The effect of bromocriptine on the intermediate lobe of the rat pituitary: an electron-microscopic,morphometric study

Summary The morphological effect of chronic synthetic and secretory inhibition of the intermediate lobe of the rat pituitary induced by bromocriptine treatment was studied using morphometric techniques in combination with electron microscopy. On the basis of granule diameters, a heterogeneous cell population was shown in the normal intermediate lobe. Bromocriptine treatment did not induce any change in the volume fraction, number or location of electron-dense secretory granules. Instead, there was a shift toward a more homogeneous cell population containing smaller granules, the mean granule volume being reduced by 30%. The volume fraction of electron-lucent granules or vacuoles was markedly reduced, indicating a functional significance of these organelles. The volume of the Golgi apparatus was not significantly altered, but the number of condensing granules within the Golgi area was reduced. The volume of the intermediate lobe was decreased, apparently due to a decrease in the mean cell volume.     

Gonadectomy induces laminin biosynthesis and basement membrane assembly in anterior pituitary glands of adult rats

Summary Laminin biosynthesis and basement membrane assembly in anterior pituitary glands of gonadectomized rats were studied by immuno-electron microscopy and radioimmunoassay. Three weeks after gonadectomy, rats received intravenous injections of sheep anti-laminin IgG conjugated to horseradish peroxidase, and glands were fixed and processed for microscopy 1 h later. Peroxidase reaction product uniformly labeled all perivascular and glandular epithelial basement membranes. In addition, reaction product was also found in abnormally multi-layered basement membranes seen especially beneath gonadotrophs, and unusual basement membrane-like structures projecting between gonadotrophs were also labeled. Pituitary sections from gonadectomized rats labeled with pre-embedding immunoperoxidase and post-embedding immungold techniques also localized intracellular laminin within biosynthetic organelles and light body vesicles of gonadotrophs. Neither abnormal basement membrane structures nor intracellular laminin were detected in pituitaries of nongonadectomized, control rats. Radioimmunoassays of pituitary homogenates showed nearly twice as much soluble laminin ( 15 ng/gland) in gonadectomized rats than in controls ( 8 ng/gland), which paralleled gland growth, but serum laminin concentrations did not differ ( 10 ng/ml in both groups). When anterior pituitary glands of gonadectomized rats that received injections of anti-laminin IgG-HRP were fixed 5 days after injection, lengths of unlabeled basement membrane were distributed between labeled lengths. This indicated that new basement membrane was spliced into old by a process similar to that seen in normal development. Supplementation of gonadectomized rats with testosterone, however, arrested laminin biosynthesis and basement membrane assembly and reversed glandular hypertrophy. These results indicate that, in an absence of sex hormone feedback, renewed synthesis of basement membrane components occurs in the anterior pituitary and is probably necessary to support the additional growth and differentiation of gonadotrophs and other pituitary cells.     

Maturation-related changes in mass and elemental contents of secretory granules as measured by electron-microprobe

Summary The relationship between granule density, protein content, and Ca and S contents were studied in two secretory granule fractions, from parotid glands of the rat, previously shown to constitute different stages in granule maturation. The density of the lighter fraction was between 1.133 and 1.142 g/ml, while that of the heavier fraction was greater than 1.142 g/ml. The mean protein content of the denser granules was 12% greater than that of the lighter granules (P<0.03), while the dry-mass elemental concentrations in the two granule fractions were unchanged. These results indicate that protein is added to granules during the maturation process (presumably by vesicular traffic), and that the resulting increase in granule density is not driven simply by decrease in water content and/or increased concentrations of inorganic Ca or S in the granules. The elemental concentration values also indicate that the diffusible elements permeate the granule membrane during the fractionation procedures.     

Control of peptide release from cells of the intermediate lobe of the rat pituitary

Summary When rats were injected with 6-hydroxydopamine the catecholaminergic nerve terminals in their intermediate lobes exhibited distinct signs of degeneration. Morphometric examination of the Golgi apparatus in cells of the intermediate lobe of these rats showed significant enlargement of Golgi cisternae. The release of adrenocorticotropin, -endorphin/lipotropin and -melanotropin from intermediate-lobe cells in vitro was measured by radioimmunoassay. The high basal peptide release was inhibited by dopamine and stimulated by methyl-isobutyl-xanthine. In contrast, -aminobutyric acid, serotonin, histamine and noradrenaline, or corticotropin-releasing hormone, rat hypothalamic extract and vasopressin had no or only very weak effects. These observations indicate that the synthetic apparatus of intermediate-lobe cells is constantly depressed by dopaminergic nerves. We were not able to stimulate peptide release from intermediate-lobe cells by use of the abovementioned endogenous agents.Supported by Deutsche Forschungsgemeinschaft: Ausbildungsstipendium Sto 162 (G.S.), SFB 87/B2, and Heisenberg Stipendium (K.H.V.)     

