LH-producing cells in the ovine pituitary

Summary An immunocytochemical technique using the peroxidase-antiperoxidase complex (PAP) was applied to identify and characterize the LH-secreting cells in the ovine pituitary at the ultrastructural level. These cells, round or oval in shape, possessing flattened cisternae of the rough endoplasmic reticulum, contain one class of secretory granules (mean diameter 250 nm) and large dense bodies (600 to 800 nm in diameter). LH molecules and the two subunits LH and LH were localized on the secretory granules and on the small granules near the Golgi complex. The large dense bodies, the cisternae of the endoplasmic reticulum and the saccules of the Golgi complex showed no reaction product.Abbreviations used in this Article O-LH ovine luteinizing hormone - b-LH bovine luteinizing hormone - p-LH porcine luteinizing hormone - p-LH porcine LH subunit - p-LH porcine LH subunit - O-FSH ovine follicle stimulating hormone - b-TSH bovine thyrotropic hormone - A-b LH antiserum to bovine LH - A-pLH antiserum to porcine LH subunit - A-pLH antiserum to porcine LH subunit     

Unique features of secretory granules observed in the pituitary growth hormone-secreting (GH) cells of the musk shrew (Suncus murinus L.)

Summary Unique rod-shaped secretory granules were observed among oval or spherical secretory granules in GH cells of the anterior pituitary gland of musk shrew using the protein A-gold procedure combined with electron microscopy. The rod-shaped and spherical secretory granules were both immunoreactive by the immuno-gold method using antiserum to sheep GH. The rod-shaped secretory granules, which seem to be formed directly from the Golgi vesicles, extend from several hundred to several thousand nm in length. They often touch each other and fuse. The spherical secretory granules are also unique in that they may also fuse with loss of dense contents to leave empty circular membrane profiles in the cytoplasm. Both the rodshaped and spherical secretory granules are secreted from the cell by exocytosis.     

Localization of microfilaments in prolactin cells of the rat anterior pituitary gland

Summary Prolactin cells from anterior pituitary glands of normal non-lactating female rats, and lactating animals, some of which were separated from their pups for 48 hours, were examined ultrastructurally for the presence of microfilaments. Microfilaments were found in specific intracellular locations in all cells examined. They were in association with the nuclear envelope, the Golgi complex, the endoplasmic reticulum, small vesicles of the endoplasmic reticulum, and secretory granules. The possible role of microfilaments in the movement of intracellular organelles is considered.This investigation was supported by the National Institutes of Health grants AM 12583 and TW 02023.The authors wish to express their gratitude to Mr. M. G. Williams and Miss Pauline Cisneros for their excellent technical assistance.     

Effects of Neuropeptide Y (NPY) on the release of anterior pituitary hormones in the rat

Neuropeptide Y (NPY) has been recently localized in several hypothalamic nuclei in the mammalian brain. In order to investigate the possible role of NPY on neuroendocrine function, we have investigated the effects of the peptide on the release of anterior pituitary hormones in the rat. Both intravenous (300 μg) or intraventricular (2 to 15 μg) injection of NPY produced in gonadectomized male rats a significant and long-lasting decrease of plasma LH levels. A short duration stimulating effect on prolactin plasma levels was also observed after the intravenous but not after the intraventricular injection of NPY. Plasma levels of the other pituitary hormones were not significantly modified after NPY injection. When incubated in vitro with anterior pituitary cells in monolayer culture, NPY produced no significant change in release of pituitary hormones. Thus NPY seems to exert a selective effect on LH release. Since this effect can be observed after both intravenous and intraventricular injection, it might be hypothesized that NPY could affect LHRH release in two areas which lack blood-brain barrier: the organum vasculosum of the lamina terminalis (OVLT) which contains LHRH cell bodies and NPY fibers and the median eminence which contains both LHRH and NPY fibers. The effect on prolactin release needs to be carefully evaluated in different experimental conditions.     

