Experimental identification of a hypothalamic gonadotropic centre

Summary In a large group of adult female and male specimens of Rana temporaria all nervous pathways to the median eminence and hypophysis were extirpated without disturbing the normal blood supply of the median eminence and hypophysis. The total interruption of the nervous pathways persisted in all animals. In all animals gametogenesis and seasonal development of the gonads and of the secondary sexual characteristics were absent. In another large group of animals, the pars ventralis of the tuber cinereum of the hypothalamus was isolated from the brain. The pars ventralis retained its normal connections with the median eminence and hypophysis. The normal blood supply of the whole isolated region (pars ventralis tuberis + median eminence + hypophysis) was preserved. Four months after operation, in the majority of the animals, the isolation was still complete. In all animals, normal gametogenesis and seasonal development of the gonads and of the secondary sexual characteristics had occurred.In accordance with previous experiments, the present experiments showed that the pars ventralis tuberis contains an important gonadotropic centre. The activity of the gonadotropic centre of the pars ventralis tuberis is, at least, mainly exerted by its influence on the gonadotropic function of the pars distalis of the hypophysis. This is shown morphologically by a gradient of nuclear volume and by the mean nuclear volume of the P.A.S. positive cells of the pars distalis. The influence of the gonadotropic centre on the pars distalis is exerted by A.F.-negative nerve fibres to the median eminence. It is highly probable that all these nerve fibres end on the primary capillary network of the hypophysial portal system. The experiments strongly suggested that the axon endings of the gonadotropic centre would influence the gonadotropic activity of the pars distalis of the hypophysis by releasing of gonadotropic releasing factors into the blood capillaries of the median eminence.The gonadotropic centre of the pars ventralis tuberis is necessary for the normal gametogenesis and for the seasonal development of the gonads and of the secondary sexual characteristics. So far as concerned the gametogenesis and the seasonal development of the gonads, the function of the gonadotropic centre is largely autonomous.Exclusion of the function of a part of the pars ventralis tuberis produces quantitative, but no qualitative changes in gametogenesis.     

The origin of the aldehyde-fuchsin-negative nerve fibres of the median eminence of the hypophysis: a gonadotropic centre

Summary In a large number of adult female and male specimens of Rana temporaria, the pars ventralis of the tuber cinereum of the hypothalamus was completely isolated from the brain. The pars ventralis retained its normal connections with the hypophysis. The vascular supply of the whole isolated region was left intact. Two and a half months after operation, in some animals, the interruption of the nervous pathways was still complete. In these animals it could be shown that all or nearly all A. F.-negative nerve fibres of the median eminence are axons of nerve cells localized in the area periventricularis of the pars ventralis tuberis. According to our results in Rana, the same A. F.-negative nerve fibres of the higher vertebrates are probably also axons of homologous nuclei of the tuber cinereum.In accordance with previous experiments, it could be concluded that the nerve cells of which the A. F.-negative fibres are axons, form an important centre that, via the pars distalis of the hypophysis, is responsible for the seasonal development of the ovaries, eggs and oviducts in the females and for the seasonal development of the thumb pads in the males.     

The origin of somatostatin fibers in the median eminence and neural lobe of Rana temporaria

Summary A light microscopic immunocytochemical study of the brain of frogs with hypothalamic lesions was performed in order to obtain evidence concerning the origin of somatostatin fibers in the median eminence and neural lobe of the hypophysis. The results indicate that the somatostatin fibers of the neural lobe originate from somatostatin perikarya located in the prechiasmatic part of the hypothalamus and possibly also in the telencephalon. The somatostatin neurons of the pars ventralis tuberis cinerei do not send axons to the neural lobe. The frog median eminence contains axon terminals of somatostatin neurons located in the pars ventralis of the tuber cinereum. Many other somatostatin fibers of the frog median eminence originate from somatostatin neurons located outside the tuber cinereum. Most of these neurons probably lie in the preoptic hypothalamic region.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Identification of adenohypophysiotropic neurohormone producing neurosecretory cells in Rana temporaria

Summary By means of electron microscopy, in the median eminence of Rana temporaria, the terminal arborizations of axons of six different types of neurosecretory cells, located in the pars ventralis of the tuber cinereum, were identified. In addition, phenomena connected with the release of neurosecretory material from the axon terminals of these neurosecretory cells into the blood capillaries of the median eminence are described.Preliminary results suggested the existence, in the median eminence, of additional different neurosecretory axon types which could also belong to corresponding neurosecretory cell types probably located in the apical part of the pars ventralis of the tuber cinereum. Moreover, in the external region of the normal median eminence, separate monoaminergic nerve fibres were tentatively identified. Arguments are adduced which plead (1) against the assumption that the ependyma or the pituicytes of the median eminence could produce adenohypophysiotropic hormones; (2) against the inference that the ependymal cells of the median eminence might be involved in the transport of adenohypophysiotropic hormones from the cerebrospinal fluid into the blood capillaries of the median eminence.     

