Claude Tougard

Immunocytochemical localization of glycoprotein hormones in the rat anterior pituitary. A light and electron microscope study using antisera against rat bet subunits: a comparison between preembedding and postembeeding methods.

The binding sites of antisera (anti) to the beta (beta) subunits of rat follicle-stimulating hormone (rFSH), rat luteinizing hormone (rLH), and rat thyroid-stimulating hormone (rTSH) have been localized in rat anterior pituitaries by immunocytochemistry using light and electron microscopy. With the light microscope, LHbeta and FSHbeta were found in the same cells, which were violet after the alcian blue-periodic acid Schiff (AB-PAS) staining. TSHbeta was found in polygonal or stellate cells that were blue after AB-PAS. With the electron microscope, the thyrotropic cells contained very small secretory granules. LHbeta and FSHbeta were found in various types of cells (types A and B and their intermediate forms), which had previously been identified as gonadotropic cells. On serial ultrathin sections using the postembedding method the same cells and even some granules inside these cells were stained by both anti-rLHbeta and anti-rFSHbeta. A comparison of binding sites of anti-rLHbeta was performed using the preembeeding and the postembeeding methods. Antigenicity was observed on secretory granules whatever the method used. However, binding sites of anti-rLHbeta were detected inside the cisternae of the rough endoplasmic reticulum only with the preembedding method.

A cell culture approach to the study of anterior pituitary cells.

Publisher Summary This chapter discusses a cell culture approach to study the anterior pituitary cells. Pituitary cells maintained in vitro for months or years differ from pituitary organ cultures in which tissue organization is maintained, and from tissue culture starting from small fragments of tissue which grow and can display selection in some cell populations. Among cell cultures one must distinguish (1) primary cultures that start from normal adult fully differentiated anterior pituitary cells, and (2) continuous cell lines that consist of homogeneous populations of pituitary glandular cells which continuously grow in vitro. The chapter discusses the functional and morphological features of pituitary cells grown in these two situations. The effects on these models of factors that regulate the secretion of anterior pituitary hormones are analyzed and discussed. Anterior pituitary continuous cell lines were found to be a useful model for the analysis of mechanisms of action of agents that quantitatively regulate their hormonal secretion. In several cases they appear to be very close to the in vivo situation, e.g., control of prolactin (PRL) secretion by TSH-releasing hormone (TRH) and estrogens, control of growth hormone secretion by thyroid hormone, and control of adrenocorticotropic hormone (ACTH) secretion by glucocorticoids. Because of their cellular homogeneity, they have the advantage in regard to primary culture of allowing investigations at the molecular level, which are undergoing rapid progress.

Cytogenesis of immunoreactive gonadotropic cells in the fetal rat pituitary at light and electron microscope levels

Abstract The cytogenesis of immunoreactive gonadotropic cells in the fetal rat pituitary was analyzed at the light and electron microscope levels using the indirect peroxidase-labeled antibody method and antisera against ovine FSH (A-oFSH) and ovine LH (A-oLH), and its two subunits (A-oLHβ and A-oLHα). At the light microscope level, the first immunoreactive cells were detected on the seventeenth day postcopulation (dpc) with A-oLHβ. Cells immunochemically stained with A-oLHα and A-oLH were generally observed 24 hr later. At the electron microscope level, the first immunoreactive cells were detected on 16 dpc with A-oLHβ. These first immunoreactive cells were small, but already displayed some small secretory granules (80–120 nm). On 17 dpc, gonadotropic cells were stained with A-oLHβ as well as with A-oLHα and A-oFSH. On 18 dpc, the number and the size of immunoreactive cells began to increase. By 19 dpc, they displayed an important development of ergastoplasmic cisternae and Golgi zone. At term, nevertheless, the ultrastructural features of fetal gonadotropic cells still differed from those of adult gonadotropic cells.

