Hypothalamic neurosecretion and metamorphosis in Xenopus laevis

Summary Normal and propylthiouracil (PTU) treated Xenopus laevis tadpoles were fixed during all stages of metamorphosis and sagittal sections were stained with aldehyde fuchsin (AF) or pseudoisocyanine (PIC). Whereas AF positive neurosecretory material could only be demonstrated in the preoptic nucleus from late prometamorphosis, increasing amounts of PIC positive material were found in cells of the dorsal part of the preoptic region from early premetamorphosis. The development of these cells correlated with that of the thyrotropic cells and the thyroids. Likewise, signs of hyperactivity in thyroids and thyrotropic cells of PTU treated larvae were accompanied by a depletion of the dorsal PIC positive cells. In the ventral preoptic region PIC positive cells developed from late prometamorphosis in control larvae, but failed to do so in PTU treated animals. It is argued that the differentiation of the PIC positive cells is largely dependent on thyroid hormones; that the dorsal PIC positive cells may produce a thyrotropin releasing factor; and that the function of these dorsal cells is inhibited by thyroid hormones.The authors thank Dr. P. G. W. J. van Oordt for his active interest and helpful advices, Miss Tineke Aafjes for technical assistance and Mr. H. van Kooten for making the photographs.     

Hypothalamic neurosecretion and metamorphosis in Xenopus laevis

Summary Tadpoles of Xenopus laevis were treated with propylthiouracil from the second half of prometamorphosis. Sagittal sections of the head region were stained a.o. with pseudoisocyanine. The goitrogen caused a degranulation of neurosecretory cells in the dorsal part of the preoptic region of the hypothalamus, suppressed the development of ventral neurosecretory cells and of the outer zone of the median eminence, stimulated the thyrotropic cells in the adenohypophysis, caused a hypertrophy of the thyroids, and impaired metamorphosis. Returning the animals to tap water had reciprocal effects and restored the normal activity of the hypothalamus, adenohypophysis and thyroid glands. It is concluded that thyroid hormones exert a morphogenetic influence upon hypothalamic centres and the outer zone of the median eminence and that a negative feed back relation exists between the thyroids on the one hand and the dorsal neurosecretory cells and the thyrotropic cells on the other.The author thanks Prof. Dr. P. G. W. J. van Oordt for his active interest and helpful advice, and Miss Tineke Aafjes for technical assistance.     

The reaction of the preoptic nucleus of Xenopus laevis tadpoles to osmotic stimulation

Summary The development of the preoptic nucleus of Xenopus laevis tadpoles during metamorphosis was studied and the effect of osmotic stimulation on this process investigated. The development of this region was not affected by treatment for one or more days in hypertonic media. It was found that at the end of metamorphosis the neurosecretory cells in the preoptic nucleus are localized in three regions: the rostro-dorsal, the caudo-dorsal and the ventral region. After osmotic stimulation only the neurosecretory cells of the caudo-dorsal region appeared to have reacted, as indicated by their loss of neurosecretory (PIC positive) material. It is concluded that the cells of this region may be involved in the synthesis of the posterior lobe hormones.The author thanks Prof. Dr. J. C. van de Kamer and Dr. F. C. G. van de Veerdonk for their interest and many helpful discussions, Dr. L. Boomgaart and Dr. A. P. van Overbeeke for correcting the English text and Miss C. M. G. van Bemmel for technical assistance.     

The thyrotropin secreting basophils in the adenohypophysis of Anoptichthys jordani

Summary In order to identify the TSH cells in the pituitary gland of Anoptichthys jordani, the development of the adenohypophysial cell types was studied in relation with the differentiation of the thyroids. In addition experiments were performed in young and adult animals with propylthiouracil (PTU) and thyroxine. At the time of the first thyroid development two meso-adenohypophysial cell types in the pituitary gland of Anoptichthys jordani are distinctly differentiated: orangeophilic cells and Alcian Blue (AB)-positive basophils. Because thyroxine and PTU could evoke changes in activity of the latter but had no effect on the orangeophils, it is concluded that the angular AB-positive basophils in the meso-adenohypophysis of Anoptichthys are responsible for the TSH production. PTU and thyroxine were unable to affect TSH cell activity in adult animals.This study was partly supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).I wish to thank Prof. Dr. P. G. W. J. van Oordt for his constant interest and helpful suggestions. I am indebted also to Miss M. I. Willems and Miss Tjitske van Soelen, who both gave conscientious technical assistance and to Mr. E. van der Vlist for making the photographs.     

