Ontogenesis of the α-MSH, β-MSH and ACTH cells in the foetal hypophysis of the rat. Correlation with the growth of the adrenals and adrenocortical activity

Pituitary sections from 15 to 21 day-old rat foetuses have been studied with the immunofluorescence technique, using antibodies anti alpha-MSH, anti beta-MSH and anti beta (1-24) ACTH. The first ACTH cells appear on day 17 of pregnancy in the pars distalis of the hypophysis and only on day 18 in the pars intermedia. beta-msh cells have been observed on day 16 in the pars anterior and on day 17 in the pars intermedia, while alpha-MSH cells appear only on day 18 and exclusively in the pars intermedia. The cytodifferentiation of the beta-MSH and ACTH cells occurs in the pars intermedia with about a 24 hours delay in comparison to that of the pars distalis. The first revealed cells are always located in the posterior half of the pituitary gland. The corticostimulating activity of the hypophysis has been tested with the fluorescence intensity of the corticotrophs, the adrenal weight, the adrenal content in corticosterone and the plasma corticosterone level. The fluorescence of the corticotrophs increases on day 18, shows a maximum on day 19 and decreases until term. The adrenal weight rises regularly between day 16 to day 20, thereafer the increase subsides. Adrenal and plasma corticosterone concentrations reach a peak on day 19 of pregnancy. These data suggest that hypophyseal corticostimulating activity is very high between days 18 and 19 and decreases between days 19 and 21.     

Quantitative fluorescence histochemistry of combined formaldehyde-chloral-induced fluorescence of amino-terminal tryptophyl-peptide in model experiments and in the pars intermedia of the rat hypophysis

The relationship between the intensity of combined formaldehyde-chloral vapour-induced fluorescence and the concentration of amino-terminal tryptophyl-peptide in model experiments was found to be non-linear. At a certain concentration the intensity began to increase more slowly than the concentration, and when the concentration further increased the intensity even began to decrease. Based on the studies previously reported and on the above findings it seems that fluorescence induced by combined formaldehyde-chloral vapour, glyoxylic acid vapour and possibly also other combined formaldehyde and carbonyl compounds in the hypophyseal cells containing amino-terminal tryptophyl-peptides is quenched in normal conditions due to the high local concentration. Thus, small to moderate changes in the amounts of amino-terminal tryptophyl-peptides cannot be observed by measuring the fluorescence intensity. In tissue experiments the intensity of combined formaldehyde-chloral vapour-induced fluorescence in the rat pars intermedia was measured after reserpine treatment, which decreases the number of hormone storage granules as demonstrated electron microscopically. The fluorescence intensity measurements were combined with an estimation of the amounts of amino-terminal tryptophyl-peptides extracted from hypophyses and separated in thin-layer chromatography, and subsequently demonstrated by combined formaldehyde-chloral vapour and a protein stain (amido black). Reserpine treatment decreased the fluorescence intensity in the pars intermedia and in thin-layer chromatography, and the staining of the fluorescent band with amido black was also decreased. Amino-terminal tryptophyl-peptides appeared to be depleted from the pars intermedia cells together with endorphins and other hormones of the ACTH/MSH cells containing tryptophan.     

Observations on the functional cytochemistry of pars intermedia of the rat hypophysis

Summary Light and electron microscopic histochemical reactions were studied in the cells of pars intermedia of the rat. The possible correlations between enzymatic reactions and endocrine functions of these cells were discussed. By combined formaldehyde and chloral vapour treatment the cells of the pars intermedia exhibited a strong yellow fluorescence suggesting the presence of a peptide or peptides with NH₂-terminal tryptophan. Masked metachromasia after acid hydrolysis was probably due to these peptides. Only a weak or no α-glycerophosphate dehydrogenase and nonspecific esterase activity was observed in the cells of pars intermedia compared to the cells of pars distalis suggesting low production rate of hormone synthesis. Specific and non-specific cholinesterases were demonstrated light and electron microscopically constantly in the cells bordering the lobules. These cells probably represent a certain type of glial cells. In the other cells the enzymatic activities varied markedly in intensity and distribution showing different ultrastructural localizations. Thus cholinesterase activities in the cells of pars intermedia reflect possibly different functional stages of the cells in their hormone production, storage and secretion processes.     