Subcellular Distribution of Secretogranins I and II in GH3 Rat Tumoral Prolactin (PRL) Cells as Revealed by Electron Microscopic Immunocytochemistry

The GH3 rat pituitary cell line which secretes prolactin (PRL) is characterized by the paucity and small size of secretory granules. We looked for the presence, in these cells and in normal PRL cells, of two acidic tyrosine-sulfated proteins which are widely distributed in dense-core secretory granules of endocrine and neuronal cells, secretogranins I and II, using immunofluorescence and electron microscope immunoperoxidase techniques. Both secretogranins were detected in secretory granules of GH3 cells and of normal cells. Moreover, with our pre-embedding approach, secretogranins were localized within some RER cisternae and within all sacules of the Golgi stacks in both PRL cell models. A few small vesicles, large dilated vacuolar or multivesicular structures, and some lysosome-like structures were also immunoreactive. Double localization of secretogranins and PRL performed on GH3 cells by immunofluorescence indicated that all cells contained secretogranins I and II, whereas only 50-70% of the cells contained PRL. Moreover, in the case of hormone treatment known to increase the number of secretory granules, most if not all mature secretory granules were immunoreactive for secretogranins, whereas in certain cells some of the granules were apparently not immunoreactive for PRL. These immunocytochemical observations show that GH3 cells, which under normal conditions form only a small number of secretory granules, produce secretogranins and package them into these granules.

Secretion of Protease Nexin-1 by C6 Glioma Cells Is under the Control of a Heterotrimeric G Protein, Go1

Heterotrimeric Go proteins have recently been described as regulators of vesicular traffic. The Goα gene encodes, by alternative splicing, two Goα polypeptides, Go1α and Go2α. By immunofluorescence and electron microscopy, we detected Go1α on the membrane of small intracellular vesicles in C6 glioma cells. After stable transfection of these cells, overexpression of Go1α but not Go2α was followed by a rise in the secretion of a serine protease inhibitor, protease nexin-1 (PN-1). This secretion was enhanced as a function of the amount of expressed Go1α. Metabolic cell labeling indicated that this increase in PN-1 secretion was not the result of an enhancement in PN-1 biosynthesis or a decrease in its uptake, but revealed a potential role of Go1α in the regulation of vesicular PN-1 trafficking. Furthermore, activators of Go proteins, mastoparan and a peptide derived from the amino terminus of the growth cone-associated protein GAP43, increased PN-1 secretion in parental and Go1α-overexpressing cells. Brefeldin A, an inhibitor of vesicular traffic, inhibited both basal and mastoparan-stimulated PN-1 secretions. These results indicate, that in C6 glioma cells, PN-1 secretion could be regulated by both Go1α expression and activation.

Heterogeneity in the pattern of distribution of the specific hormonal product and secretogranins within the secretory granules of rat prolactin cells.

We investigated the subcellular distribution of secretogranins I, II (Sg I, Sg II), and prolactin (PRL) by double immunogold electron microscopy in GH3B6 rat pituitary tumor cells grown in different culture conditions and in normal PRL cells in adult male rat anterior pituitary. Co-localization of Sg I or Sg II with PRL was observed in most secretory granules in GH3B6 cells and normal PRL cells, except for some secretory granules containing only Sgs in GH3B6 cells and containing only PRL in normal PRL cells. In GH3B6 cells treated with thyrotropin-releasing hormone (TRH) for 2 hr, the newly formed small secretory granules within the Golgi zone contained preferentially immunoreactive PRL. Interestingly, when co-localized with PRL, Sgs (particularly Sg I) were observed at the periphery of the matrix of secretory granules in GH3B6 cells as well as in normal PRL cells, suggesting their possible interaction with the secretory granule membrane. The present study indicates a heterogeneous subcellular distribution of PRL, Sg I, and Sg II in individual secretory granules of GH3B6 cells and of normal PRL cells, pointing out the formation of different types of aggregates during the condensation of secretory products in these two PRL cell models.

