian gonadotropin releasing hormone (GRH) treatment in the male brown trout as determined by radioimmunoassay. Endocr. Res. Comm. 1:101-110. D'Angelo, D., A. Gordon, and H. Charipper. 1941. The role of the thyroid and pituitary gland in the anomalous effect of initiation on amphibian metamorphosis. J. Exptl. Zool. 87:259-277. Deery, D. J. 1974. Determination by radioimmunoassay of the luteinizing hormone-releasing hormone (LHRF) content in the hypothalamus of the rat and some lower vertebrates. Gen. Comp. Endocr. 24:280-285. Deery, D. J. and A. C. Jones. 1975. Effects of hypothalamic extracts, neurotransmitters, and the synthetic hypothalamic releasing hormones on REFERENCES adenyl cyclase activity in the ventral lobes of the pituitary of the dogfish, (Scyliorhinus canicula L.). J. Endocr. 64:49-57. Allen, B. H. 1916. The results of extirpation of the anterior lobe of the hypophysis and the thyroid of Dent, J. N. 1968. Survey of amphibian metamorRanaptpiens larvae. Science 44:755-757. phosis. In W. Etkin and L. I. Gilbert (eds.), Alpert, L. C , J. M. Brouwer, J. M. O.Jackson, and S. Metamorphosis pp. 271-311. North Holland Publ. Reichlin. 1976. Localization of LH-RH in neurons Co., Amsterdam. in frog brain (Rana pipiens and Rana catesbiana). Dierickx, K. 1967. The function of the hypophysis Endocrinology 98:910-922. without preoptic neurosecretory control. Z e, C , B. Ekengren, B. Fernholm, and G. FriedZellforsch. 78:114-130. telencephalon. The oval or round perikarya are small and rather scarce. Immunoreactive cytoplasm is found in a thin layer around the nucleus and in a protrusion, which apparently is the beginning of an axon. Axons from these perikarya were observed to run rather diffusely in a ventro-caudal direction in the lateral walls of the diencephalon, passing over and under the horizontal commissure and reaching the area dorsal to the pituitary stalk. It is not yet clear how and where these axons actually end (Goos and Murathanoglu, 1977). Examination of brain tissue from some other teleosts, e.g., of a barbel species (Barbus conchonius), head and tail-lights (Hemigrammus caudovitatis) and the goldfish (Carassius auratus) did not give an answe;. A possible explanation is that anti-mammalian-LH-RH does not crossreact with GRH from these species. Another possibility is that the physiological condition of the animals causes the supply of immunoreactive material to be too small (Goos and van Oordt, 1978). In conclusion, it appears that in amphibians and in teleosts at least two centers in the brain are involved in regulating the gonadotropic function of the pituitary. Additional experimental studies will be required to prove that neurons, showing an immunological reation with antimammalian-LH-RH, indeed produce a substance that acts as gonadotropinreleasing hormone in amphibians and teleosts. HYPOPHYSIOTROPIC CENTERS IN AMPHIBIANS AND FISH _ P 409 Dierickx, K. 1974. Identification of adenohytreatment. Z. 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