Evidence for corticotropin releasing factor (CRF) synthesis in the preoptic nucleus of Xenopus laevis tadpoles

Summary For the study of the hypothalamo-hypophysial system of Xenopus laevis tadpoles, hypothalamic lesions were made by means of the electrocoagulation technique. Lesioning of the ventral region of the preoptic nucleus resulted in a decrease of the number of ACTH cells in the pars distalis of the pituitary gland and in a diminution of the PAS-positive reaction of these cells. In addition, regeneration of the neurosecretory cells of the ventral region of the preoptic nucleus observed 6 weeks after lesioning was accompanied by the reappearance of normal PAS-positive ACTH cells in the pars distalis. It is suggested that the neurosecretory cells of the ventral region of the preoptic nucleus of Xenopus laevis tadpoles are related to the ACTH synthesizing cells, probably by producing CRF.Dedicated to Prof. Dr. med. W. Bargmann on the occasion of his 70th birthdayThe authors thank Prof. Dr. J.C. van de Kamer for his interest, Prof. Dr. P.G.W.J. van Oordt for his many helpful comments, and Messrs. H. van Kooten, E. van der Vlist, J.J. van der Vlis and M.C.A. van Pinxteren for preparing the illustrations     

Hypothalamo-hypophyseal system and thyroid gland of Acipenser güldenst?dti sturgeon fry during temperature changes

In the parr of the sturgeon (body length varies from 6.1 to 14.0 cm) histometric studies have been made on the elements of hypothalamo-hypophyseal neurosecretory system, tireotropic cells (TC) of the hypophysis and the thyroid gland (TG) during the decrease of water temperature from 24--27 degrees to 10--14 degrees. It was shown that in all size groups of the fish an increase in the number, growth and differentiation of neurosecretory cells in the dorsal part of the preoptic nucleus are taking place. Correlation was found between the activity of neurosecretory cells of the dorsal part of the preoptic nucleus and hypophyseal TC. The response to the decrease in water temperature follows the pattern of stress reaction, being expressed to different extent in various size groups of the parr depending on the initial condition of the functional complex investigated.     

The function of the hypophysis without preoptic neurosecretory control

Summary In a large number of adult female and male specimens of Rana temporaria, a total extirpation of the magnocellular neurosecretory preoptic nuclei was performed. Several months after operation, no regeneration of the neurosecretory nuclei had occurred. The results showed 1. that all A.F.-positive neurosecretory nerve fibres of the hypothalamo-hypophysial region originate from nerve cell bodies localized in the magnocellular preoptic nuclei; 2. that the magnocellular preoptic nuclei do not play an important part in the inhibitory control of the activity of the pars intermedia of Rana temporaria; 3. that the possible role of the magnocellular preoptic nuclei in the seasonal development of the gonads and of the secondary sexual characteristics of Rana, could be only of secondary importance; 4. that, in accordance with previous experiments, in Rana, ovulation is dependent on preoptic hypothalamic structures. Some results of the present and of other experiments can be interpreted as supporting the hypothesis of a possible dual higher control over the gonadotropic centre (Dierickx, 1965b, 1966, 1967) of the pars ventralis of the tuber cinereum.A part of these studies has been reported previously in abstract form (Dierickx, 1963a, 1963b, 1965a).     

