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.     

Elementary granules,small vesicles and exocytosis in the rat neurohypophysis after acute haemorrhage

Summary Neural lobes of rats subjected to severe acute haemorrhage under sodium pentobarbitone anaesthesia were examined electron microscopically and the ultrastructure compared with that in anaesthetised and unanaesthetised controls. Changes in the localisation and numerical distribution of elementary granules and small vesicles in the neurohypophysial nerve endings of bled rats were consistent with the occurrence of exocytosis. The occurrence of exocytotic profiles was observed more frequently in freeze-etched tissue samples as compared with the material fixed for conventional electron microscopy. The ratio of small vesicles: elementary granules was shown to be significantly increased (P<0.005) in the nerve endings of neural lobes from bled rats. Equally, the numbers of exocytotic profiles related to 1000 m² of neurohypophysial tissue area were significantly greater (P<0.005) in bled rats.The study was supported by Medical Research Council of Canada. The authors are grateful to Dr. W. Costerton, Biology Department, The University of Calgary, for use of facilities for freeze-etching, and to Miss Y. Carter for technical assistance.Research Associate, Consejo Nacional de Investigationes Cientificas y Tecnicas, Argentina.Associate, Medical Research Council of Canada.     

Membrane routing during exocytosis and endocytosis in neuroendocrine neurones and endocrine cells: use of colloidal gold particles and immunocytochemical discrimination of membrane compartments

Summary The hypothesis that the retrieval of membranes of neurohypophysial neurosecretory granules (NSG) and small electron-lucent microvesicles occurs by different routes was tested by incubating neurohypophysial neurosecretosomes with colloidal gold particles of various sizes. Neurosecretosomes derived from normal Long Evans rats and incubated in media of normal ionic composition endocytosed a few small (<25 nm) gold particles into 40–50 nm electron-lucent microvesicles. After depolarisation, more small gold particles were found in microvesicles, and small and large (>25 nm) gold particles in vacuoles. Oxytocin-containing neurosecretosomes derived from Brattleboro rats, which contain 160 nm-diameter NSG, endocytosed gold particles in a pattern indistinguishable from that of neurosecretosomes from Long Evans rats. However, neurosecretosomes derived from defective vasopressin neurones of Brattleboro rats, which contain microvesicles, small vacuoles, and a few 100 nm dense-cored vesicles, but not 160 nm NSG, endocytosed only small colloidal gold particles. Early after depolarisation the gold particles were present only in microvesicles, but later some could be found in vacuoles and lysosome-like structures. Immunogold cytochemistry using a polyclonal antiserum raised against microvesicle-rich neurosecretosomes derived from Brattleboro rats labelled microvesicles in the posterior pituitary strongly, NSG weakly, and vacuoles to a variable extent. These data together indicate that, after exocytosis, the membranes of NSG are recaptured as large vacuoles. Microvesicles are exocytosed and endocytosed separately.     

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.     

Canada goose posterior lobe pituicytes: seasonal ultrastructural changes in relation to axonal release of neurosecretion

Changes in ultrastructure of the posterior lobe pituicytes during six phases (Spring premigratory, Spring postmigratory, breeding, moulting, Fall premigratory and Fall postmigratory) of the annual life cycle of the migratory Canada goose, were studied. Pituicyte nuclear volume in females was significantly greater than that in males during the Spring premigratory and moulting phases. In the Fall postmigratory phase, nuclear volume in males was greater than that in females. During Spring premigratory phase, the axonal endings in males contained fewer neurosecretory granules (NSG) than those in the Fall postmigratory phase. In females, however, the number of NSG was relatively more than that in males in the Spring premigratory phase but fewer in the moulting phase. Nuclear volume is correlated with pituicyte function in releasing NSG containing arginine vasotocin (AVT) from axonal endings. The significance of AVT release is discussed in relation to initiation of reproductive and migratory behaviour, and to osmotic and metabolic regulation.     

