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.     

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.     

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 gonadotropic centre of the tuber cinereum hypothalami and ovulation

Summary In adult female specimens of Rana temporaria, the pars ventralis of the tuber cinereum of the hypothalamus was isolated from the brain. The isolated 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.The results showed (1) that, in accordance with previous experiments, as far as concerned gametogenesis, seasonal development of the gonads and of the secondary sexual characteristics, the function of the gonadotropic centre of the pars ventralis tuberis is largely independent of nervous connections between the pars ventralis of the tuber cinereum and the brain; (2) that, in animals in which the pars ventralis tuberis was isolated from the brain at the beginning of the seasonal development of the ovaries, the L.H. secretion is disturbed during the ovulation period; (3) that consequently, in such animals, the stimulating activity of the isolated gonadotropic centre on the L.H. secretion of the hypophysis is too weak to cause ovulation; (4) that nervous connections between the isolated region and the preoptic region of the brain are indispensable for ovulation.     

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.     

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     

Electron-microscopic investigation of the innervation of the pars distalis of the hypophysis in adult Rana temporaria L.

Summary In the pars distalis of the hypophysis of adult Rana temporaria, three types of nerve-fiber profiles were found at two distinct sites, in both lateral parts of the bordering regions of the anterior lobe with the intermediate lobe of the hypophysis. The first type of nerve-fiber profile consists of bundles of very fine axonal elements (diameter: <0.7 m). The second type is formed by larger nerve fibers (diameter up to 4 m) containing a few neurosecretory granules of approximately 100 nm. The third type of nervefiber profile resembles the second type but these nerve fibers make synaptoid contacts on at least two different types of glandular cells. The possible functional significance of these nerve fibers in the pars distalis is discussed.No nerve fibers were found (1) in the central part of the bordering region of the pars distalis with the intermediate lobe, (2) at the bordering region with the median eminence and (3) with the neurohypophysial stalk, and (4) in all other parts of the pars distalis.     

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.     

The structure and activity of the hypophysis of Rana temporaria in normal and in experimental conditions

Summary Volumetric, karyometric and histological studies were done on the pars distalis of the hypophysis of Rana temporaria, in normal and in experimental conditions.In the normal pars distalis, a gradient of activity of the P.A.S.-positive cells is described which shows a close relation with the blood supply. This relation strongly suggests the presence of a gradient of humoral factors which would influence the activity of the pars distalis. This gradient is not dependent on the influence of A.F.-positive neurosecretory fibres. In the P.A.S.-negative cells (O.G.-cells) a similar gradient could not be found. In transplants of pars distalis the gradient of activity is absent.There exists a striking difference between autotransplants and homotransplants of pars distalis in the eye, and also between autotransplants on the iris and autotransplants in the cornea.A parallelism between autotransplants of pars distalis on the iris and the pars distalis of animals deprived of their median eminence could be established. In both groups the pars distalis shows a similar decrease in volume. The glandular cells are well differentiated, but show a low activity. This low activity is most pronounced in the P.A.S.-cells. The normal variability in staining of the P.A.S.-cells has diminished: in general, the great majority of the P.A.S.-cells are P.A.S.-purples. The gradient of activity of the pars distalis has disappeared. The seasonal development of the gonads and of the secundary sexual characteristics is absent. This absence is probably not caused by a decrease of the total number of P.A.S.-cells of the pars distalis. It is probably due to the low activity of the P.A.S.-cells, which cannot be ascribed to the absence of the neurosecretory influence of the magnocellular preoptic nuclei.A preliminary note on the gradient of activity of the pars distalis has been published (Dierickx 1963c).     

Seasonal changes of the ultrastructure of the pars tuberalis of the hypophysis of Rana temporaria

Summary In the pars tuberalis of the hypophysis of Rana temporaria, which shows the ultrastructural characteristics of a polypeptide hormone secreting endocrine gland, seasonal changes of the ultrastructure are described. In accordance with the literature, these seasonal changes of ultrastructure are interpreted as the morphological expression of seasonal changes of endocrine activity of the pars tuberalis.     