Hypophysiotropic Centers in the Brain of Amphibians and Fish

The subject is the localization of three different hypophysiotropiccenters in the brain of amphibians and fish. The thyrotropic hormone-releasing hormone (TRH) in Xenapus mayoriginate from the dorsal magno-cellular neurons of the preopticnucleus. This hypothesis is based on correlative changes betweenthese cells and alterations in thyroid activity during metamorphosis.Experimental data are in support of a functional relationshipbetween certain preoptic neurons and the thyrotropic activityof the pituitary. The MSH inhibiting activity of the hypothalamus is effectedby means of an aminergic innervation of the pars intermediain amphibians, teleosts and elasmobranchs. In amphibians theaminergic fibers originate from the caudal part of the paraventricularorgan (PVO); in elasmobranchs probably from the nucleus mediushypothalamicus(NMI); in teleosts the origin still has to beinvestigated. Two centers producing gonadotropic hormone-releasing hormone(GRH) have been demonstrated. Lesion experiments lead to thehypothesis that GRH is produced in the caudal hypothalamus,i.e., in the nucleus infundibularis ventralis of amphibiansand in the nucleus lateralis tuberis of fishes. ImmunoHuorescencestudies indicate in both groups the presence of neurons, infront of the preoptic area in the telencephalon, and these neuronsare immuno-reactive with anti-mammalian LH-RH.     

Cell types in the pituitary of the eel,Anguilla anguilla L., at different stages in the life-cycle

Summary Striking differences in certain cell-types in the pituitary of the eel at different stages of the life-cycle indicate their possible function. One type, suspected of GTH function, was not present in the elver but present in the young adults and vacuolated in older adults just prior to migration; these changes accompany stages in the development of the gonads. Others, characterised as STH and TSH cells behaved in a manner consistent with the view that they are concerned in the regulation of metabolic activity and metamorphosis.Stainable neurosecretory material was not detected in the tracts leading to the anterior lobes of the pituitary in the elver, but was present in these tracts in the adult.Dedicated to Professor W. Bargmann on his 60th birthday.     

Distribution of lamprey gonadotropin-releasing hormone-III (GnRH-III) in brains of larval lampreys (Petromyzon marinus)

Two immunoreactive forms of gonadotropinreleasing hormone (GnRH), lamprey GnRH-I and lamprey GnRH-III, were found in neurons in larval sea lampreys (Petromyzon marinus). Using antisera preferentially directed against either lamprey GnRH-I or-III, dense reaction product was seen in cell bodies in the rostral hypothalamus and preoptic area. Reaction product was also dense in fibers to and within the neurohypophysis, in addition to numerous fibers which projected caudally, beyond the neurohypophysis through the mesencephalon. The majority of immunoreactive GnRH was lamprey GnRH-III, and when lamprey GnRH-I was seen, it was in cells that appeared to contain both forms of GnRH. A small number of cells found in the caudal hypothalamus contained only immunoreactive lamprey GnRH-III, and these may constitute a functional subgroup within the population of GnRH neurons. In animals undergoing metamorphosis there was a large increase in reaction product in all GnRH-containing cells and fibers. A striking change within the distribution of GnRH cells was localized to a distinct group of GnRH-immunoreactive cells (GnRH-I and-III) in the ventral anterior hypothalamic area. These cells were minimally detectable in larvae, but during metamorphosis became densely filled with immunoreactive product in perikarya and distal processes. The results are consistent with the hypothesis that lamprey GnRH-III is an important form of GnRH during the maturation of GnRH cells and fibers, and further indicates that these cells have attained their normal positions in the preoptic area and hypothalamus before metamorphosis.     

The evolution of neurosecretory centers in bilaterian forebrains: insights from protostomes

Forebrain neurosecretory systems are widespread in the animal kingdom. This review focuses on recent molecular data from protostomes, discusses the original complexity of the bilaterian forebrain neurosecretory system, provides an evolutionary scenario for the emergence of the vertebrate preoptic area/hypothalamus/neurohypophysis and suggests a possible function for an ancient set of sensory-neurosecretory cells present in the medial neurosecretory bilaterian forebrain.     

Inhibitory effect of large doses of propylthiouracil and methimazole on an increase of thyroid radioiodine release in response to thyrotropin.