Observations on the functional cytochemistry of pars intermedia of the rat hypophysis.

Light and electron microscopic histochemical reactions were studied in the cells of pars intermedia of the rat. The possible correlations between enzymatic reactions and endocrine functions of these cells were discussed. By combined formaldehyde and chloral vapour treatment the cells of the pars intermedia exhibited a strong yellow fluorescence suggesting the presence of a peptide or peptides with NH2-terminal tryptophan. Masked metachromasia after acid hydrolysis was probably due to these peptides. Only a weak or no alpha-glycerophosphate dehydrogenase and nonspecific esterase activity was observed in the cells of pars intermedia compared to the cells of pars distalis suggesting low production rate of hormone synthesis. Specific and non-specific cholinesterases were demonstrated light and electron microscopically constantly in the cells bordering the lobules. These cells probably represent a certain type of glial cells. In the other cells the enzymatic activities varied markedly in intensity and distribution showing different ultrastructural localizations. Thus cholinesterase activities in the cells of pars intermedia reflect possibly different functional stages of the cells in their hormone production, storage and secretion processes.     

Quantitative fluorescence histochemistry of combined formaldehyde-chloral-induced fluorescence of amino-terminal tryptophyl-peptide in model experiments and in the pars intermedia of the rat hypophysis

Summary The relationship between the intensity of combined formaldehydechloral vapour-induced fluorescence and the concentration of amino-terminal tryptophyl-peptide in model experiments was found to be non-linear. At a certain concentration the intensity began to increase more slowly than the concentration, and when the concentration further increased the intensity even began to decrease. Based on the studies previously reported and on the above findings it seems that fluorescence induced by combined formaldehyde-chloral vapour, glyoxylic acid vapour and possibly also other combined formaldehyde and carbonyl compounds in the hypophyseal cells containing amino-terminal tryptophyl-peptides is quenched in normal conditions due to the high local concentration. Thus, small to moderate changes in the amounts of amino-terminal tryptophyl-peptides cannot be observed by measuring the fluorescence intensity. In tissue experiments the intensity of combined formaldehyde-chloral vapour-induced fluorescence in the rat pars intermedia was measured after reserpine treatment, which decreases the number of hormone storage granules as demonstrated electron microscopically. The fluorescence intensity measurements were combined with an estimation of the amounts of amino-terminal tryptophyl-peptides extracted from hypophyses and separated in thin-layer chromatography, and subsequently demonstrated by combined formaldehyde-chloral vapour and a protein stain (amido black). Reserpine treatment decreased the fluorescence intensity in the pars intermedia and in thin-layer chromatography, and the staining of the fluorescent band with amido black was also decreased. Amino-terminal tryptophyl-peptides appeared to be depleted from the pars intermedia cells together with endorphins and other hormones of the ACTH/MSH cells containing tryptophan.This study was supported by grant from J.K. Paasikivi Foundation.     

The pars intermedia of the mink,Mustela vison

Summary The pars intermedia of intact and experimental female minks has been studied by light, electron and fluorescence microscopy. The general structure of the mink intermediate lobe is described. Two main cell types are recognized. One, termed glandular cell, predominates in number and is characterized by the presence of electron-dense granules about 200 nm in diameter and numerous large vesicles up to 300 nm in diameter. The other, termed stellate cell, is devoid of cytoplasmic vesicles and granules and possesses microfilaments, junctional complexes and elongated processes inserted between the glandular cells. Different treatments (photostimulation and administration of hypertonic saline and metopirone) induced morphological reactions in the glandular cells. The significance of these changes and the possibility of a functional relation between the pars intermedia and ACTH secretion are discussed.Numerous nerve fibres and axon terminals containing electron-dense granules (60–120 nm) and electron-lucent vesicles (30–40 nm) are observed throughout the pars intermedia.With the histochemical fluorescence method of Falck-Hillarp a rich system of delicate fluorescent varicose fibres, sometimes provided with irregular swellings or droplets, is observed in the pars intermedia and also in the pars nervosa. Microspectrofluorometrically these fibres exhibit the spectral characteristics of catecholamines. All the cells of the pars intermedia and a large number of cells in the pars distalis show a yellowish weak fluorescence, which becomes much stronger after combined formaldehyde-ozone treatment. This cellular fluorophore shows the same microspectrofluorometric characteristics as does the fluorophores of tryptamine and of certain peptides with NH₂-terminal tryptophan.Supported by the Swedish Fur Breeders' Association and the Swedish Natural Science Research Council (grant No. 2124). Thanks are due to Miss W. Carlsson and Miss Y. Lilliemarck for their helpful technical assistance.Supported by the Harald and Greta Jeanssons Stiftelse and by the Ford Foundation. The skilful technical assistance of Mrs. Eva Svensson and Miss Annika Borgelin is greatfully acknowledged.     