The hypothalamo-hypophysial system of the frog Rana temporaria

Summary The median eminence (ME) of the adult frog, Rana temporaria, was studied by means of electron microscopy including quantitative electron-microscopic autoradiography. In frogs captured in May and June numerous peptidergic neurosecretory fibres extending via the internal zone to the pars nervosa display large swellings containing few granules, mitochondria, neurotubules and cisternae of the smooth endoplasmic reticulum. In addition, few secretory globules up to 1.5 m in diameter occur in these varicosities. In animals collected during the autumn period many of these neurosecretory swellings filled with neurosecretory granules and polymorphic inclusions resemble Herring bodies. Three types of granule-containing neurosecretory fibres were observed in the external zone (EZ) of the ME of adult R. temporaria. Peptidergic A₁- and A₂-type fibres are characterized by granules 150–220 nm and 100–160 nm in diameter, respectively. Monoaminergic fibres of type B with granules approximately 100 nm in diameter represent 50% of all neurosecretory elements in the EZ of the frog ME; 12% of the total number of granule-bearing axons in the EZ actively taking up radiolabelled 5-hydroxytryptophan are thought to be serotoninergic terminals. Neurosecretory terminals of all types and glial vascular endfeet establish direct contacts with the perivascular space of the primary portal capillaries. Some neurosecretory terminals are separated from the lumen of the third ventricle by a thin cytoplasmic lamella of tanycytes. The possible physiological significance of this structural pattern is discussed.     

On the relations between the hypothalamus and the anterior pituitary in Rana temporaria

Summary Dawson (1952) showed that, in Rana, many nerve terminals end round the capillaries of the venous portal system of the median eminence. Our observations not only confirm these facts but show as well that, besides the posterior lobe, the outer zone of the median eminence is also a centre of accumulation of neurosecretory material derived from the pre-optic nucleus. The amount of this neurosecretory material is moreover subject to a certain variation in connection with the seasons and with experimental conditions. The idea is put forward that the outer zone of the median eminence of Rana has to be considered as an analogical organ of the posterior lobe. Indeed, in the median eminence, the same morphological facts can be found, which are advanced for the conception that the posterior lobe is a centre of accumulation and a place of release of neurosecretory material originating from the pre-optic nucleus. On the basis of our observations we presume that the hypothalamo-hypophyseal neurosecretory system of Rana can be divided in two sub-units. System I ends in the posterior lobe; system II in the outer zone of the median eminence. The data of the literature indicate that the two systems also probably exist in the higher vertebrates. From these facts it is obvious that the neurosecretory material is transported from the pre-optic nucleus, via the tract, towards the outer zone 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).     

Immuno-enzyme cytochemical demonstration of mesotocinergic nerve fibres in the pars intermedia of the amphibian hypophysis

Summary Immuno-enzyme cytochemical investigations showed that the whole amphibian pars intermedia of the hypophysis is innervated by an intercellular network of peptidergic varicose nerve fibres which contain mesotocin or (and) parts of the mesotocin molecule. The pars intermedia does not contain vasotocinergic fibres. The mesotocinergic fibres are branches of axons leaving the pituitary stalk and the neural lobe. In animals of which the hypothalamic magnocellular neurosecretory preoptic nuclei had been completely removed, the immuno-reactive mesotocinergic fibres of the pars intermedia had totally disappeared. From this result, it is concluded that the mesotocinergic fibres of the pars intermedia of the amphibian hypophysis are axons of neurosecretory perikarya located in the hypothalamic magnocellular neurosecretory preoptic nuclei.Dedicated to Professor Berta Scharrer on the accasion of her 70th birthdayThis investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Myelinated Herring bodies in the median eminence of the cat.

An electron-microscopic study was carried out on the median eminence of cats during post-natal development. From the moment of birth (observations performed 12 hours later) Herring bodies were seen in the fibrillary layer of the median eminence. At 45 days after birth, myelinated nerve fibres could be observed, some of them containing neurosecretory granules. The number of myelinated fibres in the median eminence increased with age and at 90 days some Herring bodies appeared surrounded by myelin sheaths; these mainly contained neurosecretory granules and a few mitochondria.     

Fine structure of the median eminence and the pars nervosa of the bullfrog,Rana catesbeiana