Thyroidal radioiodine release increased shortly after a single injection of small doses of PTU, while moderate doses of MMI produced a similar increase of thyroidal radioiodine release with a latency of 7-9 hr. Large doses of PTU and MMI failed to augment thyroidal radioiodine release for at least 29 to 34 hr after the initial administration of goitrogens, although plasma TSH increased significantly because of goitrogen administration. An increase of thyroid hormone release in response to exogenous TSH was depressed by PTU and MMI in rats and mice treated with T4. Since this depression of TSH action only continued for a short period in spite of continuous administration of goitrogens, and since final thyroidal radioiodine release rate was similar to that produced by small doses of PTU, the effects mentioned were not simply due to general toxic action of goitrogens. It is suggested that large doses of PTU and MMI not only block thyroid hormone synthesis but also interfere with the action of TSH on thyroid hormone secretion.     

Therapeutic exploration of betulinic acid in chemically induced hypothyroidism

Hypothyroidism is a chronic condition characterized by abnormally low thyroid hormone production. The decreased serum level (>5.1 mIU/l) of thyroid-stimulating hormone (TSH) in blood indicates hypothyroidism. The study was an attempt to access the effect of betulinic acid on chemically induced hypothyroidism in female albino rats. Betulinic acid is a naturally occurring pentacyclic triterpenoid, which has antiretroviral, antimalarial, and anti-inflammatory properties, as well as anticancer potential, by inhibiting topoisomerase. Hypothyroidism was induced in female albino rats using propylthiouracil (PTU) at a dose of 60 μg/kg body weight orally for 1 month. Induction of hypothyroidism was confirmed by increased TSH level. At the end of second month, blood was collected, centrifuged and serum was analyzed for TSH, T3, and T4 level and protocol was terminated by killing of animals. The animals exposed to PTU were treated with pure standard drug thyroxine at a dose of 10 μg/kg of body weight by oral route and the test drug betulinic acid 20 mg/kg by oral route through force feeding in their respective groups. Treatment was carried out for a period of 2 months. Group with PTU-induced hypothyroidism showed an elevation in serum TSH and reduction level, which was restored by the betulinic acid in treated female albino rats. Betulinic acid also reduced the damage caused in the thyroid tissues by PTU, thus minimizing the symptoms of hypothyroidism. Histopathological examinations of the thyroid tissue showed changes in the thyrocytes of PTU-treated group while thyroxine group showed normal thyroid follicles cell architecture and the group treated with betulinic acid also showed marked improvement in the follicles integrity which shows that betulinic acid has some protective activity. This study shows that the betulinic acid has thyroid-enhancing potential by lowering down the TSH levels and reducing the damage caused in the thyroid tissues, thus minimizing the symptoms of hypothyroidism when used anaphylactically in rats.     

Comparison of potential protective effects of melatonin, indole-3-propionic acid, and propylthiouracil against lipid peroxidation caused by potassium bromate in the thyroid gland

Potassium bromate (KBrO3) is a prooxidant and carcinogen, inducing thyroid tumors. Melatonin and indole-3-propionic acid (IPA) are effective antioxidants. Some antioxidative effects of propylthiouracil (PTU)--a thyrostatic drug--have been found. The aim of the study was to compare protective effects of melatonin, IPA, and PTU against lipid peroxidation in the thyroids, collected from rats treated with KBrO3, and in homogenates of porcine thyroids, incubated in the presence of KBrO3. Wistar rats were administered KBrO3 (110 mg/kg b.w., i.p., on the 10th day of the experiment) and/or melatonin, or IPA (0.0645 mmol/kg b.w., i.p., twice daily, for 10 days), or PTU (0.025% solution in drinking water, for 10 days). Homogenates of porcine thyroids were incubated for 30 min in the presence of KBrO3 (5 mM) plus one of the antioxidants: melatonin (0.01, 0.1, 0.5, 1.0, 5.0, 7.5 mM), or IPA (0.01, 0.1, 0.5, 1.0, 5.0, 7.5, 10.0 mM), or PTU (0.01, 0.1, 0.5, 1.0, 5.0, 7.5, 10.0 mM). The level of lipid peroxidation products (MDA + 4-HDA) was measured spectrophotometrically in thyroid homogenates. In vivo pretreatment with either melatonin or with IPA or with PTU decreased lipid peroxidation caused by KBrO3--injections in rat thyroid gland. Under in vitro conditions, PTU (5.0, 7.5, and 10.0 mM), but neither melatonin nor IPA, reduced KBrO3-related lipid peroxidation in the homogenates of porcine thyroids. In conclusion, melatonin and IPA may be of great value as protective agents under conditions of exposure to KBrO3.     

