Secretory granules of prolactin cells of neonatally thyroidectomized female rats

In the anterior pituitary glands of neonatally thyroidectomized female rats sacrificed at 30 days of age, the prolactin granules were small and spherical in shape. The administration of thyroxine to neonatally thyroidectomized rats produced an obvious increase in the number and size of secretory granules in prolactin cells; comparatively large, pleomorphic secretory granules were frequently observed in these cells. These enlarged and pleomorphic granules closely resembled those observed in the prolactin cells of sham-operated control rats. These results may indicate that thyroxine stimulates the basic metabolism or cellular function of prolactin cells of neonatally thyroidectomized rats and leads to the formation of prolactin granules that are similar to those of sham-operated control rats.     

Ginkgo biloba extract enhances male copulatory behavior and reduces serum prolactin levels in rats

The aim of this study was to investigate the effects of Ginkgo biloba extract (EGb 761) on male copulatory behavior in rats. EGb 761 (1 mg/ml) induced significant production of testosterone (T) in rat Leydig cells in vitro. Its effects on sexual behavior were then tested in Long-Evans male rats after 7, 14, 21, or 28 days of oral gavage of vehicle (distilled water) or EGb 761 at doses of 10, 50, or 100 mg/kg. Administration of 50 mg/kg of EGb 761 for 28 days and of 100 mg/kg for 14 or 21 days significantly increased intromission frequency compared to controls on the same day. An increase in ejaculation frequency was seen after treatment with 50 mg/kg of EGb 761 for 14, 21, or 28 days when compared to either the control group on the same day or the same group on day 0. A reduction in ejaculation latency was only seen after administration of 50 mg/kg of EGb 761 for 14 days compared to the vehicle-treated group. After treatment for 28 days, no significant difference was seen in mount latency, intromission latency, serum T levels, reproductive organ weight, sperm number, or levels of the metabolite of dopamine, 3,4-dihydroxyphenylacetic acid in the brain with any dose of EGb 761, but significantly reduced serum prolactin levels and increased dopamine levels in the medial preoptic area and arcuate nucleus were seen at the dose of 50 mg/kg. These findings show that EGb 761 (especially at the dose of 50 mg/kg) enhances the copulatory behavior of male rats and suggest that the dopaminergic system, which regulates prolactin secretion, may be involved in the facilitatory effect of EGb 761.     

Dynamics of secretory granules in somatotrophs of rats after stimulation with growth hormone-releasing factor: a stereological analysis

The anterior pituitary tissue of male rats injected with growth hormone-releasing factor (GRF) was either processed for stereology at the light-and electron-microscopic levels, or homogenized for growth hormone (GH) assay 2–60 min after GRF injection. Secretory granules of somatotrophs became smaller but increased in numerical density 2 min after GRF injection. Their volume density began to increase at 5 min. The frequency of exocytosis of the granules was most prominent as early as 2 min after GRF injection and reduced thereafter. GH levels in the tissue were lowest at 2–5 min, and returned to the control value by 60 min. Serum GH levels were highest at 15 min; even at 60 min, this value was higher than in the controls. These findings suggest that secretory granules in somatotrophs are stimulated to divide by GRF, resulting in a decrease in size and an increase in number. The discrepancy between the earlier formation of new secretory granules and the later restoration of intracellular GH levels implies that GRF first stimulates the synthesis of constituents of granules other than GH, and only later the synthesis of GH, and that newly formed small secretory granules contain less GH. From the clearance rate of serum GH and the frequency of granule exocytosis, it can be estimated that about a half million granules are released to maintain 1 ng/ml of serum GH in rats.     

Co-operative binding of concanavalin A to A glycoprotein in lipid bilayers

Lectin-binding curves are reported for a concanavalin A receptor glycoprotein in lipid bilayers and intact cells. The results are consistent with previous studies of the structurally dissimilar transmembrane glycoprotein, glycophorin. High-affinity lectin binding to model membranes was influenced by the presence of apparently unrelated macromolecules, which we suggest is an example of receptor modulation by local interactions. Furthermore, high-affinity binding to the model membranes displayed characteristics, including positive cooperativity, similar to those seen with intact cells.     