Summary The hypothalamic neurosecretory system of the bullfrog, Rana catesbeiana, was studied with light- and electron microscopy. The median eminence is roughly divided into two portions. The upper portion mostly consists of ependymal cells, glial cells and preoptico-hypophysial nerve tract, whereas in the lower portion, neurosecretory axons, glial cells, processes of glial and ependymal cells, and fine blood vessels of the hypothalamic portal vein are located. A part of the neurosecretory axons of the preoptico-hypophysial tract proceeds to the lower portion of the median eminence. These axons are arranged perpendicularly to the capillaries of the hypothalamic portal vein. The glial cells are densely located in the area of the median eminence where neurosecretory material is abundant. The neurosecretory material in the neurosecretory cells, their axons, the median eminence and the pars nervosa of the bullfrog shows a positive reaction to PAS treatment.The neurohemal area of the median eminence is occupied by many neurosecretory and non-neurosecretory axons, containing neurosecretory granules and/or synaptic vesicles. The axonal portions with the synaptic vesicles which are considered to be the nerve endings abut on the capillaries of the portal system. The size of synaptic vesicles in the axon terminals containing few neurosecretory granules is larger than those in the endings with many neurosecretory granules. Infrequently glial and ependymal processes are interposed between the nerve endings and the capillary wall.In the hilar region of the infundibulum, synapses are frequently observed between the thin fibers with or without neurosecretory granules and dendrites of non-neurosecretory neurons. The probable functions of these synapses are briefly discussed on the basis of our findings. Both in the hilar region of the infundibulum and in the pars nervosa, electron-dense neurosecretory granules of two different sizes were observed. The median eminence contains only one type of granules.The fine structure of the pars nervosa shows similar structures to those of the median eminence. Both in the median eminence and the pars nervosa, the fenestrated endothelium of the capillaries was frequently observed. The thick perivascular connective tissue space containing fibroblasts and collagen fibrils was observed both in the median eminence and the pars nervosa. Vesicles in the cytoplasm of the endothelial cells which appear to take a part in the transendothelial transport were observed.This investigation was supported in part by United States Public Health Service Research Grant, No. A-3678, to Hideshi Kobayashi from the National Institute of Arthritis and Metabolic Diseases and partly by a grant for Fundamental Scientific Research from the Ministry of Education of Japan. The authors wish to express their thanks to Prof. K. Takewaki for his kind encouragement.     

Immunohistochemical demonstration of alpha-1-antitrypsin in the islet cells of human pancreas

Summary Using the unlabeled antibody peroxidase-antiperoxidase complex (PAP) technique at the light microscopic level, it has been shown that, in the dipnoan preoptico-hypophysial neurosecretory system, vasotocin and mesotocin are synthesized in separate neurons. In the preoptic nuclei, the perikarya of these two types of neurosecretory neurons are not located preferentially. The two types of neurosecretory perikarya give rise to separate vasotocinergic and mesotocinergic axons, respectively. The dipnoan median eminence and neural lobe contain separate vasotocinergic and mesotocinergic nerve fibres, the general distribution of which is described. In the pars distalis and the pars intermedia of the hypophysis, neurohypophysial hormone-containing nerve fibres have not been found.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk OnderzoekThe authors are greatly indebted to Prof. Dr. Hyder, Department of Zoology, University of Nairobi, Kenya for kindly supplying us with the fixed material used in this study     

On the histochemical differences of aldehyd-fuchsin positive material in the fibres of the hypothalamo-hypophyseal tract of Rana temporaria

Summary Using a modified Alcian blue/Alcian yellow method, differences in stainability in the fibres of the preoptic-hypophyseal tract of Rana temporaria were observed. Near the preoptic nucleus most of the fibres stained yellow, in the tract the fibres stained lightgreen or showed green inclusions on a yellow background, whereas at the fibre-endings a colour switch to blue-green was found. This colour switch was the most pronounced in the neural lobe, where the neurosecretion stained blue-green, whereas in the fibre-endings in the outer zone of the median eminence a colour switch to green was noticed.The results can best be explained with the assumption that a modification or transformation of the neurosecretion occurs during its transport from the preoptic nucleus to the fibre-endings.Literature related to this hypothesis is discussed.     

The vascularization and monoaminergic structures of the organon vasculosum laminae terminalis of Rana temporaria

Summary In Rana temporaria, the organon vasculosum laminae terminalis is a highly vascularized organ which contains catecholaminergic cells and nerve fibres. Its ependymal cells are characterized by the presence of typical autofluorescent cytoplasmic granules. Intraependymal vacuolar spaces are described.     

Ultrastructural Identification of gastrin-like immunoreactive nerve fibres in the brain of Xenopus laevis by means of colloidal gold or ferritin immunocytochemical methods

Summary By use of an anti-gastrin serum and colloidal gold- or ferritin-labelled sheep anti-rabbit -globulins, nerve fibres and nerve terminals containing a gastrin-like substance were characterized at the ultrastructural level in the median eminence of Xenopus laevis. These immunoreactive fibres contain neurosecretory granules displaying medium to high electron density and a mean diameter of 75 nm. Labelling intensity varies from granule to granule. This is the first demonstration at the ultrastructural level of the precise location of a gastrin-like hormone in the median eminence of a vertebrate.Supported by the D.G.R.S.T., Contrat no 80.7.0242