Ultrastructure of melatonin-responsive cells in the ovine pars tuberalis

Summary Functional receptors for melatonin have been localized and characterized on the pars tuberalis (PT) of a number of mammalian species, but the cell-type responsive to melatonin is unknown. The ultrastructure of the ovine pars tuberalis has been examined and these findings correlated with the functional response of the gland to melatonin. This study revealed that two secretory cell types predominate in the ovine PT, which differ in the abundance of dense-core granules. The most abundant of the cells are either agranular or very sparsely granulated and represent 90% of the total population, with the remaining 10% being composed of cells with abundant dense-core vesicles. Few follicular cells were observed. This ratio of secretory cell-types persisted in primary culture, with the two types non-separable by Percoll gradient centrifugation. Using forskolin, as a non-specific stimulant of adenylate cyclase, melatonin was shown to inhibit the formation of cyclic AMP by 80–90% in cells both before and after Percoll centrifugation. The results demonstrate that the agranular secretory cells of the ovine pars tuberalis are the melatonin responsive cell-type of this gland.     

The thyrotropic cells of the guinea pig pituitary

Summary Three different immunocytoenzymatic techniques were used to identify and characterize the thyrotropic cells in the pituitary of normal guinea pigs at the ultrastructural level (superimposition technique, immunocytochemical technique using P.A.P. and indirect immunohistoenzymatic method before embedding).These cells are characterized by a dark cytoplasm with granules ranging from 1500 to 2000 Å in diameter. The appearance of these granules is very variable: some display a marked electron density and are homogeneous but some have a less marked electron density with a more electron dense peripherally situated region.The TSH molecules are essentially confined to the granules but when the immunocytochemical reactions are carried out before embedding, positive staining is also seen in the cytoplasm and the outer surface of most of the rough endoplasmic reticulum membranes. These results are discussed.We thank D. Quief for technical assistance. This work was supported by a grant from U.E.R. III LilleAttaché de Recherche INSERM     

A possible phagocytic role for folliculo-stellate cells of anterior pituitary following estrogen withdrawal from primed male rats

Summary Ultrastructural changes suggesting a phagocytic role for the nongranular folliculo-stellate cells of the anterior pituitary are investigated in estrogen-primed male rats after withdrawal of estrogen. Morphological changes in mammotropes following the removal of a subcutaneous estradiol-containing Silastic implant include the formation of intracellular lipid bodies. These lipid bodies appear to be associated with enhanced estrogen-dependent prolactin secretion in mammotropes. Seven and 24 h after estrogen withdrawal intracellular lipid within mammotropes seems to be released into the intercellular space. Seventy-two h after estrogen withdrawal, lipid droplets are almost entirely cleared from mammotropes while folliculo-stellate cells become packed with lipid globules. Folliculo-stellate cells also undergo dramatic hypertrophy 7 and 24 h after the removal of E2-containing implants. Extensive intercellular junctions including zonulae adhaerentes, desmosomes, and putative gap junctions are formed. Intercellular junctions delineate extravascular channels into which numerous microvilli project. Folliculo-stellate cells appear capable of accumulating many lipid droplets, presumably related to mammotrope metabolism. What appear to be large secondary lysosomes as well as the lipid droplets are observed within folliculostellate cells; lipid, therefore, may be degraded through a lysosomal pathway in folliculo-stellate cells.     

Quick-freeze,deep-etch visualization of exocytosis in anterior pituitary secretory cells: localization and possible roles of actin and annexin II