ISOLATED NERVE ENDINGS (NEUROSECRETOSOMES) FROM THE POSTERIOR PITUITARY : Partial Separation of Vasopressin and Oxytocin and the Isolation of Microvesicles

Subcellular fractions of the bovine posterior pituitary, including one composed almost exclusively of pinched-off nerve endings (neurosecretosomes), were characterized electron microscopically, hormonally, and enzymically. 15% of the nerve terminals in the gland were isolated as neurosecretosomes, as estimated from determinations of lactic dehydrogenase, a soluble, cytoplasmic enzyme. Neurosecretosomes were subdivided into three fractions by density-gradient centrifugation. The three subfractions, each shown to be nearly homogeneous populations of neurosecretosomes by means of electron microscopic and enzymic criteria, differed from each other in their vasopressin/oxytocin (VP/OT) ratios. The VP/OT ratio increased from the lightest to the densest fraction, indicating that VP is localized to denser and OT to lighter neurosecretosomes; similar results have been obtained previously for subfractions of neurosecretory granules (NSG). No morphological differences were apparent in neurosecretosomes among the three subfractions. Although complete separation of VP and OT was not achieved, the findings suggest that VP and OT are each stored in a different species of nerve ending and support the hypothesis that a given neurosecretory cell synthesizes, stores, and secretes only one of the peptide hormones. Microvesicles, 40–80 mµ diameter and contained in typical neurosecretory cell terminals, are believed to be degradation products of membrane ghosts of depleted NSG; electron micrographs indicative of this transformation are presented. A fraction rich in microvesicles, but containing some NSG membranes, was prepared by density-gradient centrifugation of an osmolysate of neurosecretosomes. Smaller, apparently nonneurosecretory nerve endings, lacking NSG but filled with small vesicles, are occasionally seen in sections from whole gland. The vesicles in these atypical posterior pituitary nerve endings may be true neurohumor-containing, "synaptic" vesicles.     

Smooth endoplasmic reticulum proliferation in mouse enterocytes induced by fructose feeding

Summary Adult male mice receiving a fructose diet show, after three days, a large proliferation of the smooth endoplasmic reticulum in the jejunal absorptive cells. This extension is a secondary stage of the trememdous increase of the glucose-6-phosphatase activity and seems to result from a crypt regulation. Glucose feeding does not provoke the same morphologic changes. This work was supported by a research grant from the Medical Research Council of Canada. Dr. D. Maestracci Ph. D. is a recipient of a Centennial Fellowship of the Medical Research Council of Canada. Mr. D. Ménard is a recipient of a studentship of the Medical Reserach Council of Canada. The authors are greatly indebted to Mrs. S. Sinave for her skillful technical assistance.     

Effect of TRH (thyrotropin-releasing hormone) on the fine structure and replication of TSH and prolactin cells in the rat

Summary In intact male rats after TRH administration for 7 and 14 days, TSH cells showed similar morphological changes to those observed after thyroidectomy. These changes were paralleled by small numerical increases in TSH cell counts. After 34 days of TRH treatment, however, most of the TSH cells had a normal appearance and the number of TSH cells also had returned to normal. TRH treatment for 7, 14 and 34 days caused morphological changes in Prolactin cells similar to those obtained after a suckling stimulus. In the three groups these changes were also paralleled by small numerical increases in Prolactin cell counts. The cell replication after TRH for 7 and 14 days, as measured by incorporation of tritiated thymidine to obtain a labeling index, was slightly but significantly increased.This work was supported by grants MA-552 and MT-2701 from the Medical Research Council of Canada. The authors wish to thank Dr. D.A.J. Ives, Connaught Medical Research Laboratories, Toronto, for providing the TRH, and Mr. G. Penz for technical assistance.Fellow of the Medical Research Council of Canada.     

The ultrastructure of dentinogenesis and amelogenesis in rat molar tooth germs grown as organ cultures in vitro

Summary Tooth germs from foetal rats of 17 days post-insemination were maintained in vitro for 12 days. Odontoblasts and ameloblasts differentiated and secreted their respective matrices in which mineralization occurred. The ultrastructure of the cells was qualitatively similar to that observed in normal development. Odontoblasts contained more lysosome-like bodies and were found to degenerate in some sites. Mantle dentine was formed but few von Korff fibres were observed. Calcospherites were rarely seen and the mineralizing front of dentine was predominantly linear, associated with numerous small early foci of mineral formation. Enamel showed prism formation associated with the Tomes' process of the ameloblast but some local disturbances in the pattern of enamel formation were observed.Work at the Strangeways Research Laboratory was carried out under the supervision of Dame Honor Fell, F.R.S. with the support of the Medical Research Council.     

Does dehydration contribute to retarded fetal growth in rats exposed to alcohol during gestation?