Orai-2 is localized on secretory granules and regulates antigen-evoked Ca mobilization and exocytosis in mast cells

The increase in intracellular Ca²⁺ through the Ca²⁺ channel is an indispensable step for the secretion of inflammatory mediators by mast cells. It was recently reported that Orai-1 is responsible for the Ca²⁺ influx that is activated by depletion of stored Ca²⁺. There are three isoforms of Orai: Orai-1, Orai-2, and Orai-3; however, isoforms other than Orai-1 are poorly understood. We found that Orai-2 is expressed and localized on secretory granules in RBL-2H3. Ca²⁺ release from Ca²⁺ store, induced by antigen stimulation, was significantly attenuated by knockdown of Orai-2, while that induced by thapsigargin was not affected. Furthermore, exocytotic release induced by antigen stimulation was inhibited in knockdown cells. This observation suggests a new role of Orai isoforms in secretory cells.     

Deglycosylation of a lectin intermediate during assembly of ConA

Summary Metabolic labelling of immature jackbeans (Canavalia ensiformis) has been used in a pulse-chase study to determine changes in the glycosylation pattern of polypeptides during the assembly of Concanavalin A. In an analysis that allowed the identification of 7 intermediates, only the first precursor form of the lectin was labelled with D-[U-¹⁴C]-glucosamine. These results indicate that processing of the lectin involves a novel deglycosylation event in which an N-linked oligosaccharide is removed from a protein in the absence of proteolysis.Abbreviations endo H endo -N-acetylglucosaminidase H - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - ConA Concanavalin A     

Effects of serotonergic agents on plasma prolactin levels in pyridoxine-deficient adult male rats

Plasma concentration of prolactin was significantly reduced in pyridoxine-deficient as compared to control (pyridoxine-supplemented) adult male rats. Administration of pyridoxine to deficient rats resulted in a significant increase in plasma prolactin. The reduction in plasma prolactin in pyridoxine-deficient rats corresponded with the significantly reduced hypothalamic contents of pyridoxal phosphate and serotonin in pyridoxine-deficient rats. Plasma prolactin concentrations were also measured in response to serotonergic agents in both groups of rats. The administration of the 5HT_1A agonist (8-hydroxy 2-n-dipropylamino tetralin) resulted in a significant increase in plasma prolactin and that of the specific 5HT_1A antagonist spiroxatrine had the opposite effect. The results suggest that the hypothalamic serotonergic regulation of prolactin release is impaired in pyridoxine deficiency.     

Lectin cytochemical evaluation of somatosensory neurons and their peripheral and central processes in rat and man

Summary Paraffin sections of the trigeminal nerve root of the rat, and human spinal nerve root and trigeminal ganglion were stained with a battery of lectin-horseradish peroxidase conjugates to localize and characterize glycoconjugate (GC) in situ. In the rat the myelin sheath of the peripheral segment contained GC with sialic acid most probably linked to the penultinate disaccharide galactose(1 4)-N-acetylglucosamine (Gal(1 )-GlcNAc), and complex type N-glycosidic side chains. The myelin sheath in the central segment differed in containing little if any of the GC named above and in containing GC with terminal -Gal linked to N-acetylgalactosamine (GalNAc), terminal GalNAc and fucose. Schwann cells stained for GC with GlcNAc or mannose whereas oligodendroglia stained for GC with the terminal disaccharide Gal-(1 3)-GalNAc and N-glycosidic side chains, especially in presumed Golgi zones, but also in processes continued as the outer myelin sheath. The human myelin sheath in the central segment differed from that of the rat in not staining with lectins specific for fucose and terminal GalNAc. Sialic acid and terminal -Gal were seen in the human central segment but these sugars appeared to bind to astroglial structures rather than to the myelin sheath as in the rat. Astrocytes in both rat and man were stained by two fucose-binding lectins. Several lectins revealed affinity for GC in the neurilemmal sheath, and staining of this structure was stronger in the human specimens. Neurons in the human trigeminal ganglion ranged from unstained to strongly positive for fucoconjugate in cytoplasmic bodies and plasmalemma. Positive ganglion cells gave rise to unmyelinated fibers which also stained for fucoconjugate. Remak fibers and their extensions into the substantia gelatinosa of the human spinal cord stained strongly for content of fucose.The stronger lectin affinity for N-glycosidic core sugars in the peripheral as compared with the central segment suggests that lectins localize P_o protein in peripheral myelin. The reactivity for several sugars in the central segment can possibly be attributed to myelin-associated glycoprotein (MAG) of central myelin, but lectin staining for GalNAc shows in addition a biochemically unrecognized GC with O-glycosidic linked oligosaccharides in myelin. The lectin cytochemistry indicates that the 170 K Dalton glycoprotein with PNA affinity obtained from rat sciatic nerves occurs in nodes of Ranvier.This research was supported by NIH Grants AM-10956, HL-29775 and United Health and Medical Research Foundation of South Carolina, Inc. Grant No. 79     

