Pattern of crypt cell proliferation in the pre- and post-closure ileum of the neonatal rat: Effects of sympathectomy

Summary The ultrastructural localization of adenylate cyclase was accomplished cytochemically in the neurointermediate lobe of Sprague-Dawley rats. The main concentration of the reaction product was found on the plasmalemma of neurosecretory nerve fibers, their terminals and plasma membranes of pituicytes. Positive reaction for adenylate cyclase was found less regularly in endothelial cells, pericapillary spaces and processes of the basal lamina. The septum between the pars nervosa and the pars intermedia showed heavy deposits of the reaction product, especially around the neurosecretory nerve fibers but also around other types of nerve fibers. Reaction for adenylate cyclase was not seen in the cells of the pars intermedia. When the substrate (ATP) was omitted, no reaction product was found. These findings support the suggestion of an involvement of cyclic AMP in the release mechanism of neurohypophysial hormones from the neurosecretory nerve terminals, and possibly also their transfer into blood vessels and perivascular channels.Supported by M.R.C. (Canada)Carreer Investigator of the Medical Research Council of Canada     

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.     

Distribution of opioid peptides in the pituitary: a new hypothalamic-pars nervosa enkephalinergic pathway

The distribution of the endorphins, beta-endorphin and enkephalin (Met5-enkephalin and Leu5-enkephalin), was determined in the pars distalis, intermedia, and nervosa of the rat pituitary using both immunocytochemical and radioimmunological methods. Immunoreactive (ir) beta-endorphin was found in pars distalis and pars intermedia. On gel filtration of the pars distalis extracts, beta-endorphin immunoreactivity was eluted in three peaks corresponding to pro-opiocortin (5%), beta-lipotropin (75%), and beta-endorphin (20%). beta-Endorphin was the only component in the pars intermedia. Enkephalin was found in high amount in the pars nervosa. A new enkephalinergic hypothalamic-pars nervosa pathway was observed. Dehydration experiments on normal rats and analysis of the genetically polyuric Brattleboro rat suggest that this enkephalinergic pathway may modulate neurohypophyseal neurosecretion.     

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.     

Ultrastructural localization of acid phosphatase in the posterior pituitary of the dehydrated rat

Summary Acid phosphatase (AcPase) was localized by means of electron-microscopic histochemistry and estimated biochemically in the posterior pituitary of rats deprived of water, given 2% NaCl ad libitum, or given tap water ad libitum over 6 days. Autophagic vacuoles, some of which gave a positive AcPase reaction, often contained neurosecretory granules (NSG) in nerve endings of control animals on tap water. Nerve endings of water-deprived or salt-treated rats were depleted of NSG, but frequently contained dense membranous residual bodies, some of which appeared to enclose microvesicles. Smooth endoplasmic reticulum located in axons and terminals appears to be a source of hydrolytic enzymes for neurohypophysial lysosomes. The total amount of AcPase per posterior lobe increased progressively to 40% above control levels after 6 days of water deprivation or salt administration, and this increase may reflect accelerated production of neuronal components in neurohypophysial cells whose secretory rate has been stimulated by elevated body osmolarity.Supported by the Medical Research Council of Canada.Medical Research Associate of the Medical Research Council of Canada.     

Ultrastructural localization of exocytotic release sites in immunocytochemically characterized cell types

Summary Preembedding visualization of exocytosis by tannic acid treatment and postembedding immunocytochemical identification of cell types were combined to demonstrate the release of secretory products by exocytosis of characterized cell types. Treatment with tannic acid was carried out by perfusion with Ringer containing tannic acid, followed by fixation, dehydration and embedding. For electron microscopical immunocytochemistry protein A-gold was used as marker. In this study, exocytotic release was demonstrated for prolactin by cells in the pars distalis, and for oxytocin by axon terminals in the pars nervosa of the pituitary gland of the rat.     

Presence of immunoreactive alpha-endorphin in rat pituitary gland.

Utilizing radioimmunoassay for α-endorphin, we attempted to demonstrate immunoreactive α-endorphin in acid extracts of pars distalis and combined pars intermedia and pars nervosa of the rat pituitary gland after chromatography on Sephadex G-25. β-Lipotropin, β-endorphin and γ-endorphin were not converted into α-endorphin during the extraction and gel chromatographic procedures. Concentrations of immunoreactive α-endorphin determined after gel chromatography of extracts from pars distalis and combined pars intermedia and pars nervosa were 1.1±0.6 and 130±17 ng/mg wet tissue (mean±SE), respectively. Serial dilution of these extracts gave parallel lines to the standard curve of synthetic α-endorphin, but not to that of γ-endorphin or δ-endorphin. These results suggest the existence of immunoreactive α-endorphin indistinguishable in molecular size from synthetic α-endorphin in the rat pituitary gland.     