Cellular mechanisms of estrogen-and dopamine-induced control of glandular kallikrein in the anterior pituitary of the rat

Glandular kallikrein (GK, a trypsin-like serine protease) exhibits estrogen induction and dopamine repression in rat pituitary lactotrophs. Steroid induction may reflect primary actions to increase selectively the synthesis of specific proteins, or may be part of broad cellular responses secondary to steroid-induced phenotype transitions. This study examined the cellular mechanisms underlying estrogen and dopaminergic control of lactotroph GK using a quantified immunocytochemical approach. Pituitaries from ovariectomized rats exhibited little GK staining. Estradiol treatment for 10 days produced dose-dependent increases in pituitary mass, the percentage of lactotrophs (indicating lactotroph proliferation) and the percentage of GK-positive cells. Also, GK staining intensity was dependent upon estradiol dose, increasing 4-fold between 5 g and 50 g/48 h. Dopamine receptor blockade with haloperidol (2.5 mg/kg/24 h) elicited weak GK immunostaining in 46% of the lactotrophs in the absence of estradiol, and markedly potentiated GK staining intensity elicited with low but not high doses of estradiol. The results suggest that GK induction is a primary estrogen effect, and is not secondary to a phenotype transition: the induction is enhanced by estrogen-induced lactotroph proliferation. Dopaminergic systems strongly inhibit GK induction by low estradiol levels. This dopaminergic modulation may shift the induction of lactotroph GK to physiological events associated with high estradiol levels or low dopaminergic tone.     

Cystlike structures derived from the marginal cells of Rathke's cleft in rat pituitary grafts

Summary Hemipituitary glands from 30 rats were isotransplanted under renal capsules. At 1, 2, 4, 7 and 20 days after transplantation, the grafts were examined by light and electron microscopy. Two days after transplantation, the central area of graft showed necrosis; however, the peripheral area, where marginal cells of Rathke's cleft were ingesting the remnants of necrotic glandular cells, survived. Four days after transplantation, mitotic figures of marginal cells were observed. Seven days after transplantation, the damaged area of the graft disappeared, and Rathke's cleft was completely lined by marginal cells. Remnants of necrotic glandular cells were not seen in intercellular spaces or in the cytoplasm of marginal cells. Cystlike structures formed in the grafts; some were connected to Rathke's cleft by narrow cavities. The cavity-lining cells of the cysts were agranular and similar to those that lined Rathke's cleft. At 20 days after transplantation, granular cavity-lining cells appeared. It is suggested that marginal cells of immature rats can differentiate into granular cells.     

Sexual differences in the Golgi apparatus of rat hepatocytes: three-dimensional analysis

Lipid metabolism takes place in the Golgi apparatus, but at a higher rate in female than in male rats. I therefore examined the Golgi apparatus by morphometric means for differences between the sexes at the light-and electron-microscopic level. The Golgi apparatus was stained in situ by a zinc-iodide-osmium method. The counts of the Golgi apparatus in cross-sections in female hepatocytes by light microscopy were approximately twice that in male hepatocytes. Upon ovariectomy, these counts were greatly reduced but were reestablished after estrogen supplement. To clarify this phenomenon, three-dimensional reconstructions of the Golgi apparatus were produced from electron-microscopic images of serially cut 160-nm sections. The Golgi apparatus of both male and ovariectomized females had the shape of a small ring, whereas it took the form of a large elongated cylinder in normal females and in castrated males after treatment with estrogen. The numerical difference in Golgi apparatus counts by light microscopy of in males and females is, therefore, apparently attributable to the size and shape of the Golgi apparatus, and is controlled by the estrogen level.     