Activation of AMPA receptors inhibits prolactin and estradiol secretion and delays the onset of puberty in female rats

Previous experiments have evidenced the neuroendocrine role of AMPA receptors. Present studies were carried out to obtain information on the role of these receptors in the control of the onset of puberty. To this end, female rats were i.c.v. injected with vehicle or AMPA (agonist of AMPA receptors: 0.1 or 0.5 nmol/day) between 26 and 30 days (Experiment 1), or 30 and 34 days (Experiment 2) of age. Serum concentrations of PRL, LH and estradiol were measured before drug administration, 10 min after the last injection, at vaginal opening (VO) and at first estrus (FE) presentation. In both experiments, AMPA administration inhibited PRL and estradiol secretion without affecting LH release. When AMPA was administered between 26 and 30 days a significant delay in the day of vaginal opening was observed. These results confirmed the inhibitory effect of AMPA on PRL secretion and suggests a role of AMPA receptors in the control of puberty onset.     

Evidence for a coupling of prolactin secretion and synthesis by rat pituitary cells in culture

The relationship between the release and the synthesis of prolactin by rat pituitary cells in culture was studied using a microtubule-disrupting drug, vinblastine. (1) Prolactin secretion was inhibited by vinblastine in short-term incubations. Vinblastine did not act via the dopamine pathway, since a potent anti-dopaminergic drug, fluphenazine, was unable to reverse the inhibiting action of the antimicrotubular agent. (2) Continuous treatment by vinblastine induced a progressive decrease of the rate of incorporation of [³H]leucine in prolactin. The half-inhibition time was about 2 days. This inhibition of prolactin synthesis was selective, since total protein synthesis remained unaffected. (3) Measurements of radioimmunoassayable prolactin showed that the inhibition of hormone release by vinblastine led to a transient increase of the intracellular content of prolactin. The phase of over-accumulation was followed by a progressive reduction of the total (cell + medium) prolactin. This result is in agreement with the observed inhibition of de novo synthesis of prolactin and indicates that a degradation process takes place in pituitary cells in culture. In conclusion, the use of vinblastine allows us to demonstrate that the rate of prolactin synthesis is dependent upon the secretory status of the cell.     

Isolation and characteristics of bovine pituitary secretory granules

Secretory granules containing primarily growth hormone and prolactin were isolated from bovine anterior pituitaries. Marker enzyme analysis and electron microscopy indicated that the secretory granule fraction did not contain measurable amounts of other intracellular organelles. Such isolated granules were resistant to a variety of chemical and physical challenges including variations in osmolarity, ionic strength, EGTA, sonication, boiling, etc. The only treatments that were found to routinely result in granules lysis were alkaline pH and 0.5% SDS. Nonspecific leakage of both groth hormone and prolactin was less than 9% of total hormone pool even after a 60-min incubation. The release of prolactin but not growth hormone could be increased by lowering the free calcium concentration. Conversely, 10⁻⁵ M ionophore A23187 caused a decrease in nonspecific hormone leakage. This raises the possibility that a nonexocytosis secretory pathway might be involved in pituitary hormone release. The initial secretory granule fraction was further purified using discontinuous sucrose gradient ultracentrifugation to yield a subfraction highly enriched in prolactin granules. These granules had the same stability characteristics as the original secretory granule fraction. The use of such granules should prove useful in our efforts to understand how calcium regulates cellular secretion.     