The exocytotic process in the anterior pituitary secretory cells was studied using quick-freeze deep-etch electron microscopy, fluorescein-isothiocyanate-phalloidin staining, heavy meromyosin decoration, and immuno-electron microscopy. The subcortical actin filaments are distributed unevenly in the peripheral cytoplasm. Few secretory granules are seen beneath the plasma membrane in the region where the peripheral cytoplasm is occupied by numerous subcortical actin filaments. On the contrary, in the region free of the subcortical actin filaments, many secretory granules lie in contact with the plasma membrane. Thus, the subcortical actin filaments may control the approach of the secretory granules to the plasma membrane in these cells. The granule and plasma membranes that lie in close proximity are linked by intervening strands. Unfused portions of both membranes remain linked by these strands during membrane fusion and opening. These strands may be involved in membrane contact, fusion and opening during exocytosis. Annexin II (calpactin I) has been demonstrated immunocytochemically to be localized at the contact sites between the granule and plasma membranes, and is therefore a possible component of the intervening strands. Membrane fusion starts within focal regions of both membranes less than 50 nm in diameter. The plasma membrane shows inward depressions toward the underlying granules immediately before fusion. The disappearance of intramembranous particles from the exocytotic site of the membrane has not been observed.     

Proliferating cells in the rat anterior pituitary during the postnatal period: immunoelectron microscopic observations using monoclonal anti-bromodeoxyuridine antibody

Proliferating cells in the male rat anterior pituitary at 1, 3, 5, and 8 weeks of age were labeled with bromodeoxyuridine (BrdU) and studied by light and electron microscopic immunocytochemistry using anti-BrdU. They decreased in number from 402±31/mm² at 1 week to 50±1.5/mm² at 8 weeks, while their cell area increased by about twofold during this period. They had a slightly higher nucleus/whole cell (N/C) ratio than non-proliferating cells. According to their ultrastructure we classified them into granular and agranular cells. The percentage of granular cells ranged from 73% to 82% of all the proliferating cells during the period studied. They had many granules of various sizes and shapes, and some contained growth hormone and prolactin. Agranular cells, constituting 18–27% of proliferating cells, were small and had a high N/C ratio, indicating their immaturity. Moreover, they showed several features of folliculo-stellate (FS) cells: they showed no secretory granules in the cytoplasm, extended thin cytoplasmic processes, and sometimes they constructed a follicle among them. These results suggest: (1) the majority of proliferating cells were mature cells producing anterior pituitary hormone(s) and (2) most of the agranular proliferating cells maybe FS cells. The possibility of the latter is discussed.     

The prolactin and growth-hormone producing cells of the guinea-pig pituitary

Summary Different immunohistochemical techniques were used to identify the prolactin and growth hormone producing cells (STH cells) in the pituitary of normal guinea pigs at the ultrastructural level.Prolactin cells revealed two main aspects: 1. Cells with granules from 2500 to 3500 Å in diameter some of which are irregularly shaped. These granulations are scattered throughout the whole cytoplasm. The rough endoplasmic reticulum is well developed and organized in parallel lamellae. 2. In cells of the second type the granules are less numerous and smaller in diameter (1800 to 2500 Å) and the rough endoplasmic reticulum is less well developed.The cytoplasm of the STH cells contains many more round granules (generally grouped) which also range from 2500 to 3500 Å in diameter.The prolactin molecules and the STH molecules are essentially confined to the granules but with the immunocytochemical technique before embedding a slightly diffuse reaction appeared in the entire cytoplasm. These results are discussed.This work was supported by a grant from U.E.R. III LilleChercheur I.N.S.E.R.M.     

The effects of gastric inhibitory polypeptide (GIP) on the release of anterior pituitary hormones

Several members of the secretin family of hormones have been demonstrated to alter anterior pituitary hormone secretion. Here we report the action of gastric inhibitory polypeptide (GIP) on gonadotropin and somatotropin release. Intraventricular injection of 1 microgram (0.2 nmole) GIP (2.5 microliters) produced a significant decrease in plasma FSH at 30 (p less than 0.02) and 60 min after its injection (p less than 0.01). The FSH-lowering effect of a higher dose of 5 micrograms (1 nmole) of GIP was already developed at 15 min (p less than 0.01) and was prolonged until the end of the experiment (60 min, p less than 0.05). No change in plasma LH was detected at any time during the experimental period. If 5 micrograms of estradiol-benzoate were given SC 48 hr prior to experiment, the initial values of FSH and LH were markedly decreased. In these animals GIP failed to influence plasma FSH and LH. When dispersed anterior pituitary cells from OVX rats were cultured overnight and incubated in vitro with GIP, the peptide was found to induce both FSH and LH release. Highly significant release occurred with the lowest dose tested of 10(-7) M and there was a dose-response effect for both hormones. The slope of the dose-response curve was similar for both FSH and LH release. GIP was less potent than LHRH which produced a greater stimulation of both FSH and LH release at a dose of 10(-9) M than did 10(-7) M GIP. The two peptides had an additive effect on the release of both FSH and LH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine-structural and immunohistochemical study of anterior pituitary cells of Snell dwarf mice