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).     

The nerve fibres controlling the gonadotropic activity of the hypophysis of Rana temporaria

Summary In 27 adult female specimens of Rana temporaria, the total interruption of the nervous pathways to the median eminence and the hypophysis was performed, without disturbing the normal blood supply of the hypophysis. In eight cases this complete interruption persisted three months after operation. In these animals there occurred no seasonal development of the eggs, ovaries and oviducts. In the 19 other operated animals a varying degree of regeneration of the interrupted nerve fibres occurred. In these animals a varying number of ripe eggs was present, but in many of them there existed a significant difference between the weight of their ovaries compared with that of the ovaries of the control animals. In all control animals the seasonal development of the ovaries and oviducts was normal. From these and from previous experiments it could be concluded that the A. F. negative nerve fibres of the median eminence, not originating from the magnocellular neurosecretory preoptic nuclei, are responsible for the normal seasonal development of the ovaries and oviducts in Rana temporaria. It must be these nerve fibres that release gonadotropic releasing factors into the blood capillaries of the median eminence. The A. F. positive neurosecretory fibres, the portal circulation of the pars distalis and the pituicytes of the median eminence play only a secondary part during this period of control of the gonadotropic activity of the hypophysis.     

Immuno-cytochemical demonstration,in the external region of the amphibian median eminence,of separate vasotocinergic and mesotocinergic nerve fibres

Summary By means of single and double immuno-enzyme cytochemical staining techniques, it was shown that the external region of the amphibian median eminence contains separate vasotocinergic and mesotocinergic nerve fibres. Moreover, it was demonstrated that the vasotocinergic fibres also contain neurophysin. In animals in which the hypothalamic magnocellular neurosecretory preoptic nuclei had been completely removed, the immuno-reactive vasotocinergic and mesotocinergic fibres of the median eminence had disappeared. From this result, it is concluded that, at least the great majority of the vasotocinergic and mesotocinergic fibres of the external region of the amphibian median eminence are processes of neurosecretory perikarya located in the hypothalamic magnocellular preoptic nuclei. On the other hand, our results do not exclude the possibility that a minority of these neurosecretory fibres originate from small immuno-reactive perikarya which were found in the tuber cinereum. The observation that both kinds of processes accumulate around blood capillaries of the hypophysial portal system strongly suggests that they play a role in the control of the activity of the pars distalis of the hypophysis.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

A gradient of activity in the hypophysis of Rana temporaria

Summary In normal pars distalis of Rana temporaria, a gradient of activity of the P.A.S.-positive cells is always present. In animals, with complete and permanent interruption of all nervous pathways to the median eminence and hypophysis and without disturbance of the normal blood supply of the hypophysis, a subtotal disappearance of the gradient of activity occurred and the total activity of the P.A.S.-cells of the pars distalis was lowered. The activity of the P.A.S.-positive cells regressed in a typical way, suggesting a humoral mechanism.In animals with marked regeneration of the interrupted nerve fibres, a normal gradient of activity was present. From these and previous experiments, it is concluded that the gradient of activity of the P.A.S.-positive cells of the pars distalis of Rana temporaria is largely dependent on the A.F.-negative fibres of the median eminence, not originating from the magnocellular neurosecretory preoptic nuclei. It is suggested that these nerve fibres exert their influence on the activity of the P.A.S.-positive cells via substances released in the blood capillaries of the median eminence.The gradient of activity and its variations are mainly related to the gonadotropic function of the pars distalis.I am indebted to miss M. T. Bakeeff and miss C. Everaert who were so kind to check the karyometric results.     

The vascularization of the neural stalk and the pars nervosa of the hypophysis in the toad,Bufo bufo (L.) (amphibia,anura)

The angioarchitecture of the neural stalk and the encephaloposthypophysial portal system of the hypophysis of the toad, Bufo bufo (L.), was studied using three different methods. The neural stalk is mainly supplied by branches of the arteria infundibularis superficialis which form a widemeshed vascular network. Dorsally this network continues into the plexus of the pars nervosa. The vascularization of the pars nervosa is made up of the encephalo-posthypophysial portal system. This portal system consists of a hypothalamic branch (=portion), a mesencephalic and a mesencephalicbulbar branch (=portion). The hypothalamic branch was found to drain the pars ventralis of the tuber cinereum as well as more dorsal regions of the diencephalon. The mesencephalic-bulbar trunk enters the hypothalamic branch. The resulting common stem of the encephalo-posthypophysial portal vein the curves around the retroinfundibular communicating artery, crosses its ventral side and runs caudally. The secondary capillary plexus of the pars nervosa is characterized by well defined capillary plexus of the pars nervosa is characterized by well defined capillary networks which are located at the periphery of the parenchyma of the pars nervosa, thus forming a rostral, dorsal and ventro-caudal net. The central region of the parenchyma of the pars nervosa is supplied only by main branches of the encephalo-postpophysial portal vein. The venous drainage of the pars nervosa is via the vena hypophysea transversa.     

