Changes of Cell Morphology and Prolactin Secretion Induced by 2-Br-«-Ergocryptine, Estradiol, and Thyrotropin-releasing Hormone in Rat Anterior Pituitary Cells in Culture Downloaded from http://rupress.org/jcb/article-pdf/86/2/377/1074283/377.pdf by guest on 26 September 2021 TONY ANTAKLY, GEORGES PELLETIER, FUSUN ZEYTINOGLU, and FERNAND LABRIE Medical Research Council Group in Molecular Endocrinology, Le Centre Hospitalier de l'Université Laval, Québec G1V 4G2, Canada . Dr. Antakly's present address is the Institute of Cancer Research, College of Physicians and Surgeons of Columbia University, New York, New York 10032. Dr. Zeytinoglu's present address is the Department of Toxicology, School of Public Health, Harvard University, Boston, Massachusetts 02115. ABSTRACT The secretion of prolactin in cultured pituitary cells was studied in correlation with the cellular changes induced by stimulatory or inhibitory agents . The techniques used in this study were: radioimmunoassay, immunocytochemistry, scanning (SEM) as well as transmission (TEM) electron microscopy. Prolactin secretion was stimulated by 17,8-estradiol (10 nM) as well as thyrotropin-releasing hormone (TRH) (3 nM) and inhibted by 2-Br-a-ergocryptine (CB-154) (1 ftM) . The total prolactin (release and cell content) increased between 2 and 8 d of estradiol treatment, indicating an increase of both synthesis and release of prolactin . This finding was in agreement with TEM observations because, in estradiol-treated prolactin cells, the Golgi saccules were distended and Golgi elements were increased, thus indicating increased synthetic activity of these cells. The addition of TRH over a 4-h period resulted in a significant degranulation of prolactin cells. In contrast, prolactin secretory granules became accumulated in the cells after CB-154 treatment for a period ranging from 4 to 24 h. In agreement, light microscope immunocytochemistry showed an increased reaction for prolactin after short-term (<24 h) incubation with CB-154. Because prolactin cells represent --70% of the glandular cell population as revealed by immunocytochemistry, it was then possible to observe the changes of cell surface by SEM. In most cells, estradiol and TRH led to an increase in the number and prominence of microvilli and blebs, whereas CB-154 treatment resulted in a slightly decreased number of microvilli and an increased occurrence of membrane foldings. This report thus provides morphological evidence for the stimulatory effects of estradiol and TRH, and the inhibitory effects of CB-154 on prolactin secretion in pituitary cells in primary culture. These data, moreover, show that acute changes in secretory activity of prolactin-secreting cells are accompanied by marked changes of their morphological characteristics. The secretion of prolactin in the anterior pituitary gland is ergot derivatives are potent inhibitors of prolactin secretion in under predominant inhibitory control by the hypothalamus vivo as well as in vitro (16, 26, 31, 33) . (30) . Recent data indicate that dopamine may be the main or The stimulatory influence of the hypothalamus can, at least even the only inhibitory substance of hypothalamic origin partly, be mediated by thyrotropin-releasing hormone (TRH). involved in the control of prolactin secretion (11, 29, 37) . This This tripeptide is known to stimulate prolactin secretion in role ofdopamine is also supported by the finding that 2-bromo- vivo (7, 14) and in vitro (15, 38) . Prolactin secretion is also well a-ergocryptine (CB-154), a potent dopamine agonist, and other known to be increased after estrogen treatment in man (10, 39) THE JOURNAL of CELL i31OLOGV " VOLUME 86 AueusT 1980 377-387 © The Rockefeller University Press " 0021-9525/80/08/0377/11 $1 .00 377 and the rat (13). Recently, estrogens have been found not only Na2CO3 with 2 mM EDTA was added to the Petri dishes. Subsequently, the Petri prolactin secretion but also to reverse the inhibitory dishes were rinsed with 1 ml of acidified DMEM, and the pooled suspensions to stimulate were frozen, thawed, centrifuged at 100 g for 7 min at 4°C, and the supernate effects of dopamine agonists (including CB-154) by a direct was stored at -20°C until assayed. action at the pituitary level (26, 34). Pituitary cells in primary culture (25, 38) offer an attractive Hormone Assays system for the study of cells by scanning electron microscopy and permit a precise correlation with changes of pituitary Prolactin was measured in duplicate by a double-antibody radioimmunoassay (15) using rat prolactin I-2 and rabbit antisera (anti-rat prolactin-S-3) kindly hormone secretion induced by various stimulatory or inhibitory supplied by Dr. A. F. Parlow fortheNational Instituteof Arthritis and Metabolic agents. In preliminary reports (1, 2), we have recently described Diseases, Rat Pituitary Hormone Program. Radioimmunoassay data were ana- a good correlation between the surface morphology of anterior lyzed with a Hewlett-Packard calculator (model 9830, Hewlett-Packard Co ., Palo pituitary cells in primary culture and prolactin and