Summary Snell dwarf mice display remarkable retardation of growth after birth and are known to lack prolactin (PRL), thyroid stimulating hormone (TSH) and growth hormone (GH). The aim of this study was to determine the reason for these hormonal deficiencies. We examined the fine structure of the gland and its immunohistochemical staining pattern with respect to antisera raised against PRL, TSH, GH, adrenocorticotrophic hormone (ACTH) and luteinizing hormone (LH). The gland of control mice reacted immunohistochemically against all antisera used, whereas only ACTH-producing cells (ACTH cells) and LH-producing cells (LH cells) were distinguished in the dwarf mice. ACTH cells in dwarf mice varied in cell shape, although they were similar in size to those of controls. The distribution of secretory granules in the cytoplasm varied from cell to cell. LH cells in the dwarf mice showed immature features, having poorly developed rough endoplasmic reticulum and Golgi apparatus. The cells were about half the size of controls, and secretory granules were smaller. In dwarf mice, non-granulated cells were encountered in addition to granulated ACTH and LH cells. Some of them formed small clusters, characteristic cell junctions being found between the cells; they thus appeared to be follicular cells. The above results suggest that hormone deficiency in Snell dwarf mice is a result of a defect in the hormoneproducing cells in the gland.     

Localization of sialic acid-containing hormones in GTH cells and ACTH cells of the rat anterior pituitary

Summary The localization of sialic acid-containing substances in the rat anterior pituitary gland has been studied by light and electron microscopy, using a peroxidase-labeled lectin (Limulus polyphemus agglutinin: LPA) which binds specifically to sialic acid residues. LPA stains two types of anterior pituitary cells: (1) round or ovoid cells which are also positively stained with anti-hCG (GTH cell), and (2) small, stellate cells which are unstained with anti-hCG (ACTH cell). All of the LPA-positive cells can be distinguished from TSH cells which are identified by the use of anti-hTSH. On ultrathin sections directly stained with LPA using the postembedding method, the reaction is confined to the secretory granules in GTH cells, and ACTH cells. Of two types of secretory granules in GTH cells, the larger one is intensely stained, whereas the smaller type shows only weak staining with LPA. Since follicle-stimulating hormone (FSH) is known to have high sialic acid contents, the results suggest possible detection of FSH with a technique other than immunohistochemistry. Furthermore, if the sialic acid-containing substances in GTH cells represents FSH, then these results support the hypothesis that LH cells and FSH cells are one cell type.This research was supported by grants from the Ministry of Education of Japan     

Ultrastructural and immunocytochemical studies on the cytoskeleton in the anterior pituitary of rats,with special regard to the relationship between actin filaments and secretory granules

Summary As previously reported, in anterior pituitary cells of the rat, secretory granules are linked with adjacent granules, cytoorganelles, microtubules, and plasma membrane by thin filaments, 4–10 nm in diameter. The quick-freeze, deep-etching method revealed that some of the filaments linking adjacent secretory granules show 5 nm-spaced striations on their surface which are known to be characteristic of actin. Immunocytochemistry showed that actin is localized in the cytoplasm beneath the plasma membrane, and around or between secretory granules. The heavy meromyosin decoration method demonstrated that actin filaments are mainly located in the cytoplasm beneath the plasma membrane, while some actin filaments are connected with the limiting membrane of the secretory granules. The actin filaments associated with the secretory granules are considered to be involved in the intracellular transport of the granules, while those localized in the peripheral cytoplasmic matrix might control the approach of the secretory granules to the plasma membrane and their release.This study was supported in part by grants from the Research Fund of the Ministry of Education, Science, and Culture, Japan     