Atrial-specific granules in the hearts of normal and water-deprived rats

Summary The morphology of atrial-specific granules, which contain atrial natriuretic polypeptide (ANP), was studied in the cardiac tissue of untreated controls and water-deprived rats by means of conventional and immunoelectron microscopy. Immature secretory vesicles or granules appeared to become buded off from the Golgi cisternae and then fused to form specific A-granules. An electron-dense plate with a fuzzy coat was frequently found on the limiting membrane at the end of such fusion. Pale specific B-granules, which were less electron-dense, larger, and more granular than A-granules, were found in small numbers in the left atrial cardiocytes, but rarely in the right ones. Very pale granules with a less granular matrix, considered to be B-type granules which had lost their electron-density, and which had less immunoreactivity for ANP, were numerous in the cardiac tissue after water deprivation. This morphological change, which is interpreted as an indication of granule degradation, was in agreement with the noted increase of natriuretic activity in the atrial tissue of water-deprived specimens.     

Subcellular distribution of laminin and prolactin in stimulated and blocked prolactin cells in the pituitary of lactating rats

Summary Laminin (LAM), a glycoprotein component of basement membranes, has been previously detected within several subcellular compartments of prolactin (PRL) cells in the pituitary gland. The present work was aimed at comparing the subcellular localization of PRL, a specific secretory product, with that of LAM, in relation to the secretory activity of PRL cells. LAM and PRL were located in parallel, by ultrastructural immunocytochemistry, in PRL cells of lactating female Wistar rats, either stimulated by suckling, or blocked by weaning, or reactivated by suckle following short-term weaning. Variations in physiological conditions were correlated with a redistribution of PRL immunoreactivity within morphologically modified compartments. The Golgi apparatus became hypertrophied, and PRL impressively accumulated within saccules of the Golgi stacks of blocked cells. On the contrary, no apparent changes occurred in LAM distribution, at least at the Golgi level. Only a slight increase of LAM immunoreactivity was observed in rough endoplasmic reticulum after a long weaning period. PRL could be detected in most of the secretory granules and particularly in forming elements, whereas LAM was observable at the peripheral edge of some mature granules. Such a labeling was not markedly influenced by the physiological state. The prominent structures, indicative of crinophagic activity, characteristic of blocked cells, contained masses of dense material, which were always immunopositive with antibodies to PRL, but never to LAM. These observations could suggest that, in PRL cells, intracellular transport and exportation of LAM are controlled by mechanisms independent from those involved in the regulation of PRL secretion.     

Association of glycoproteins and pepsinogen in the secretory granules of fundic epithelial cells isolated from guinea pig stomach

Epithelial cells were isolated from the fundic portion of the guinea pig stomach. Cells were separated by velocity sedimentation at unit gravity in a Ficoll 70 gradient and pooled in three fractions. By morphological and biochemical criteria, each fraction was characterized as a population highly enriched in one of the three main functional types: oxyntic cell; chief cell and mucus-secreting cell. Measure of the pepsinogen content and specific stainings of the secretory granules for light and electron microscopy led to the definition of two types of mucus-secreting cells in nearly equal quantity; mucous cells with smaller secretory granules entirely glycoproteic in nature and muco-peptic cells containing larger heterogeneous secretory granules. These granules were made of a proteic core containing pepsinogen surrounded by a thin membrane and a voluminous cap, both containing carbohydrates. The cap appeared as if built of orderly packed layers of glycoproteins. Secretory granules of chief cells were also surrounded by a membrane containing glycoproteins and occasionally a small glycoproteic cap. Pepsinogen content was estimated to be three times higher in a single chief cell than in a muco-peptic cell.     