Accumulation of secretory granules in pituitary clonal cells derived from the epithelium of Rathke's pouch

Summary 2A8 clonal cells derived from the epithelium of Rathke's pouch of the fetal rat are essentially agranular when grown in vitro, in spite of their active secretion of hormones, i.e., ACTH, prolactin and growth hormone. These cells do not produce detectable amounts of thyrotrophic or gonadotrophic hormones in vitro. When the growth medium (Ham's F10) was supplemented with rat median eminence extract (MEE), l-thyroxin and fresh serum from hypophysectomized rats, some of the 2A8 cells accumulated and stored secretory granules which were characteristic of the cells of the intact pituitary gland. Typical thyrotrophic and gonadotrophic cells also became recognizable and all six anterior pituitary hormones were released into the culture medium. Growth of 2A8 cells in this modified culture medium resulted in an increased production of all hormones with increasing time in culture.These results indicate that the processes that lead to the accumulation of typical mature secretory granules which characterize the individual pituitary cell types are initiated or promoted by some unidentified factor or factors which are present in fresh rat serum. It is also apparent that fresh rat serum can promote the differentiation of gonadotrophs and thyrotrophs in vitro.Supported by USPHS Grant Am 12583 and Institutional Research Grant of The University of Texas Health Science Center at San Antonio, TexasThe authors wish to thank Mrs. Pauline Polette for her skillful technical assistance     

In vivo correlation between c-Fos expression and corticotroph stimulation by adrenocorticotrophic hormone secretagogues in rat anterior pituitary gland

In the anterior pituitary gland, c-Fos expression is evoked by various stimuli. However, whether c-Fos expression is directly related to the stimulation of anterior pituitary cells by hypothalamic secretagogues is unclear. To confirm whether the reception of hormone-releasing stimuli evokes c-Fos expression in anterior pituitary cells, we have examined c-Fos expression of anterior pituitary glands in rats administered with synthetic corticotrophin-releasing hormone (CRH) intravenously or subjected to restraint stress. Single intravenous administration of CRH increases the number of c-Fos-expressing cells, and this number does not change even if the dose is increased. Double-immunostaining has revealed that most of the c-Fos-expressing cells contain adrenocorticotrophic hormone (ACTH); corticotrophs that do not express c-Fos in response to CRH have also been found. However, restraint stress evokes c-Fos expression in most of the corticotrophs and in a partial population of lactotrophs. These results suggest that c-Fos expression increases in corticotrophs stimulated by ACTH secretagogues, including CRH. Furthermore, we have found restricted numbers of corticotrophs expressing c-Fos in response to CRH. Although the mechanism underlying the different responses to CRH is not apparent, c-Fos is probably a useful immunohistochemical marker for corticotrophs stimulated by ACTH secretagogues. This work was supported by the Jichi Medical University young investigator award.     

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.     

Response of PAS-positive cells of the pituitary pars intermedia in the teleost Carassius auratus to acid water

Summary The PAS-positive or PIPAS cells in the pars intermedia of goldfish are activated after reduction of the pH of the ambient freshwater from 7.5 to 3.5. The cells increase in number and exhibit a five-fold increase in cell volume. Granular endoplasmic reticulum occupies most of the cytoplasm. Goldfish PIPAS cells (also termed calciumsensitive cells) are thought to have a hypercalcemic function. Therefore, their activation in acid water may be caused by the severe drop in plasma calcium concentration following exposure of the fish to low water pH. However, activation of the PIPAS cells in response to acidification of the water is not prevented when the calcium concentration of the water is increased to levels that result in hypercalcemia instead of hypocalcemia. Activation of the PIPAS cells occurs also in fish exposed to acidified freshwater enriched with NaCl to an osmolarity similar to that of the blood. This prevents the reduction in plasma osmolarity and Na⁺ and Cl^- concentrations that follow exposure of goldfish to acidified normal freshwater. Our observations do not support the hypothesis that the PIPAS cells in goldfish produce a hypercalcemic hormone, or indeed any hormone involved in calcium metabolism or osmoregulation. The cells may be implicated in acid-base regulation (a characteristic of many types of fish when exposed to acidified water) but the evidence is indirect.     

Binding and internalization of native gonadoliberin (GnRH) by anterior pituitary gonadotrophs of the rat

Summary To identify anterior pituitary cell types containing GnRH binding sites and to study the internalization process of this peptide by target cells under physiological conditions, autoradiography was performed on rat anterior pituitaries removed at specific time intervals (2–60 min) after intravenous injection of mono-radioiodinated ¹²⁵I-GnRH into intact males. At electron-microscopic level, gonadotrophs and lactotrophs appeared to contain silver grains. Concomitant administration of an excess of unlabeled GnRH with the radioiodinated hormone prevented this localization indicating the specificity of the reaction. The time-course study in gonadotrophs showed that 2 min after injection silver grains could be found over the plasma membrane, secretory granules and nuclear membrane. Similar results were observed 5 and 15 min after injection. Extensive label was observed over the nucleus and nuclear membrane 15 to 60 min after injection. The injection of a radioiodinated GnRH agonist [D-Trp⁶, Pro⁹ (Net), DesGly¹⁰]-GnRH produced comparable results. In contrast, the injection of ¹²⁵I-[D-pGlu¹, D-Phe², Trp^3,6]-GnRH, an antagonist of GnRH, produced positive labeling only at the plasma membrane without internalization. These results indicate that, after binding with receptors on the plasma membrane, GnRH is rapidly internalized, accumulating in secretory granules, and localizing over the nuclear membrane and later, in the nucleus. Association of radioactivity with secretory granules could be related to a specific action of GnRH at this level or to receptor recycling, and presence of label in the nucleus may be related to stimulation of neosynthesis of LH and GnRH receptors.     