Ultrastructural observation of paracrystalline aggregates of microtubules in anterior pituitary cells of the chinchilla (Chinchilla laniger)

Summary Unusual paracrystalline aggregates of microtubules which have not been described in any other mammalian species were observed in cultured anterior pituitary cells of normal chinchillas as well as in situ in the pituitary glands of these animals. These aggregates appeared as regularly arranged tubular structures in the longitudinal plane, and as a checkerboard pattern of closely and regularly packed microtubules when examined in transverse section. Supplementation with vinblastine, colcemide or colchicine in the culture medium did not change these structures morphologically. Each unit of tubules consisted of an outer wall or parellelogram profile and an inner wall composed of a single hexagonal doublet or in a figure 8 form. The outer wall of the parallelogram was 35×28 nm in length for both sides, while the diagonal of the inner wall was 18×28 nm. These paracrystalline aggregates of microtubules in the chinchilla pituitary cells are morphologically distinct from the paracrystalline assembly of cytoplasmic microtubules induced by vinblastine or other alkaloids.The function and significance of these paracrystalline aggregates in anterior pituitary cells of the chinchilla are uncertain.Supported by USPHS Grant HD 11826     

Mutagenicity of bisphenol A and its suppression by interferon-α in human RSa cells

Bisphenol A is used as a monomer in the production of polycarbonate plastic products. The widespread use of bisphenol A has raised concerns about its effects in humans. Since there is little information on the mutagenic potential of the chemical, the mutagenicity of bisphenol A was tested using human RSa cells, which has been utilized for identification of novel mutagens. In genomic DNA from cells treated with bisphenol A at concentrations ranging from 1×10⁻⁷ to 1×10⁻⁵ M, base substitution mutations at K-ras codon 12 were detected using PCR and differential dot-blot hybridization with mutant probes. Mutations were also detected using the method of peptide nucleic acid (PNA)-mediated PCR clamping. The latter method enabled us to detect the mutation in bisphenol A-treated cells at a dose (1×10⁻⁸ M) equivalent to that typically found in the environment. Induction of ouabain-resistant (Oua^R) phenotypic mutation was also found in cells treated with 1×10⁻⁷ and 1×10⁻⁵ M of bisphenol A. The induction of K-ras codon 12 mutations and Oua^R mutations was suppressed by pretreating RSa cells with human interferon (HuIFN)-α prior to bisphenol A treatment. The cells treated with bisphenol A at the concentration of 1×10⁻⁶ M elicited unscheduled DNA synthesis (UDS). These findings suggested that bisphenol A has mutagenicity in RSa cells as well as mutagens that have been tested in these cells, and furthermore, that a combination of the PNA-mediated PCR clamping method with the human RSa cell line may be used as an assay system for screening the mutagenic chemicals at very low doses.     

Functional differentiation of the anterior pituitary cells in the fetal pig

Summary The fetal porcine pituitary was investigated by means of ultrastructural immunocytochemistry (1) to identify the first cells synthesizing the adenohypophyseal hormones, (2) to follow their differentiation during fetal development, and (3) to compare their ultrastructural characteristics with those of mature adult cells.The first ACTH-cells, which produced and stored ACTH, -LPH, -MSH, and - and -endorphin in the same granules, were very numerous at day 34 and displayed a uniform morphology. At day 50 and thereafter, until the end of gestation, the ACTH-cells differed in their appearance probably reflecting various stages of differentiation of one cell type. The GH-cells gained rapidly ultrastructural features comparable to those of mature GH-cells. In contrast, in the case of PRL-cells, which appeared only at the end of the gestation period as immature elements containing very small secretory granules, the morphological maturation seemed to take place only after birth. The first cells synthesizing the glycoprotein hormones (LH, LH, FSH and TSH) displayed ultrastructural features of immature cells. At day 50, their ultrastructural organization started to show a different pattern. At the end of gestation, the TSH-cells and the gonadotropic cells displayed the ultrastructural features of mature cells.     