Identification of Multiple Subunits of Heterotrimeric G Proteins on the Membrane of Secretory Granules in Rat Prolactin Anterior Pituitary Cells

The subcellular distribution of multiple subunits of heterotrimeric GTP-binding proteins has been investigated in rat anterior pituitary cells in primary culture, and more precisely in prolactin cells, by immunocytochemistry and subcellular fractionation followed by immunoblotting or ADP ribosylation, using polyclonal affinity-purified antibodies directed against Gi3 alpha, Gs alpha, Go1 alpha, Go2 alpha, and G beta. As expected, all these subunits were detected on the plasma membrane. They were, however, also detected on the membrane of several intracellular compartments involved in the secretory pathway, particularly on the secretory granule membrane. Differences appeared between the precise subcellular distribution and the local concentration of each subunit. The main subunits present on the secretory granule membrane were Gi3 alpha and Gs alpha. Go1 alpha, Go2 alpha, and G beta were detected, to a lesser extent, on parts of the membrane of a few secretory granules located near the plasma membrane. Domains of the rough endoplasmic reticulum cisternae were immunolabeled with anti-Gs alpha and anti-Go1 alpha. In the Golgi zone, the membrane of some vesicles was stained only with anti-Gs alpha and anti-Go2 alpha. The association of this set of heterotrimeric G protein subunits on the membrane of the secretory granules suggests that these subunits could be involved in the regulation of formation, storage, targeting, and/or exocytosis of these organelles.

Proteolytic Processing of Sulfated Secretogranin II in the trans-Golgi Network of GH3B6 Prolactin Cells

Secretogranin II (SgII) is a protein specific to the matrix of the secretory granules in neurons and neuroendocrine cells. We have already demonstrated the precursor-product relationship between sulfated SgII and four N-terminal derived peptides in GH3B6 prolactin cells. In this study, we have investigated the subcellular compartment in which the cleavage of SgII is initiated by taking advantage of its tyrosine sulfation in the trans-Golgi network (TGN). In order to prevent export of radiosulfated SgII from the TGN, we used brefeldin A (BFA) as well as incubation at 20°C. BFA completely inhibited the cleavage of SgII when added immediately post-pulse. BFA added a few minutes post-pulse or after a 20°C incubation, however, permitted the cleavage of SgII in the presence of the drug. These SgII-derived peptides generated in the presence of BFA could not be released upon stimulation of the cells by either thyroliberin, a physiological secretagogue, or KCl. These results demonstrate that SgII can be cleaved in the TGN. They also evidence that the cleavage occurs in a distal compartment of the TGN different from the sulfation site. The transfer of SgII from the sulfation site to this distal compartment of the TGN involves BFA-sensitive membrane dynamics.

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

SummaryLaminin (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.

PRODUCTION AND SECRETION OF N-TERMINAL SECRETOGRANIN II DERIVED PEPTIDES IN GH3B6 PROLACTIN CELLS

Chromo/secreto-granins are proteins specific of neuroendocrine cells. Chromogranin B (CgB) and secretogranin II (SgII) are both present in normal and in tumoral (GH3B6) prolactin cells, in which they are colocalized in the same secretory granules. These proteins contain multiple dibasic cleavage sites and are considered as potential precursors of active peptides, though their exact function remains unknown. SgII is sulfated on tyrosine-126. We took advantage of this feature to study its post-translational processing in anterior pituitary cells in primary culture and in GH3B6 cells. Pulse-chase experiments with [35S]sulfate demonstrated the precursor-product relationship between SgII and four N-terminal-derived peptides. Kinetic experiments showed the sequential cleavage of SgII from the C-terminus to the N-terminus. Mature SgII and the derived peptides were secreted by both cell models, and their release was stimulated by TRH (30 nM), a secretagogue of prolactin. These data show that SgII is proteolytically processed into different secreted peptides in prolactin cells and demonstrate that GH3B6 cells provide a good cell model for studying the maturation of SgII in anterior pituitary.