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

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

Immuno-electron-microscopic study of the prolactin cells in the pituitary gland of male Wistar rats during aging

Summary Prolactin cells were identified by means of immunocytochemistry with protein-A gold as a marker on ultrathin sections of the pituitary gland of young (3–4 months), middle-aged (16–19 months), and aged (26–30 months) male Wistar rats. Point-counting volumetry revealed that the prolactin (PRL) cell-volume density in middle-aged rats was significantly increased in comparison to the volume densities in young and aged rats. Within the PRL-cell population, four types of PRL cells were distinguished on the basis of the shape and size of their secretory granules. During aging, dramatic changes occurred in the relative volumes of the four cell types. The volume percentage of cells with round granules (type I, granule diameter 150–250 nm, and type IIA, granule diameter 250–350 nm) increased from ±30% in young rats to ±90% in old rats. The volume percentage of cells with round and polymorphic granules (type IIB; granule diameter 350–400 nm and type III; granule diameter 500–600 nm) decreased from ±70% in young rats to ±7% in old rats. Age-related changes in serum PRL levels were not found. It is concluded that although during the life span of the male Wistar rat considerable changes in PRL-cell volume densities and in the ratios of PRL-cell types occur serum, PRL levels remain more or less constant.     

A C-type lectin associated and translocated with cortical granules during oocyte maturation and egg fertilization in fish

Oocyte maturation and egg fertilization in both vertebrates and invertebrates are marked by orchestrated cytoplasmic translocation of secretory vesicles known as cortical granules. It is thought that such redistribution of cellular content is critical for asymmetrical cell division during early development, but the mechanism and regulation of the process is poorly understood. Here we report the identification, purification and cDNA cloning of a C-type lectin from oocytes of a freshwater fish species gibel carp (Carassius auratus gibelio). The purified protein has been demonstrated to have lectin activity and to be a Ca(2+)-dependent C-type lectin by hemagglutination activity assay. Immunocytochemistry revealed that the lectin is associated with cortical granules, gradually translocated to the cell surface during oocyte maturation, and discharged to the egg envelope upon fertilization. Interestingly, the lectin becomes phosphorylated on threonine residues upon induction of exocytosis by fertilization and returns to its original state after morula stage of embryonic development, suggesting that this posttranslational modification may represent a critical molecular switch for early embryonic development.     

Isolation and partial characterization of two populations of secretory granules from rat parotid glands

Summary A method is described for the isolation of two populations of secretory granules from rat parotid glands utilizing differences in their sedimentation characteristics. The granule preparations were analyzed for homogeneity by electron microscopy and chemical analyses. The soluble contents of both types of granules were obtained by hypotonic lysis, and the proteins compared by SDS-PAGE and ion exchange-gel filtration chromatography. Both populations of secretory granules appear to have the same protein composition as that of the parotid saliva. The secretory granules with the smaller apparent buoyant density became labelled with radioactive leucine earlier than the heavier granules when a pulse of this amino acid was supplied to a gland slice system. The lighter granules appear to represent an earlier stage in maturation.     

Morphometric classification of the neurosecretory granules in the rat pars nervosa

Summary The distribution of the diameters of the neurosecretory granules in the rat pars nervosa (measured from electron micrographs taken at 40 000 × ) was compared among axons by nonparametric statistical methods and the axons were classified into five groups with median granule diameters of 143, 155, 167, 180 and 193 nm. We suggested that these five axon types carried different secretory substances contained in the pars nervosa. This investigation is supported by a grant from the Population Council, New York and grant from the Ministry of Education. Authors are grateful to Japan Electron Optics Laboratory Company for their technical assistance with the electron microscopy and to Miss Kazue Yamamoto for her help in preparing the figures.