Galanin-like immunoreactivity in the brain and pituitary of the "four-eyed" fish, Anableps anableps

The distribution of galanin (GAL)-like immunoreactivity was investigated in the brain and pituitary of the "four-eyed" fish, Anableps anableps. GAL-immunoreactive (GAL-ir) perikarya were located in the area ventralis telencephali pars supracommissuralis, nucleus preopticus periventricularis, nucleus preopticus pars parvocellularis, nucleus preopticus pars magnocellularis, nucleus lateralis tuberis ventralis, nucleus lateralis tuberis lateralis, and nucleus lateralis tuberis posterior. A few scattered, GAL-ir neurons were also observed in or adjacent to the nucleus recessus lateralis, nucleus recessus posterioris and lobus facialis (VII). GAL-ir fiber networks were widespread in the brain, with a comparatively higher density in the ventral telencephalic, preoptic and infundibular regions. The neurohypophysis showed GAL-ir innervation and there were GAL-ir cells in the adenohypophysis. The presence of GAL-ir cells in the hypothalamus and in the pituitary is an important asset for the supposed role of GAL-like peptide in neuroendocrine regulation of brain and pituitary functions.     

Immunohistochemical Localization of C-RFamide, a FMRF-related Peptide, in the Brain of the Goldfish, Carassius auratus

Carassius RFamide (C-RFa) is a novel peptide found in the brain of the Japanese crucian carp. It has been demonstrated that mRNA of C-RFa is present in the telencephalon, optic tectum, medulla oblongata, and proximal half of the eyeball in abundance. Immunohistochemical methods were employed to elucidate the distribution of the peptide in the brain of the goldfish (Carassius auratus) in detail. C-RFaimmunoreactive perikarya were observed in the olfactory bulb, the area ventralis telencephali pars dorsalis and lateralis, nucleus preopticus, nucleus preopticus periventricularis, nucleus lateralis tuberis pars posterioris, nucleus posterioris periventricularis, nucleus ventromedialis thalami, nucleus posterioris thalami, nucleus anterior tuberis, the oculomotor nucleus, nucleus reticularis superior and inferior, facial lobe, and vagal lobe. C-RFa immunoreactive fibers and nerve endings were present in the olfactory bulb, olfactory tract, area dorsalis telencephali pars centralis and medialis, area ventralis telencephali, midbrain tegmentum, diencephalon, medulla oblongata and pituitary. However, in the optic tectum the immunopositive perikarya and fibers were less abundant. Based on these results, some possible functions of C-RFa in the nervous system were discussed.     

Immunoreactive material resembling ovine prolactin in perikarya and nerve terminals of the rat hypothalamus

Summary The presence of prolactin (PRL)-like material is demonstrated in the brain of rats with the aid of anti-ovine PRL (oPRL) IgG as primary antibody in the unlabeled antibody-enzyme method. Immunoreactive deposits are visualized as an intraneuronal constituent with a widespread distribution in the hypothalamus and neural lobe of the pituitary. Dense networks of reactive nerve terminals derived from two prominent fibre tracts, a ventral (VHT) and a dorsal hypothalamo-neurohypophysial tract (DHT) are seen. The VHT is confined to the median eminence and pars oralis tuberis, the DHT to the pars caudalis tuberis. Both fibre tracts pass through the infundibular stalk into the neural lobe. The origin of the immunoreactive nerve terminals can be elucidated only to some extent. The VHT gives off beaded fibres entering the ependymal and glandular layer of the median eminence. Immunoreactive perikarya are observed in the supraoptic nucleus, the paraventricular nucleus, the anterior hypothalamic nucleus, the anterior commissural nucleus, the preoptic nucleus and the interstitial nucleus of the stria terminalis. A few of the immunoreactive perikarya are observed in close connection with brain vessels and the ependymal cells of the third ventricle. The results indicate that the anti-oPRL has a unique region specificity implying that only a segment of the mammalian PRL molecule is present in these nuclei of the brain. Fragments of PRL may function as neuromodulators or neurotransmitters in the rat brain.We are indebted to Dr. Mogens Hammer, Rigshospitalet, Copenhagen for the gift of Arg-VP and anti-VP, and to NIAMDD for the gift of ovine PRL, ratPRL, anti-rPRL, anti-hPRL and bovineSTH     