Studies on secretory activity in the pars intermedia of Xenopus laevis 1: Fine structural changes related to the onset of secretory activity in vivo

Changes in the fine structural organization of the pars intermedia related to the onset of secretory activity within the gland have been studied. It is shown that during the first seven days, following the onset of secretory activity, there is an extensive membraneogenesis within the cytoplasm of the parenchymatous cells of the pars intermedia which results in the formation of a well-organized array of endoplasmic reticulum and an increased development of the Golgi complex. Simultaneously the large population of secretory granules present in the cells in the inactive condition is reduced, the granules fusing with the plasma membrane and releasing their contents into the extracellular space. During the process of intracellular reorganization, in addition to the elaboration of those components of the Golgi complex which are believed to be concerned with the formation of the secretory granules, a second distinctive cisternal element develops within the Golgi area. This component which remains confined to the Golgi area for only a short time (days 2-4) appears to be responsible for the production of membrane-bound dense bodies with a finely granular content. The dense bodies, in turn, become transformed into the larger heterogeneous structures which are a prominent feature of actively secreting pars intermedia cells.     

Structural and microspectrofluorometric studies on glial cells from the periventricular and arcuate nuclei of the rat hypothalamus

Summary Fluorescence spectroscopy and electron microscopic techniques have been employed to investigate a class of glial cells that is characterized by the presence in their cytoplasm of large fluorescing inclusions that stain with paraldehyde-fuchsin or chrome-hematoxyline-alum. In the periventricular nucleus the cells have been identified as a population of astrocytes whose inclusions emit an orange-red fluorescence. In the arcuate nucleus there are, in addition to an overwhelming majority of such astrocytes, also some microglial cells with similar characteristics. The ability of the latter to emit any kind of fluorescence has not yet been established. The fluorescence maximum of these astrocytic inclusions was found to be at 640 nm when excited at 405 nm. The data obtained suggest that the fluorescence observed is due to the presence of porphyrins in the astrocytic inclusions. In the majority of our electron microscopic pictures the inclusions lack a bounding membrane. By contrast, neuronal lipofuscin has an outer membrane. In cryostat sections, the lipofuscin emits a yellow fluorescence when excited at 400–410 Onm.     

Demonstration of stellate cells of the pars intermedia of the pituitary gland using a new silver impregnation technique

The pars intermedia of the pituitary gland in the rat and other species contains, in addition to secretory cells, stellate cells first characterized by using electron microscopy. The distribution and relationships of these cells is difficult to assess at the ultrastructural level. We have developed an ammoniacal silver nitrate method for stellate cells of the pars intermedia. Staining is carried out in 15 micron sections of buffered formalin fixed, paraffin embedded pituitaries. This method shows that in the rat pars intermedia, stellate cells showing numerous cytoplasmic projections are abundant in, and evenly distributed throughout, pars intermedia lobules.     