An earlier study showed that pregnant rats given ethanol in drinking water exhibited a significant degree of dehydration. The objective of the present study was to determine whether dehydration alone contributes to fetal growth retardation in alcohol treated rats. Female Sprague-Dawley rats were divided into 4 dietary groups. Group 1 (alcohol) received 20% ethanol in drinking water for four weeks prior to mating and 30% alcohol in drinking water throughout pregnancy and a stock diet $\text{ad libitum}$. Group 2 (pair-fed) was given an amount of food equal to that consumed by the alcohol group with the alcohol isocalorically substituted by corn starch. Water was available $\text{ad libitum}$. Group 3 (pair-water) was given an amount of food and water equal to that consumed by the alcohol animals. Group 4 ($\text{ad libitum}$) was given food and water $\text{ad libitum}$. On day 21 of gestation body weights of the alcohol exposed fetuses were significantly lower than those of the other three treatment groups. The difference in fetal body weights between the pair-fed and pair-water groups was not significant. Placentas were significantly heavier in the alcohol group than in the pair-fed and pair-water groups. Maternal plasma osmolality was significantly higher in the alcohol treated rats when compared to the pair-fed and $\text{ad libitum}$ controls but not the pair-water group. No significant differences were seen in fetal plasma osmolality among the four treatment groups. It is concluded that dehydration does not contribute significantly to retarded fetal growth in rats given alcohol in drinking water as the sole source of fluid prior to and during gestation.     

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     

Isolation and characterization of neurosecretory granules of the rat posterior pituitary gland

Summary Neurosecretory granules (NSG) of rat posterior pituitary glands were prepared by differential centrifugation techniques mainly according to the procedure as described by Barer, Heller and Lederis (1963). As revealed by electron microscopy, the recovery of neurophysin and the contents of enzymes, purified NSG were obtained in a pellet at 30 000 g/60 min (0.44 M sucrose). Eighteen h after injection of (³⁵S) cysteine into the supraoptic nucleus 60% of the recovered radioactivity in the neural lobe was found in the NSG, whereas 20% was found in the final supernatant (100 000 g/120 min). Sixteen days after injection the NSG and the final supernatant fraction contained fairly equal amount of (³⁵S) cysteine (approximately 40%). It is suggested that after a period of intragranular maturation neurophysin is extruded into an extragranular pool of neurosecretory material.With the use of conventional polyacrylamide-gel electrophoresis it was shown that the predominating proportion of radioactivity in the NSG after a hypothalamic injection of (³⁵S) cysteine was located within the neurophysin fraction A and in fraction B. Fraction B is suggested to be partly bound to the NSG membranes. When the NSG soluble and NSG insoluble proteins, obtained after lysis of NSG, were separated on polyacrylamide gels in the presence of sodium dodecylsulphate, the highly radioactive soluble protein was shown to consist of two components with average molecular weights of 12 300 and 14 600. Most of the proteins in the lysate were found in the NSG membranes, though less radioactive. A component with a mol.wt. of 37 000 was enriched in the membrane fraction. At longer times after isotope injection the high mol.wt. proteins, particularly those of the NSG membranes, contained increased amounts of radioactivity.Abbreviations NSG neurosecretory granule - NSM neurosecretory material - SON supraoptic nucleus The present investigation was supported by grants from Svenska Livförsäkringsbolags nämnd för medicinsk forskning from Svenska Sällskapet för Medicinsk forskning, from the Medical Faculty, University of Göteborg, and from the Swedish Medical Research Council (B 72-12X-2543-04A).We are indebted to Mrs. Marie-Louise Eskilsson, Mrs. Wally Holmberg and Mrs. Ulla Svedin for technical assistance, and to Miss Gull Grönstedt for careful secreterial work.     

Oral administration of orthovanadate and Trigonella foenum graecum seed powder restore the activities of mitochondrial enzymes in tissues of alloxan-induced diabetic rats