Ultrastructure of the peptidergic and monoaminergic neurons in the hypothalamic neurosecretory system of Anuran Batracians

Summary In the toad Bufo arenarum Hensel the following regions of the hypothalamic — neurohypophyseal system were studied under the electronmicroscope: preoptic and paraventricular nuclei, median eminence and infundibular process of the neurohypophysis.Neuronal perikarya of the preoptic nucleus are loaded with typical neurosecretory granules of peptidergic nature having a mean diameter of 1660 Å. While most neurons of the winter toad are in a storage stage a few show signs of a more active synthetic activity. A distinctive feature of preoptic neurons is the presence of large lipid droplets. The paraventricular nucleus contains small neurons containing granulated vesicles with a mean diameter of 800-1000 Å. In the region extending between these two nuclei and the median eminence axons containing either neurosecretory elementary granules or granulated vesicles are observed.The inner zone of the median eminence is occupied by axons of the preoptic neurohypophyseal tract; two types of axons, according to the size and density of the neurosecretory granules, may be recognized. The outer zone of the median eminence contains mainly axons and nerve terminals containing granulated vesicles of probable monoaminergic nature and only a few with granules of peptidergic type.The neurohypophysis contains two kinds of axons: one with more dense granules of 1800 Å and the other with granules of lesser electron density and 2100 Å. At the ending proper small clear vesicles of synaptic type are found.A progressive increase in volume of the peptidergic granules along the axon is demonstrated. This is of the order of 218% from the preoptic perikarya down to the infundibular process. The physiological significance of the two neurosecretory systems — i.e. the monoaminergic and the peptidergic — and the probable nature of the two types of peptidergic axons is discussed.Supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-67).The authors want to express their gratitude to Mrs. Defilippi-Novoa and Mr. Alberto Sáenz for their skillful assistance.     

Ultrastructural and cytochemical effects of trypan blue on TSH stimulation of thyroid follicular cells

Summary Ultrastructural and cytochemical techniques were used to study the effects of trypan blue on the response of mouse-thyroid cells to exogenous stimulation by thyroid stimulating hormone (TSH). The dye delayed the response to TSH resulting in decreased colloid-droplet formation in the apical region of the cells. The dye did not stop the shift of trimetaphosphatase activity from lysosomes to phagolysosomes. The duration of the TSH-induced response was shorter in the dye treated thyroids. Small vesicles, with trimetaphosphatase reaction product, were found near Golgi elements, phagolysosomes, and the plasma membrane facing the intercellular space of adjacent follicle cells. Their enzyme activity was not affected by exposure to the dye. These data indicate that the primary effect of trypan blue on the response of thyroid follicle cells to TSH stimulation was reduced endocytosis in the apical region resulting in fewer colloid droplets.     

Immunofluorescence of somatostatin-producing sites in the hypothalamus of the tadpole,Alytes obstetricans Laur.

Summary Three sites of somatostatin-synthesizing perikarya, or a related antigen, were determined by immunofluorescence in the hypothalamus of the tadpole, Alytes obstetricans (Amphibia, Anura). Two sites of neurosecretory perikarya were localized in the preoptic nuclei of the anterior hypothalamus; the axons extended either to the anterior diencephalon or to the median eminence and the pituitary. The third site was found in the posterior hypothalamus. These neurosecretory cells showed a strong immunofluorescent reaction; their axons all terminated at the level of the median eminence. Somatostatin cells were only found in intact or hypophysectomized tadpoles given somatotropin (STH). The strong reaction observed in hypophysectomized tadpoles was possibly due to the loss of the terminal portion of the neurosecretory pathway (median eminence and pituitary) by which the agent is transported to the site of discharge.     