Immunocytochemical distribution of FMRFamide-like substance in the brain of the cloudy dogfish,Scyliorhinus torazame

Summary The distribution of the molluscan cardioexcitatory tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH₂) in the brain of the cloudy dogfish, Scyliorhinus torazame, was examined by immunocytochemistry. FMRFamide-like immunoreactivity was demonstrated to occur extensively in various regions of the dogfish brain, except for the corpus cerebelli. Immunoreactive neuronal perikarya were located in the ganglion of the nervus terminalis, the preoptic area, and the hypothalamic periventricular gray matter consisting of the nucleus medius hypothalamicus, the nucleus lateralis tuberis, and the nucleus lobi lateralis. some of the immunoreactive cells in the hypothalamus were identified as cerebrospinal fluid-contacting neurons. The bulk of the immunostained fibers in the nervus terminalis penetrated into the midventral portion of the telencephalon and ran dorsocaudally toward the basal telencephalon and hypothalamus, showing radial projections or ramifications. The labeled fibers were abundant in the midbasal part of the telencephalon and in the hypothalamus, where some fibers were found in loose networks around the cell bodies of the nucleus septi and hypothalamic periventricular nuclei. The fibers demonstrated in the hypothalamus terminated around the vascular wall of the primary capillary plexus of the median eminence or penetrated deeply into the pars intermedia of the hypophysis. These results suggest that, in the dogfish, an FMRFamide-like substance participates in the regulation of adenohypophysial function. This molecule may have a role as a neurotransmitter and/or neuromodulator in the central nervous system.     

Efferent projections from the retrochiasmatic area to the median eminence and to the pars nervosa of the hypophysis with special reference to the A15 dopaminergic cell group in the sheep

Anterograde tracers, viz. Phaseolus vulgaris leucoagglutinin and fluorescein dextran, were used in conjunction with tyrosine hydroxylase immunohisto-chemistry to study the projections of the A15 dopaminergic cell group towards the median eminence and pituitary in sheep. After injection of the tracers in the retrochiasmatic area, which contains the cell group A15, fibres containing anterograde tracer were observed in the internal zone of the median eminence and in the pars nervosa of the pituitary. Numerous tyrosine hydroxylase immunoreactive fibers were present in the external zone of the median eminence and in the pars intermedia and the pars nervosa of the pituitary, with characteristic patterns of organisation in each area. Most tyrosine hydroxylase-immunoreactive fibres containing fluorescein dextran were located in the pars nervosa, whereas only a few were observed in the internal zone of the median eminence. It was concluded that at least part of the dopaminergic innervation of the pars nervosa originated from the A15 group. These results provide morphological evidence for (1) the role of dopaminergic neurons of the A15 cell group in the seasonal control of prolactin secretion via the release of dopamine in the pars nervosa, and (2) putative physiological interactions between dopamine and the secretion of neurohypophysial hormones in sheep.     

The structure and vascularization of the pars tuberalis of the hypophysis of Rana temporaria

Summary The pars tuberalis of the hypophysis of Rana temporaria presents the general structural and the cytological characteristics of an endocrine gland. It is composed of elongated cells with long, branching processes ending on the external basement membrane of the pericapillary space. The pars tuberalis cells produce secretory granules which are accumulated in the pericapillary endings of the processes.Corresponding to its separate localization, the pars tuberalis of Rana temporaria has a separate vascularization of which the efferent capillaries anastomose with the capillary plexus of the median eminence. The general direction of the blood flow of the pars tuberalis is towards the capillaries of the median eminence. Also, the secretory products of the pars tuberalis pass into the blood stream of the hypophysial portal system.Several characteristics of the pars tuberalis show that its function must be different from that of the pars distalis of the hypophysis. Moreover, in contrast with the pars distalis, the activity of the pars tuberalis is not regulated by neurohumoral factors.The results show that a role of the pars tuberalis in the regulation of the activity of the pars distalis of the hypophysis is not excluded.     