Electron microscopic histochemistry of lysosomes in neurosecretory nerve endings and pituicytes of rat posterior pituitary

Summary Electron microscopic localization of acid phosphatase (AcPase) was carried out on posterior pituitary glands from rats. An estimated 5% of the neurosecretory nerve terminals contained structures which showed reaction product. Most of the lysosomes were small dense bodies, often with a membranous substructure. Other lysosomes were larger in size or were found within vacuoles. AcPase was also localized to lysosomes and the Golgi apparatus of pituicytes. Evidence is presented which would associate the large lipid droplets characteristic of pituicytes with AcPase-positive dense bodies. The present study indicates that hydrolytic activity by lysosomes occurs within the terminals of neurosecretory cells, and adds further support to the concept that this process represents a normal phenomenon of cells and their extensions in general.Supported by the Medical Research Council of Canada.Medical Research Associate of the Medical Research Council of Canada.     

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.     

Corticotrope and melanotrope cells in cat, fox, rat and human fetus adenohypophysis: studies with induced fluorescence, cytoimmunologic technics and lead hematoxyline]

Formaldehyde-induced fluorescence, acid-catalyzed or not, methods, and immunocytology with anti-ACTH (1-24), anti-ACTH(17-39), ANTI-BETA-LPH immunserums were applied on the same preparations of cat, fox, rat and human foetus pituitaries. The superpositions of results showed that the pars intermedia and pars distalis corticomelano-lipotrophic cells of fox and cat pituitary, those of human foetal pituitary, and the purely corticotrophics cells of the rat pars distalis contained a fluorogenic probably granular compound. Moreover, the granules of the same cell types were electively revealed on our lymphilized material by plombic hematoxylin. Only the anti-beta-LPH and/or anti-beta-MSH fixing celpls exhibited hypercyanophilic, PAS-positive and bleu alcian-positive caracteristics.     

Distribution of methionine- and leucine-enkephalin within the rat pituitary gland measured by highly specific radioimmunoassays

The distribution of methionine- and leucine-enkephalin within the rat pituitary gland was measured using highly specific antisera in conjunction with purification by high performance liquid chromatography. The highest concentrations were found in the pars intermedia (7 pmole/mg for methionine-enkephalin, 4 pmole/mg for leucine-enkephalin), whilst the pars nervosa contained 2.2 pmole/mg of each and the pars anterior the least (methionine-enkephalin: 0.51 pmole/mg, leucine-enkephalin: 0.36 pmole/mg).     

Organization and ultrastructural identification of the catecholamine nerve terminals in the neural lobe and pars intermedia of the rat pituitary

Summary The central catecholamine innervation of the pituitary neural lobe and pars intermedia of the rat have been identified ultrastructurally and their organization has been investigated in a combined fluorescence histochemical and electron microscopical study. The dopamine analogues, 5-hydroxydopamine and 6-hydroxydopamine, were used to label the catecholamine terminals, and to enable the direct correlation between the fluorescence microscopical and the electron microscopical pictures.The fibre type that was identified as catecholamine-containing was ultrastructurally chiefly characterized by dense-cored vesicles, 500–1200 Å in diameter, intermingled with varying numbers of small empty vesicles. 5-hydroxydopamine was selectively accumulated in these fibres and caused an increased electron density of the granular vesicles as well as of some small normally agranular vesicles, and systemically administered 6-hydroxydopamine caused a selective degeneration of these fibres, most prominently within the neural lobe. The dopaminergic terminals of the neural lobe showed frequent close contacts (80–120 Å), without real membrane thickenings, to neurosecretory axons and to pituicyte processes. It is suggested that these close contacts might signify a direct dopaminergic influence on the neurosecretory axons and/or on the pituicyte processes. The identified central catecholamine fibres were also found to make common synapse-like contacts on the pars intermedia cells, whereas the innervation by neurosecretory fibres was very rare. This suggests that the direct central nervous control of the rat pars intermedia is exerted by the catecholamine neurons. A very special feature of the catecholamine fibres in the pituitary is the occurrence of peculiar, large dopamine-filled droplet-like swellings. Electron microscopically, such large axonal swellings (more than 2 in diameter) were found to contain, in addition to the characteristic vesicles and organelles, strongly osmiophilic lamellated membrane complexes resembling myelin bodies and multivesicular bodies encircling disintegrated vesicles, suggesting that these droplet fibres represent dilated stumps of spontaneously degenerating dopaminergic axons. It is suggested that the dopaminergic neural lobe fibres are undergoing continuous reorganization through degeneration—regeneration cycles, a phenomenon previously suggested for the neurosecretory axons of the neural lobe.Supported by the Deutsche Forschungsgemeinschaft.Supported by Svenska Livförsäkringsbolags Nämnd för Medicinsk Forskning, by The Medical Faculty, University of Lund and by the Ford Foundation.     