Localization of acetylcholinesterase and choline acetyltransferase in the rat pituitary gland

Summary Experiments were conducted to determine the presence of two cholinergic biomarkers, acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in the rat pituitary. A histochemical procedure for AChE was used to provide visualization of structures containing this enzyme. Radiochemical methods provided a sensitive assay for measuring ChAT activity. Nerve fibres staining for AChE activity were observed in the neurointermediate lobe, with the greatest concentrations appearing at the junction region with the pituitary stalk. Cells staining for AChE were found in the pars distalis and pars intermedia. ChAT activity correlated well with AChE distribution in pars nervosa and pars intermedia but not in pars distalis. The greatest levels of ChAT activity were in pars intermedia and the region where the stalk joins the pituitary. Significant values were also found for the pars nervosa. The presence of AChE and ChAT in pars intermedia and pars nervosa is evidence for a cholinergic innervation to these regions. In pars distalis, where other investigators have found muscarinic receptors, intense staining for AChE and absence of ChAT activity may indicate non-innervated, acetylcholine-sensitive sites.     

Demonstration of Stellate Cells of the Pars Intermedia of the Pituitary Gland Using a New Silver Impregnation Technique

The pars intermedia of the pituitary gland in the rat and other species contains, in addition to secretory cells, stellate cells first characterized by using electron microscopy. The distribution and relationships of these cells is difficult to assess at the ultrastructural level. We have developed an ammoniacal silver nitrate method for stellate cells of the pars intermedia. Staining is carried out in 15 µl;m sections of buffered formalin fixed, paraffin embedded pituitaries. This method shows that in the rat pars intermedia, stellate cells showing numerous cytoplasmic projections are abundant in, and evenly distributed throughout, pars intermedia lobules.     

The hypothalamo-hypophysial system of the wild carp,Cyprinus carpio L.

Summary The posterior neurohypophysis (PNH)-pars intermedia complex of the wild and pond carp, Cyprinus carpio L., has been studied by light, fluorescence and electron microscopy. Gomori-positive neurosecretory fibres are abundant in the main trunk of the neurohypophysis as well as its roots penetrating the pars intermedia. Terminals of these fibres are in contact with capillaries of the general circulation and with glandular cells of the pars intermedia. Monoaminergic fibres with a weak green fluorescence, somewhat increasing after injection of nialamide into the pond carp, have largely the same distribution. Three types of neurosecretory fibres and their terminals have been recognized in the PNH-pars intermedia complex. Types-A₁ and -A₂ fibres, containing granules of 140–180 nm and 100–160 nm in diameter respectively, are peptidergic Gomori-positive. Type-A₂ fibres predominate in the PNH. The least frequent monoaminergic type-B fibres have granules of 60–100 nm in diameter. Numerous peptidergic and few monoaminergic neurosecretory terminals make contact with the capillaries located within the roots of the PNH as well as at the border between them and the pars intermedia. Both peptidergic and monoaminergic terminals make direct synaptoid contacts with the gland cells or end close to connective tissue septa, basal lamina or pituicytes. The PAS-positive gland cells and to a lesser degree the leadhaematoxylin-positive gland cells show these relationships with neurosecretory terminals. The question concerning the mode of interaction between peptidergic and monoaminergic structures in the dual control of the gland cells of the pars intermedia of teleosts is discussed.     

Monoamine-containing fibres in the pituitary neuro-intermediate lobe of the pig and rat

Summary A rich system of monoamine-containing fibres is described in the neural lobe and pars intermedia of the pig and rat. a) A rich network of delicate varicose fibres is evenly distributed throughout the parenchyma of the neural lobe and surrounds the cells of the pars intermedia. b) Droplets or clusters of droplets are scattered throughout the neural lobe. Most of them probably constitute terminal swellings or end-apparatuses of smooth or varicose fibres. The number of droplets varies from animal to animal; they are found also in the pars intermedia. c) Coarse varicose fibres are mainly localized around larger vessels. At least some of these fibres are nerve fibres of sympathetic origin. A combination of fluorescence microscopy and aldehyde-fuchsin staining on the same sections demonstrated that the majority at least of these monoamine-containing structures were not identical with aldehyde-fuchsin positive neurosecretory fibres.This research was supported by a grant from the Swedish Medical Research Council (B68-12X-712-03B) and by the Faculty of Medicine, University of Lund.     

Pituitary cytology of the chimaeroid fish Hydrolagus colliei (Lay and Bennett).