Characterization of obestatin- and ghrelin-producing cells in the gastrointestinal tract and pancreas of rats: an immunohistochemical and electron-microscopic study

Both ghrelin and obestatin are derived from preproghrelin by post-translational processing. We have morphologically characterized the cells that produce obestatin and ghrelin in new-born and adult Sprague-Dawley rats that were freely fed, fasted, or subjected to gastric bypass surgery or reserpine treatment. Tissue samples collected from the gastrointestinal tract and pancreas were examined by double-immunofluorescence staining, immunoelectron microscopy, and conventional electron microscopy. Obestatin was present in the stomach, duodenum, jejunum, colon, and pancreas. In the stomach, differences were noted in the development of obestatin- and preproghrelin-immunreactive (IR) cells on the one hand and ghrelin-IR cells on the other, particularly 2 weeks after birth. Preproghrelin- and obestatin-IR cells were more numerous than ghrelin-IR cells in the stomach, suggesting the lack of ghrelin in some A-like cells. Most obestatin-producing cells in the stomach were distributed in the basal part of the oxyntic mucosa; these cells co-localized with chromogranin A (pancreastatin) and vesicle monoamine transporters type 1 and 2, but not with serotonin or histidine decarboxylase. Immunoelectron microscopy revealed the obestatin- and ghrelin-producing cells to be A-like cells, characterized by numerous highly electron-dense granules containing ghrelin and obestatin. Some granules exhibited an even electron density with thin electron-lucent halos, suggestive of monoamines. Feeding status, gastric bypass surgery, and reserpine treatment had no obvious effect on the A-like cells. In the pancreas, obestatin was present in the peripheral part of the islets, with a distribution distinct from that of glucagon-producing A cells, insulin-producing beta cells, and cells producing pancreatic polypeptide Y. Thus, obestatin and ghrelin co-localize with an anticipated monoamine in A-like cells in the stomach, and obestatin is found in pancreatic islets. This study was supported by a grant from the Cancer Foundation of St. Olav’s Hospital, Trondheim, Norway.     

Ultrastructure and immunolabeling of gonadotrops and thyrotrops in the pituitary of the African catfish,Clarias lazera

Summary Pituitaries of the African catfish (Clarias lazera) were studied with immunocytochemical methods, at the light-microscopic and ultrastructural levels, for the characterization and localization of gonadotropic and thyrotropic cells. Two immunostaining procedures with the use of different markers were carried out: (i) with peroxidase-antiperoxidase, (ii) with protein A-gold. In routinely stained sections for light microscopy two types of basophils were identified in the proximal pars distalis: (1) large, round, purple cells, and (2) small, angular, light-blue cells. Both types were immunolabeled with antibodies against Clarias ,-gonadotropin (GTH) and salmon G100-GTH. Only the large basophils were immunolabeled with anti-carp -GTH, whereas the small basophils were the only cells immunolabeled with anti-human thyrotropin beta subunit (anti-h TSH-). It was concluded that the large basophils represent the gonadotrops and the small basophils the thyrotrops. At the ultrastructural level the immunostaining of the GTH-cells was confined to three types of inclusions: (i) secretory vesicles, (ii) globules, and (iii) electron-dense, membrane-bound irregular masses. Especially the protein A- gold method, in combination with the use of a highly diluted homologous antiserum, resulted in a distinct localization of GTH. The presence of two types of nerve fibres, synaptically contacting the gonadotrops, is discussed with regard to the presence of a peptidergic (stimulatory) and an aminergic (inhibitory) control of GTH-secretion.Part of the results were presented as a poster at the IX^th conference of the European Society of Comparative Endocrinology, Jerusalem, August 1981, and published as an abstract (Peute et al. 1982)     

Studies on secretory glycoproteins in the rat exocrine pancreas

Summary A fetuin, fucosyl transferase has been identified in the smooth microsomal fraction from the rat exocrine pancreas. This enzyme is involved in the glycosylation of secretory proteins and is bound to membranes, predominantly of the Golgi complex. Optimal in vitro conditions for the assay of the enzyme activity were established: a pH of 5.5–6.0, a temperature of 21° C and concentrations of Mg^{+ +} at 5.0 mM and ATP at 2.0 mM.Supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (Ke 113/10). Dedicated to Professor Helmut Ferner, Vienna, on the occasion of his 65^th birthday.     