Ultrastructural localization of thyrotropin (TSH) in the porcine anterior pituitary

Summary Two different immunocytochemical techniques based on specific antibodies against -subunits of porcine, rat and bovine TSH were applied at the ultrastructural level to identify the TSH cells in the porcine anterior pituitary and to compare the subcellular localization of the hormone.The post-embedding method on serial ultrathin sections revealed the localization of TSH in the granules of a specific cell type, negative for the other hormones. TSH was found in polyhedral cells characterized (i) by their content of granules that were the smallest of all the cell types examined, and (ii) by their flattened or slightly dilated RER cisternae. The pre-embedding method applied to isolated cells permitted a good penetration of antisera and the maintenance of antigenicity in sites inaccessible to the post-embedding method. Thus, immunoreactivity of TSH was detected in the secretory granules, the cytoplasmic matrix and in portions of the rough endoplasmic reticulum, in association with some membranes and inside some saccular structures.     

Cytochemical localization of adenylate cyclase activity in the rat anterior pituitary

Summary The cytochemical localization of adenylate cyclase was studied in relation to the secretory function of the anterior pituitary glands of male rats. The reaction product of adenylate cyclase was localized on the outside of plasma membranes, but was not detected intracellularly. High activity of adenylate cyclase was detected on somatotrophs and microvilli of follicular cells, whereas no activity was found on thyrotrophs or corticotrophs. Although most of the gonadotrophs showed little or no adenylate-cyclase activity, some was detected in a small number of gonadotrophs in the central portion of the gland. In somatotrophs, activity was not detected on the plasma membranes facing perivascular spaces where exocytotic extrusion of secretory granules was frequently observed, although the remaining areas of plasma membranes of the same somatotrophs were associated with high levels of adenylate-cyclase activity. These findings indicate that the association of a high level of adenylate-cyclase activity is not directly related to the ability of the plasma membranes to fuse with secretory granule membranes.     

Adenosine regulates the release of adrenocorticotropic hormone (ACTH) from cultured anterior pituitary cells

We have recently shown the presence of adenosine receptors coupled to adenylate cyclase in anterior pituitary and in the present studies we have investigated the effects of adenosine on ACTH release. The R-site specific analogs of adenosine such as N-Ethylcarboxamide adenosine (NECA), L-N⁶-phenylisopropyl adenosine (PIA), 2-chloro-adenosine (2-Cl-Ado) all stimulated ACTH release in a dose-dependent manner. NECA was the most potent analog and stimulated ACTH release by about 170% with an apparent Ka of 0.1 µM, whereas PIA and 2-Cl-Ado were less potent and stimulated the release by about 110% and 125% with an apparent Ka of 0.2 and 0.4 µ-M respectively. The stimulation of ACTH release by NECA was inhibited by 3-isobutyl-1-methylxanthine (IBMX). On the other hand, adenosine deaminase (ADA) treatment of the cells also stimulated ACTH release as well as adenylate cyclase activity by about 2-fold, suggesting that endogenous adenosine plays an inhibitory role in the release of ACTH. Other agents, such as corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP) and forskolin (FSK) also stimulated ACTH release from these cells. In addition, the stimulation by an optimal concentration of NECA was almost additive with maximal stimulation caused by VIP and FSK. These data suggest that adenosine modulates ACTH release from anterior pituitary through its interaction with adenosine receptors coupled to adenylate cyclase.Abbreviations NECA N-Ethylcarboxamideadenosine - PIA L-N⁶-Phenylisopropyladenosine - 2-Cl-Ado 2-chloroadenosine - FSK Forskolin - VIP Vasoactive Intestinal Peptide - CRF Corticotropin Releasing Factor - ADA Adenosine Deaminase - IBMX 3-Isobutyl-1-methylxanthine