Immunohistochemical localization of vasoactive intestinal polypeptide(VIP)-containing neurons in the hypothalamus of the Japanese quail,Coturnix coturnix

Summary The localization of vasoactive intestinal polypeptide (VIP) in the hypothalamus of the quail has been studied by means of light- and electron-microscopic immunohistochemistry. Numerous VIP-immunoreactive perikarya are distributed in the caudal portion of the nucleus infundibularis (n. tuberis) and nucleus mamillaris lateralis, and sparse in the preoptic area, nucleus supraopticus and nucleus paraventricularis. Dense localization of immunoreactive-VIP fibers is observed in the external layer of the median eminence, in close contact with the primary portal capillaries. The main origins of these fiber terminals are VIP-immunoreactive perikarya of the nucleus infundibularis. These neurons are spindle or bipolar and extend one process to the ventricular surface and another to the external layer of median eminence. They are CSF-contacting neurons and apparently constitute the tubero-hypophysial tract that links the third ventricle and the hypophysial portal circulation. VIP-reactive neurons in the nucleus mamillaris lateralis also project axons to the external layer of the median eminence, constituting the posterior bundle of the tuberohypophysial tract. Numerous VIP-immunoreactive perikarya occur also in the nucleus accumbens/pars posterior close to the lateral ventricle. They are also CSF-contacting neurons extending a process to the lateral ventricle. There are moderate distributions of VIP-reactive fibers in the area ventralis and in the area septalis.Ultrastructurally, the immunoreactive products against VIP are found in the elementary granules, 75–115 nm in diameter, within the nerve fibers in the median eminence.This investigation was supported by Scientific Research Grants No. 00556196, No. 56360027 and No. 56760183 from the Ministry of Education of Japan to Professor Mikami and Mr. Yamada     

Immunocytochemical devidence for hypophysial projections of the cells of the nucleus lateralis tuberis pars lateralis in the rainbow trout (Salmo gairdneri)

In the rainbow trout the pars lateralis is the most prominent part of the nucleus lateralis tuberis (NLT). To demonstrate a morphological relationship between this lateral part of the NLT and the pituitary, immunocytochemistry was applied as a staining method. Experiments were carried out on glutaraldehyde-picric acid-acetic acid-fixed brain sections of mature male and female rainbow trout using the peroxidase-anti-peroxidase immune technique with an antiserum against 27-S-methylglucagon as the first antibody. Most of the cells in the NLT/pars lateralis reacted with the antiserum. Axons from these cells enter the pituitary, extending exclusively in the numerous neurohypophysial digitations in the pars intermedia. No immunoreactive neurohypophysial protrusions were found in those parts of the adenohypophysis where the gonadotropic cells are located, indicating that the lateral part of the NLT is not directly involved in the control of gonadotropin secretion. In addition to cells of the NLT/pars lateralis only prolactin cells in the rostral pars distalis of the adenohypophysis reacted with the antiserum used.     

Immunocytochemical evidence for α-melanocyte-stimulating hormone in the hypothalamus of the frog Rana esculenta

Summary The distribution of immunoreactive -melanocyte-stimulating hormone (-MSH) within the brain of the frog, Rana esculenta, has been studied on adjacent serial sections using an indirect immunofluorescence technique. Immunoreactive cell bodies are found in the anterior part of the preoptic nucleus and in some ventral subependymal cerebrospinal fluid-contacting elements, and in the nucleus infundibularis ventralis. Numerous -MSH-like immunoreactive fibers are present in the preoptic area, in the pars ventralis of the tuber cinereum, and in the outer layer of the median eminence. This staining pattern is completely eliminated after preabsorbing the antiserum with the corresponding antigen, but blocking tests with -MSH-related peptides do not lead to any change in the immunoreaction. From these results it may be inferred that an -MSH-like system is present in the hypothalamic neurosecretory area of R. esculenta, and is probably related to its hypophysiotropic functions.The results are compared to the distribution of -MSH within the hypothalamus of reptiles and mammals.This work was supported by a grant from the M.P.I. (60%)     

Identification of adenohypophysiotropic neurohormone producing neurosecretory cells in Rana temporaria

Summary Electron microscopic investigation of neural isolated and normal pars ventralis of the tuber cinereum showed the presence, in Rana temporaria, of a tubero-hypophysial neurosecretory system. A striking structural resemblance between this parvicellular, aldehydefuchsin negative, tubero-hypophysial neurosecretory system and the magnocellular, aldehydefuchsin positive, hypothalamo-hypophysial neurosecretory system was observed. Six different neurosecretory cell types are described, characterized by different shape and size of their respective secretory granules. The nature of the secretory product of these cells is briefly discussed.Of the results of this investigation, a preliminary note has been published (Dierickx, 1971).The authors wish to thank Prof. Dr. H. Steyaert and Dr. W. Moerdijk of the Laboratory for Mathematical Statistics, Ghent University, for invaluable advice and assistance with their computers. They thank Dr. G. De Waele for his help with the scanning electron microscopy.