The origin and ultrastructural characteristics of corticotropin-releasing factor (CRF)-immunoreactive nerve fibers in the posterior pituitary of the rat

Summary A fine network of corticotropin-releasing factor (CRF)-immunopositive fibers was found in the posterior lobe of the pituitary of the rat. The intermediate and distal lobes were free of CRF-immunoreactivity. Varicose, terminal-like axons were frequently observed around capillary vessels. Surgical isolation of the paraventricular nuclei resulted in a complete disappearance of CRF-immunoreactive fibers from the posterior lobe. CRF-immunopositive fibers show the general characteristics of peptidergic axons. These ultrastructural observations support the idea that CRF is secreted into capillary vessels.     

Chondrocytes are released as viable cells during cartilage resorption associated with the formation of intrachondral canals in the rat tibial epiphysis

The development of cartilage canals is the first event of the ossification of the epiphyses in mammals. Canal formation differs from vascular invasion during primary ossification, since the former involves resorption of resting cartilage and is uncoupled from bone deposition. To learn more about the fate of resorbed chondrocytes during this process, we have carried out structural, cell proliferation, and in situ hybridization studies during the first stages of ossification of the rat tibial proximal epiphysis. Results concerning the formation of the cartilage canals implied the release of resting chondrocytes from the cartilage matrix to the canal cavity. Released chondrocytes had a well-preserved structure, expressed type-II collagen, and maintained the capacity to divide. All these data suggested that chondrocytes released into the canals remained viable for a specific time. Analysis of the proliferative activity at different regions of the cartilage canals showed that the percentage of proliferative chondrocytes at areas of active cartilage resorption was significantly higher than that in zones of low resorption. These results are consistent with the hypothesis that resting chondrocytes surrounding canals have a role in supplying cells for the development of the secondary ossification center. Since released chondrocytes are at an early stage of differentiation greatly preceding their entry into the apoptotic pathway and are exposed to a specific matrix, cellular, and humoral microenvironment, they might differentiate to other cell types and contribute to the ossification of the epiphysis.This research was supported by the Ministerio de Ciencia y Tecnología (Spain), grant no. MCT-00-BMC-0446. The Instituto Universitario de Oncología is financed by Obra Social Cajastur-Asturias, Spain. J. Alvarez receives financial support from the Ministerio de Ciencia y Tecnología (CAJAL-03-06) and L. Costales from the Ministerio de Ciencia y Tecnología (MCYT, FP2000-5486).     

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.     

Differences in claudin synthesis in primary cultures of acinar cells from rat salivary gland are correlated with the specific three-dimensional organization of the cells

Tight junctions are essential for the maintenance of epithelial cell polarity. We have previously established a system for the primary culture of salivary parotid acinar cells that retain their ability to generate new secretory granules and to secrete proteins in a signal-dependent manner. Because cell polarity and cell-cell adhesion are prerequisites for the formation of epithelial tissues, we have investigated the structure of the tight junctions in these cultures. We have found two types of cellular organization in the culture: monolayers and semi-spherical clusters. Electron microscopy has revealed tight junctions near the apical region of the lateral membranes between cells in the monolayers and cells at the surface of the clusters. The cells in the interior of the clusters also have tight junctions and are organized around a central lumen. These interior cells retain more secretory granules than the surface or monolayer cells, suggesting that they maintain their original character as acinar cells. The synthesis of claudin-4 increases during culture, although it is not detectable in the cells immediately after isolation from the glands. Immunofluorescence microscopy has shown that claudin-4 is synthesized in the monolayers and at the surface of the clusters, but not inside the clusters. Only claudin-3, which is present in the original acinar cells following isolation and in the intact gland, has been detected inside the clusters. These results suggest that differences in claudin expression are related to the three-dimensional structures of the cell cultures and reflect their ability to function as acinar cells. This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Culture, Sports, and Technology of Japan (16591868, 16791135), by a Suzuki Memorial Grant of the Nihon University School of Dentistry at Matsudo (Joint Research Grant for 2003), by a Nihon University Multidisciplinary Research Grant for 2005 and 2006, and by a Grant-in-Aid for a 2003 Multidisciplinary Research Project from MEXT.