Cholinesterase in the posterior and intermediate lobes of the pituitary

Summary Posterior and intermediate lobes of pituitary glands of cat, rabbit, beef, and rat were examined histochemically for specific (AChE) and non-specific (BuChE) cholinesterase by light and electron microscopy. Acetylthiocholine was utilized in conjunction with ethopropazine to demonstrate AChE, and butyrylthiocholine with BW 284C51 to demonstrate BuChE. Glandular cells of the intermediate lobe of cat, rabbit and rat contained variable amounts of AChE, whereas those of beef contained BuChE. In the posterior pituitary, AChE was detected in the cat, BuChE in the beef and rat, and both AChE and BuChE in the rabbit. In the posterior lobe of all species examined, cholinesterase, whether true or pseudo enzyme, as the case may be, was localized to certain pituicytes and pituicyte-neuron junctions. These histochemical studies failed to identify cholinergic neurons in the posterior pituitary. Large blood vessels of the pituitary were innervated apparently by adrenergic nerves only. Speculations on the role of pituicyte cholinesterase in posterior pituitary secretion are presented.Supported by the Medical Research Council of Canada.Medical Research Associate of the MRC of Canada.     

Histological investigation of pituitary homotransplants in the marine form (trachurus) of the threespine stickleback,Gasterosteus aculeatus L.

Summary Whole pituitary glands were autotransplanted into the tail musculature of intact sticklebacks similar in size to the donors. The structure of the grafted pituitaries and of the in situ glands in the recipient and sham-operated fish was examined. The prolactin-secreting cells, thyrotrophs and possibly the gonadotrophs showed signs of being under inhibitory hypothalamic control whereas the somatotrophs appeared to be more autonomous of hypothalamic influence. The pars intermedia and corticotrophs appeared to be dependent upon the hypothalamus for maintenance of functional acitvity.I am indebted to Drs. W. S. Hoar and T. J. Lam for their help during the period of this investigation and to Mr. L. Sharman for his technical assistance.The work was supported by a grant in aid of research from the National Research Council of Canada to Dr. W. S. Hoar.     

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)     

Sauvagine-like and corticotropin-releasing factor-like immunoreactivity in the brain of the bullfrog (Rana catesbeiana)

Summary Immunocytochemical methods were used to investigate the occurrence and distribution of sauvagine, corticotropin-releasing factor-, or urotensin I-like immunoreactivities (SVG-ir, CRF-ir, UI-ir, respectively) in the bullfrog (Rana catesbeiana) brain, using specific antisera raised against non-conjugated SVG, ovine CRF, rat/human CRF, and UI. In the hypothalamus, SVG-ir was found in the magnocellular perikarya, in the dorsal and ventral regions of the preoptic nucleus, and in the hypothalamo-hypophyseal projections to the external zone as well as the internal zone of the median eminence, to pars nervosa, and in fibres running from the pars nervosa to the pars intermedia of the pituitary. In contrast, CRF-ir was found only in parvocellular perikarya, mainly localized in the rostro-ventral part of the preoptic nucleus, with fine processes protruding through the ependyma of the third ventricle, fibre projections terminating in the anterior preoptic area and in the neuropil of the periventricular gray, and a caudal projection to the external zone of the median eminence. No CRF-ir staining was seen in the pars nervosa and pars intermedia. The use of UI-specific antisera failed to give a positive response in the frog brain. It is concluded that, in the frog brain, two anatomically different CRF-like (or SVG-like) systems co-exist, comparable to the reported co-existence of UI-ir and CRF-ir neuronal systems in fish brain.     

Endorphin in rat pituitary glands: its distribution within the gland, and age related changes in gland content in male and female rats

Endorphins were extracted with glacial acetic acid:acetone from freshly microdissected single lobes of rat pituitary and assayed for inhibition of ³H-etorphine stereospecific binding to rat brain opiate receptors. Highest tissue concentrations of endorphin were found in pars intermedia and pars distalis, with minimal activity observed in pars nervosa. In addition, mature female rats exhibited a significantly higher anterior lobe endorphin content than males, reflecting the larger gland weight, although no significant differences were found between neurointermediate lobe endorphin content of either sex. Pituitary endorphin content increased substantially with age, rising rapidly between the fifth and tenth week after birth. This elevation was still apparent at twenty-five weeks. These studies emphasize the importance of employing rats of closely similar age and weight in any experiments designed to examine pituitary endorphin function, and suggest that ontological development of pituitary endorphin may proceed in a manner similar to that of other pituitary hormones (e.g., MSH) derived from the same precursor peptide.     

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.     

CXCL14-like immunoreactivity in growth hormone-containing cells of urodele pituitaries

Immunohistochemical techniques were employed to investigate the distribution of a chemokine, namely, CXCL14-like immunoreactivity in the axolotl (Ambystoma mexicanum) and Japanese black salamander (Hynobius nigrescens) pituitaries. CXCL14-immunoreactive cells concentrated at an area of the pars distalis adjacent to the pars intermedia. We found that these cells correspond to the cells immunoreactive to an antibody against rat growth hormone (GH). Immunoelectron microscopy indicated that the CXCL14-like substance and GH coexisted on the secretory granules in the axolotl pituitary. Western blot analysis of axolotl pituitary extracts revealed the anti-human CXCL14 antibody labeled an approximately 16.6-kDa band that was not labeled by the anti-GH antibody. The CXCL14-like substance in the pars distalis may participate in GH functions in these species.