The pituitary of Hydrolagus colliei is divided into the adenohypophysis, neurohypophysis and an oral Rachendachhypophyse. The adenohypophysis is further divided into rostral and proximal pars distalis and neurointermediate lobe. The neurohypophysis is restricted to the pars intermedia only. The rostral pars distalis is composed of acidophils, chromophobes, lightly PAS+ cells and amphiphils. The amphiphils were stained with Heidenhain's iron haematoxylin and lead haematoxylin also. The proximal pars distalis is formed of cyanophils where the granules are AF and PAS positive, acidophils, chromophobes and H.Pb+ cells. The pars intermedia has perviascular amphiphils which are H.Pb+, lightly PAS+ cells and chromophobes. Few AF+ cells were also identified. All the component parts of the adenohypophysis have follicular cavities which are probably developed from the hypophysial cavity, which is well seen in the young specimen as a single cavity extending antero-posteriorly throughout the adenohypophysis.     

Immunocytochemistry of a “private” luteinizing-hormone-releasing hormone system in the pituitary

Summary Immunocytochemistry of paraffin sections of Bouin-fixed rat pituitaries with antiserum to luteinizinghormone-releasing hormone (LHRH) revealed two types of cells. Type I cells exhibit granular staining throughout their cytoplasm. The immunoreactivity of type II cells is confined to a much smaller area of the cytoplasm. Type I cells are located in the ventral margin of the pars intermedia, the region between the pars intermedia and the pars distalis, and the pars distalis adjacent to this region. Type II cells have a broader distribution in the pars distalis, but tend to concentrate in the region of the pars distalis near the pars intermedia. Type I cells are distinct from gonadotropes. Type II cells appear to comprise a subgroup of gonadotropes. Staining in type I, but not type II, cells in pituitary explants, maintained in serum-free media for seven days, is as intense as that in normal pituitary tissue. The data suggest that the type I cells are producing an intrinsic LHRH-like material and may be responsible, in part, for the regulation of some gonadotropes.Supported by NIH grants HD12932, NS15843 and NS15809 (LAS), National Science Foundation grant BNS 82-05643 (LAS), and a grant from the Phillippe Foundation (JYL)     

Life span changes in the presence of alpha-melanocyte-stimulating-hormone-containing cells in the human pituitary.

Since recent circumstantial evidence has suggested possible functions of alpha-MSH in intrauterine growth and labour, the presence of this hormone in the human pituitary was determined by means of the indirect immunofluorescence procedure during development and adulthood. Cross reaction of the antibodies with other peptides was measured after which they were purified by solid phase absorption. Experiments on the rat pituitary showed that staining of alpha-MSH- and ACTH-containing cells could be obtained well until 48 h after death. In the pars distalis the ability of ACTH-containing cells to take up stain increased during the period of post-mortem storage. In the youngest human fetus studied (15 weeks) only alpha-MSH-containing cells were found in the pars intermedia and no ACTH-containing cells were observed. In the other fetal pituitaries a distinct pars intermedia containing more alpha-MSH cells than ACTH cells was found. In the pars distalis of the fetuses more ACTH- than alpha-MSH-containing cells were observed. From birth to 19 years, progressively fewer alpha-MSH containing cells could be detected in the 'zona intermedia' and pars distalis, while in adults only a few such cells were found in either area. Irrespective of age, sex, cause of death or therapy, alpha-MSH-containing cells were found in all pituitaries throughout life. The number of ACTH containing cells gradually increased in the zona intermedia and pars distalis and reached a high adult level in the latter structure. In the pituitaries of seven anencephalics, no alpha-MSH-containing cells were present. The presence of alpha-MSH in the fetal pars intermedia, the change in the ratio of the alpha-MSH/ACTH cells during the course of development, and the absence of alpha-MSH in anencephaly all support the possibility that human fetal pituitary alpha-MSH is involved in both intrauterine growth and fetal adrenal function and thus also in parturition.     