thyroid- Alto, Calif.) using a program based on model II of Rodbard and Lewald (35). The statistical significance was determined according to the multiple-range test stimulating hormone (TSH) secretion after short- and long- of Duncan-Kramer (24) . Alldata are presented as the means t SEM of duplicate term treatment with estrogens, CB-154, triiodothyronine, TRH, measurements of triplicate dishes. and colchicine (3). In the present report, we have used immu- nocytochemical localization of prolactin, scanning and trans- Immunocytochemistry mission electron microscopy, and radioimmunoassay data for each experimental condition, to describe the effects and inter- Petri dishes were rinsed four times with Tris-buffered saline (Tris, 0.05 M, actions of l7ß-estradiol (E2), TRH, and CB-154 in this system NaCl 0.9%, pH 7.6) and fixed in situ with Bouin's fixative for 20 h at 4°C or with 4% buffered paraformaldehyde as previously described (4). Cells were subse- at the cellular level. quently processed for the immunocytochemical localization of prolactin by the Downloaded from http://rupress.org/jcb/article-pdf/86/2/377/1074283/377.pdf by guest on 26 September 2021 unlabeled antibody method and the peroxidase-antiperoxidase complex tech- MATERIALS AND METHODS nique (PAP) according to Stemberger (36) and as previously described (4). The dilution of the rabbit anti-prolactin serum (same lot as that used in the hormone Materials assays) was 1/1,000-1/2,000. Diaminobenzidine (Sigma Chemical Co ., St . Louis, Mo.) (22) was used as hydrogen donor for the localization of peroxidase. The CB-154 was kindly provided by Sandoz Pharmaceuticals (East Hanover, N.J .) specificity of the immunocytochemical reaction was tested by incubating the cells whereas TRH and Ez were purchased from Peninsula Laboratories Inc. (San either with antisera previously absorbed with excess antigen or with sera from Carlos, Calif.) and Steraloids, Inc. (Pawling, N.Y .), respectively . Media and sera unimmunized rabbits. Finally, to eliminate the possibility that endogenous per- for tissue cultures were obtained from Grand Island Biological Co. (Grand oxidases might be participating in the reaction, cells from each experimental Island, N.Y .), and glutaraldehyde (special grade) was a product of Ladd Research group were simply processed directly for the localization of peroxidase without Industries, Inc. (Burlington, Vt.). incubation with the antisera. The staining of the nuclei in the same Petri dish was done by indirect Preparation of Dispersed Anterior Pituitary Cells immunotluorescence procedure using sera from patients containing antinuclear antibodies (17) (kindly provided by the Department of Immunology of Le Centre Adult female Sprague-Dawley rats weighing 200-400 g (obtained from Ca- Hospitalier de l'Universit6 de Laval) . Observations were performed with a Leitz nadian Breeding Farms, St . Constant, Quebec) at random stages of the estrous microscope suitable for fluorescence and phase-contrast microscopy . A given cycle were used for the preparation of primary cultures of anterior pituitary cells field was first observed for fluorescence using a mercury lamp source; the as previously described (25, 38). The dissociated cells (8.5-9.0 x 10) were plated fluorescing nuclei were counted (Fig. 3). Then, the mercury lamp source was in Coming Glass Works, Science Products Div. (Corning, N.Y.), polystyrene shut off using the built-in shutter, and a usual tungsten lamp light source was Petri dishes (35 x 10 mm) in 1.5 ml of Dulbecco's modified Eagle's medium used and the number of positive cells, stained dark brown by the peroxidase (DMEM) containing 2.5% fetal calf serum and 10% horse serum (dextran-coated localization reaction, wascounted. In some cases, the same field was also observed charcoal adsorbed, 25), nonessential amino acids, 50 U/ml penicillin, and 50 pg/ by phase-contrast microscopy . A total of -800 cells was counted in each Petri ml streptomycin . Cell counts were done (using a hemocytometer) during plating dish in not less than three Petri dishes for each group in most cases. to ensure that all the Petri dishes in an experiment were seeded with the same number of cells. The cells were maintained at 37°C in a water-saturated atmos- phere of 95% OZ and 5% C02 for up to 12 d. Scanning Electron Microscopy (SEM) Petri dishes that were removed from the 37°C incubator were quickly rinsed Determination of Cell Viability three times with 0.1 M cacodylate buffer containing 5% sucrose, pH 7.4, pre- warmed at 37°C before fixation in situ for l h at 37°C with 0.15 M Na-cacodylate For a cell culture to be representative of the whole population of a tissue, cell buffer containing 1% glutaraldehyde (390 mosmol, pH 7.4) also prewarmed at viability after dissociation must be high enough so that no major fraction of the 37°C . Petri dishes were subsequently rinsed with thecacodylate buffer, postfixed cells is excluded . The percent of viable cells in the dispersed cell suspension was with 1% OS04 in 0.1 M Na-cacodylate (pH 7.4), and then dehydrated with determined by the trypan blue method .