Adenohypophysial-thyroid activity of the tiger salamander, Ambystoma tigrinum, as a function of metamorphosis and captivity

The aim of this study was to determine the timing of adenohypophysial activation during metamorphosis of the tiger salamander, Ambystoma tigrinum. It consisted of two parts: 1) determination of plasma thyroid hormone concentrations and analysis of thyroid gland histology as a function of metamorphic stage and 2) analysis of the time-course of uptake of 125I by the thyroids during metamorphosis as an indicator of endogenous thyrotropin (TSH) levels. Significant increases in both triiodothyronine (T3) and thyroxine (T4) first were evident at the onset of metamorphic climax (stage II). Maximum levels of both hormones were not observed, however, until the completion of gill resorption (stage VII). No changes in thyroid histology were observed that could be unambiguously related to metamorphic transformation. The thyroids accumulated 125I in a slow but linear fashion in premetamorphic larvae (stage I). However, uptake exhibited a rapid peak during early climax (stage II), before maximum concentrations of thyroid hormones were observed. In addition, uptake was maintained above premetamorphic levels at stage VII, in conjunction with maximum levels of T4 and T3. Captivity alone produced a small but significant increase in plasma concentrations of T3. It produced no significant effect on either thyroid histology or uptake of 125I. These results indicate that adenohypophysial activation occurs rapidly and is maximal at the onset of metamorphic climax.     

The distribution of monoamines in the hypothalamus of the Japanese quail Coturnix coturnix japonica

Summary The distribution of monoamines in the hypothalamus of the Japanese quail (Coturnix coturnix japonica) has been studied using a histochemical fluorescence technique. In the posterior hypothalamus catecholamine-containing nerve fibres are localised in the nucleus tuberis and nucleus hypothalamicus posterior medialis and are linked by fluorescent tracts running in the stratum cellulare internum. Further tracts may be traced from the nucleus tuberis around the base of the third ventricle to the sub-ependymal layer of the median eminence, where they then appear to pass through the hypothalamo-hypophysial neurosecretory tract to terminate in the palisade zone on the portal vascular bed. The innervation of the palisade layer by catecholamines is sparse. The fluorescent terminals are spread evenly throughout both the anterior and posterior divisions of the median eminence. There is no monoamine innervation of the pars nervosa. The paraventricular organ has both 5-hydroxytryptamine- and catecholamine-containing cell bodies and axons may be traced into the region of the nucleus hypothalamicus posterior medialis. In the anterior hypothalamus the neurosecretory paraventricular nucleus contains many catecholamine nerve fibres and terminals. These are linked by fibre tracts to the nucleus basalis and to the nucleus hypothalamicus posterior medialis. The supraoptic nucleus is less well innervated although a dense accumulation of fibres lies in the preoptic recess. The latter is thought to give rise to long axons which pass in association with the neurosecretory tract to end in the nucleus tuberis.Supported by a Grant (AG 24/36) from The Agricultural Research Council. We are indebted to Dr. G. A. Clayton, Institute of Animal Genetics, University of Edinburgh, for supplying the birds.     

Histological changes in the pituitary-thyroid axis during spontaneous and artificially-induced metamorphosis of larvae of the flounder Paralichthys olivaceus

Summary Histological changes in the pituitary TSH cells and in the thyroid gland of flounder (Paralichthys olivaceus) larvae during spontaneous or artificially induced metamorphosis were studied. Activity of the immunoreactive TSH cells (IrTSH cells) gradually increased during premetamorphosis, reaching the highest level in prometamorphic larvae, and the cells were degranulated in metamorphic climax. The IrTSH cells were most inactive at the post-climax stage. The thyroid gland was morphologically the most active in metamorphic climax when the degranulation occurred in the pituitary IrTSH cells, and appeared inactive at post-climax. A few weeks after metamorphosis, both the IrTSH cells and the thyroid gland appeared to be activated again in the benthic, juvenile flounder. Administration of thyroxine or thiourea revealed negative feedback regulation of the pituitary-thyroid axis in flounder larvae. These results indicate that activation of the pituitary-thyroid axis induces metamorphosis in the flounder.