An immunohistochemical study of adenohypophyseal cells in the viviparous reptile Chalcides chalcides

The morphology of the hypophysis and the immunocharacteristics of the adenohypophyseal cells in the viviparous reptile Chalcides chalcides were studied by light microscopy, using conventional staining methods and an indirect antibody technique (ABC method), respectively. The general morphology of the C. chalcides hypophysis was comparable to that of other reptiles, showing three main regions: the pars distalis, the pars intermedia and the pars nervosa. The gland appeared as an elongated body in a cephalic-caudal direction and was almost completely enclosed in the sella turcica. For this reason, the hypophysis was studied in toto with the brain in decalcified specimens. The pars distalis accounted for most of the whole organ. The pars intermedia surrounded the pars nervosa as a goblet. The pars tuberalis was lacking.The immunohistochemical identification of the adenohypophyseal cells was performed using rabbit antisera against mammalian/synthetic hypophyseal hormones. Prolactin cells were clustered in small cellular cordons in the rostral pars distalis and in the medial pars distalis in both male and female specimens. Somatotropic cells were found in the caudal pars distalis. Corticotropic cells were observed in the medio-rostral pars distalis, as well as in the pars intermedia, where melanotropic cells were also present. Melanotropic cells were confined to the pars intermedia. Gonadotropic cells were mostly distributed in the ventral and lateral portions of the pars distalis, where they were found isolated or in small clusters. Thyrotropic cells were detected in the pars distalis with a distribution similar to that of the gonadotropic cells; however, atypically, they were also found in the pars intermedia. Therefore, the cytological characteristics of the adenohypophyseal cells appeared mostly conserved.     

Peptides with NH2-terminal tryptophan in adrenocorticotrophic hormone and melanocyte-stimulating hormone granules of adenohypophysis.

Fluorescence microscopy has demonstrated formaldehyde-ozone-induced fluorescence in the pars intermedia cells (melanocyte-stimulating hormone cells) and in certain cells of the pars distalis of the mammalian pituitary. From histochemical and chemical evidence the fluorescence is believed to reflect the presence of peptides with NH2-terminal tryptophan. In the pars distalis of hamster, cat and pig pituitary, the cells that exhibit formaldehyde-ozone-induced fluorescence have now been identified as adrenocorticotrophic hormone (ACTH) cells by immunohistochemistry. Granules from pig pituitaries were purified by passage through a succession of Millipore filters followed by centrifugation on a continuous sucrose gradient. Two granular fractions were identified by electron microscopy and found to contain high concentrations of peptides with NH2-terminal tryptophan as well as high ACTH bioactivity. These fractions, when pelleted and analyzed histochemically, displayed formaldehyde-ozone-induced fluorescence and ACTH-like immunoreactivity.     

Aminergic Innervation of the Cranial and Caudal Neurosecretory Systems in the Teleost Gillichthys mirabilis

Abstract Numerous fluorescent varicosities surround most of the caudal neurosecretory neurons and also regularly occur among pars intermedia cells of the adenohypophysis in the teleost, Gillichthys mirabilis. The color of the varicosities, as well as their responses to pharmacological treatments, is diagnostic of catecholaminergic neurons and processes. No fluorescence characteristic of monamines is found in the rostral pars distalis, in the proximal pars distalis or in the cells of the nucleus lateralis tuberis (NLT), although fluorescent varicosities are found within the ventral hypothalamus in the vicinity of the NLT. Bilateral clusters of fluorescent cell bodies are located in the ventral hypothalamus (posterior to the NLT); some of these cells border the neurohypophysis. Fluorescent tracts from these cell clusters extend to a pair of elongate nuclei of nonfluorescent neurons which are surrounded by fluorescent varicosities. Alteration of osmotic conditions did not effect the fluorescence, except for the caudal neurosecretory cells of fish exposed to fresh water for long periods. Adrenergic nervous input thus seems to be an important component of both the cranial and caudal neurosecretory systems.     

Histochemical distribution of monoamines in the hypothalamo-hypophysial region of the lamprey,Lampetra japonica

The distribution of monoamine fluorescence was studied in the hypothalamohypophysial region of the lamprey. Groups of intensely fluorescent cells were observed in the lateral walls of the caudal part of the third ventricle. The anterior part of the neurohypophysis which is situated over the pars distalis showed weak fluorescence. The posterior part of the neurohypophysis which is contiguous to the pars intermedia contained highly fluorescent material in its rostral part. The distribution of monoamines in the lamprey neurohypophysis is compared with that in the higher vertebrates and their functional significance is discussed.