The effect of oral administration of sodium orthovanadate (SOV) and Trigonella foenum graecum seed powder (TSP), a medicinal plant used extensively in Asia, on the mitochondrial metabolism in the alloxan diabetic rats has been investigated. Rats were injected with alloxan monohydrate (20 mg/100 g body wt) or vehicle (Na-acetate buffer), the former were treated with either 2 IU insulin i.p., 0.6 mg/ml SOV ad libitum, 5% TSP ad libitum, and a combination of 0.2% SOV and 5% TSP ad libitum for 21 days. Selected rate-limiting enzymes of the tricarboxylic acid cycle, hydrogen shuttle system, ketone body metabolism, amino acid metabolism and urea cycle were measured in the mitochondrial and cytosolic fractions of liver, kidney and brain tissues of the experimental rats. Majority of the mitochondrial enzymes in the tissues of the diabetic rats had significantly higher activities compared to the control rats. Similarly, the activities of mitochondrial and cytosolic aminotransferases and arginase were significantly higher in liver and kidney tissues of the diabetic rats. The separate administrations of SOV and TSP to diabetic rats were able to restore the activities of these enzymes to control values. The lower dose of SOV (0.2%) administered in combination with TSP to diabetic rats lowered the enzyme activities more significantly than when given in a higher dose (0.6%) separately. This is the first report of the effective combined action of oral SOV and TSP in ameliorating the altered mitochondrial enzyme activities during experimental type-1 diabetes. Our novel combined oral administration of SOV and TSP to diabetic rats thus conclusively proves as a possible method to minimize potential vanadate toxicity without compromising its positive effects in the therapy of experimental type-1 diabetes.     

Mitochondrial myelin-like figures: A non-specific reactive process of mitochondrial phospholipid membranes to several stimuli

Summary Small concentric arrays resembling myelin figures were found in the liver cell mitochondria of fasted rats, of fasted rats given insulin and glucose, and of untreated fed rats. In addition to myelin-like figures, interdigitation of the membranes of adjacent mitochondria were common and often formed chains of mitochondria. Fusion between the outer membranes of mitochondria and protrusions of part of one mitochondrion into another were also found. Since mitochondrial myelin-like figures were even occasionally observed in control fed animals and since identical structures have been described in a number of unrelated conditions, it is concluded that they are not aetiologically specific. The myelin-like figures are considered to represent a form of focal mitochondrial phospholipid membrane degeneration under various stimuli.This work was supported by the Medical Research Council of Canada, the Banting Research Foundation and the Canada Arts Council.     

Sustained NPY Overexpression in the PVN Results in Obesity via Temporarily Increasing Food Intake

Increasing neuropeptide Y (NPY) signaling in the paraventricular nucleus (PVN) by recombinant adeno‐associated virus (rAAV)‐mediated overexpression of NPY in rats, results in hyperphagia and obesity in rats. To determine the importance of hyperphagia in the observed obesity phenotype, we pair‐fed a group of AAV‐NPY‐injected rats to AAV‐control‐injected rats and compared parameters of energy balance to ad libitum fed AAV‐NPY‐injected rats. For 3 weeks, AAV‐NPY‐injected rats, received the same amount of food as ad libitum‐fed rats injected with control rAAV They did not gain more body weight than these controls. When allowed access to food ad libitum, these AAV‐NPY‐injected rats increased food intake, which subsequently decreased when rats reached the same body weight as AAV‐NPY‐injected rats that were fed ad libitum for the entire study. These data indicate that overexpression of NPY in the PVN results in obesity by increasing food intake until a certain body weight is achieved.     

Electron microscopic observations of cell regeneration from cultured protoplasts ofAmmi visnaga

Summary Protoplasts ofAmmi visnaga initiated cell wall formation within 2 days in culture; after 13 days the new cells were enclosed by a cell wall similar to the walls on the original cultured cells. Budding occurred in protoplasts with little or no detectable cell wall. No evidence was obtained for direct participation of any organelle in cell wall formation. The cytoplasm of regenerating cells contained numerous organelles and appeared typical of actively growing plant cells; they were easily distinguished from degenerate cells and protoplasts. While coated vesicles were common, spiny vesicles occurred in only a few cells. Sustained cell division yielded multicellular aggregates. Multinucleate protoplasts, formed by spontaneous fusion, did not divide; some of them contained annulate lamellae with few pore complexes.Supported by the National Research Council of Canada, Grant A6304.     

Temperature-sensitive mutations in Drosophila melanogaster. XIV. A selection of immobile adults

Summary Improved methods for rearing and screening large numbers of flies permitted the recovery of 10 mutations exhibiting a reversible temperature-dependent adult paralysis among 1.1×10⁶ flies tested. Of the 10 mutations, two were allelic to para ^ts,two were alleles in a new locus, stoned (stn), and six fell into a third area, the shibire (shi) locus. Several of the shi alleles cause embryonic, larval and adult paralysis at 29° C as well as structural anomalies of various tissues. In addition to the ts mutations, several non-conditional mutations affecting adult movement were recovered.This work was supported by the National Research Council of Canada, grant A-1764, National Cancer Institute of Canada, grant 6051.Medical Research Council of Canada Postdoctoral Fellow.     

QUANTITATIVE LYSOSOMAL ENZYME ACTIVITY CHANGES IN THE NEURAL LOBE OF THE RAT FOLLOWING WATER DEPRIVATION AND LACTATION

—The activities of five lysosomal enzymes, acid phosphatase, β-glucosidase, β-glucuronidase, β-galactosidase and N-arylamidase (classified according to Marks (1970)) were measured by means of sensitive microchemical techniques in frozen-dried rat neural lobe tissue after experimental and physiological stimulation of hormone release from the hypothalamo–neurohypophysial system i.e. water deprivation (3 and 6 days), delivery and lactation (10 days). During all conditions of stimulation increases of 29 to 106 per cent were measured for lysosomal enzyme activity, expressed as mmol/ng DNA/h. With histochemical staining methods the acid phosphatase activity appears to be mainly localized in the pituicytes, but it was impossible to visualize the microchemically measured acid phosphatase activity increase within the two main compartments of the neurohypophysis, i.e. axonal endings and the neurohypophysial glial cells, the pituicytes.     

The influence of long-term melatonin administration on basic physiological and metabolic variables of young Wistar:Han rats

The question of effects of long-term melatonin (MEL) administration have not yet been explained sufficiently, especially its metabolic consequences in young persons and animals. The aim of the present study was to analyze the effects of MEL given during prolonged time (for 3 months) and chronically (for 6 months) at the dose of 4 μg/mL of tap water, on the selected metabolic and hormonal parameters in young female and male Wistar:Han (WH) rats. The weights of selected organs, tissues, body weight gains and food and water intake were registered. Six weeks aged rats were adapted to standard housing conditions and light regimen L:D=12:12 h, fed standard laboratory diet and drank tap water (controls) or MEL solution ad libitum; finally they were sacrificed after overnight fasting. Prolonged MEL administration decreased serum glucose concentration and increased triacylglycerol and malondialdehyde concentration/content in the liver in females. In males MEL increased concentrations of serum phospholipids, corticosterone and liver malondialdehyde. MEL treatment reduced the body weight in both sexes and weight of epididymal fat in males, without any alterations of food and water intake. Chronic MEL administration reduced serum glucose concentration and increased concentration/content of glycogen, triacylglycerol and cholesterol in the liver and glycogen concentration/content in heart muscle in males. In females, the significant rise of serum corticosterone concentration and liver malondialdehyde content was recorded. MEL significantly increased liver weight and decreased thymus weight in males. MEL administration increased temporarily water intake in males, body and epididymal fat weights were similar to that in controls. Body weight of MEL drinking females was reduced in the 1^st half of experiment only; the food and water intake did not differ from control group. The response in WH rats on MEL was more prominent as in the Sprague-Dawley strain (our previous studies). Male rats were generally more affected, probably due to higher daily and total consumption of melatonin.     

The posterior pituitary of the garden dormouse (Eliomys quercinus L.)

Summary Ultrastructural examination of the posterior pituitary of the garden dormouse (Eliomys quercinus L.) was carried out at different times in the annual cycle of this hibernating rodent. Obvious differences between experimental groups have not been observed, and the results presented here must be considered as general features of the garden dormouse posterior pituitary. Neurosecretory axons and endings can be divided into two types, according to different aspects of neurosecretory granules (NSG) and microvesicles (MV). One type contains spherical NSG with homogeneous cores and round MV. In the other type, NSG have various, often elongated, shapes. Their content shows two types of crystalline structures and most of the MV have flattened aspects. As it is very unlikely that this duality in NSG is a result of an artefact of fixation, three hypotheses are presented as explanation. The duality of NSG might be related either to their hormonal content (oxytocin or vasopressin) or to their degree of maturation. Moreover, both explanations may be valid. In the species studied, pituicytes often contain concentric lamellar structures of the endoplasmic reticulum (whorls), the significance of which remains obscure.This work was supported in part by grants of INSERM (C.R.L. n 73.1091.7 and A.T.P. n 4.74.25)