Effects of Colchicine on the Morphology and Prolactin Secretion of Rat Anterior Pituitary Cells in Monolayer Culture TONY ANTAKLY,' GEORGES F E R N A N D LABRIE
PELLETIER,' F U S U N ZEYTINOGLU AND
Medical Research Council Group in Molecular Endocrinology, le Centre Hospitalier d e I'Uniuersite Laual, Quebec GlV4G2, Canada
ABSTRACT The effects of incubation of rat anterior pituitary cells in M colchicine have been investigated during timemonolayer culture with intervals extending from 1 to 96 hours. Prolactin release, as measured by radioimmunoassay, was rapidly inhibited by colchicine, this inhibition being accompanied by increased cellular prolactin content for up to 24 hours of treatment and followed by decreased values of cellular prolactin concentration a t later time-intervals. Immunocytochemical localization showed an increased positive reaction for prolactin up to 24 hours after colchicine treatment, whereas transmission electron microscopy demonstrated, in parallel, an increased number of intracellular prolactin secretory granules during the same interval. Longer periods of treatment (24-96 hours) resulted in the appearance of more lysosomes, autophagic vacuoles and microfilaments in the cells, whereas the number of Golgi elements was decreased. Following four hours of colchicine treatment and a t later stages, microtubules could no longer be observed in the sections. Scanning electron microscopic data showed that colchicine treatment induced dramatic changes in the cell surface morphology: a t short time intervals (4 and 8 hours), the number of microvilli decreased and the cell surface became folded, whereas, later, "b1eb"-like protrusions of variable dimensions partially covered the cell surface and seemed to be released from it. These data show a good correlation between secretory activity of prolactin-producing cells and morphological changes induced by colchicine treatment. Colchicine, a drug which binds to tubulin, has been reported to interfere with the secretory activity of various endocrine and exocrine cells. For example, colchicine has been shown to inhibit the release of insulin from beta cells of the pancreas (Lacy et al., '68; Malaisse et al., '731, I'31 from the thyroid gland (Williams and Wolff, '701, catecholamines from adrenal medulla (Poisner and Bernstein, '711, very low density lipids (VLDL) and albumin from liver (Redman et al., '75) and histamine from mast cells (Gillespie et al., '68). We have also reported that vincristine inhibits prolactin and growth hormone secretion from rat hemi-pituitaries (Labrie e t al., '73a). The mechanisms responsible for the inhibitory action of colchicine on secretory processes are still largely unknown. Ultrastructural observations often associate colchicine treatAM. J. ANAT. (1979)156: 353-372.
ment with disappearance of microtubules (Pelletier and Bornstein, '71; Wolff and Bhattacharya, '75) and modifications of organelles involved in secretion (Moskalewski et al., '76). An interesting observation is that colchicine treatment leads to a n accumulation of secretory granules and newly synthesized proteins (Malaisse et al., '73; Pelletier and Bornstein, '71; Redman et al., '75, '78). Immunocytochemical studies have shown that fibrinogen synthesized in the liver accumulates in the endoplasmic reticulum, Golgi apparatus and vacuoles of hepatocytes after colchicine injecReceived Jan. 3, '79. Accepted July 27, '79. ' Present address: Institute of Cancer Research, College of Physicians and Surgeons of Columbia University, 701 West 168 St., New York, New York 10032. 'To whom requests should be sent. Present address: Laboratory of Toxicology, Harvard University, School of Public Health, Boston, Massaehueetts.
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ANTAKLY, PELLETIER, ZEXTINOGLU AND LABRIE
tion (Feldmann et al., '75). Colchicine also affects cell-membrane fluidity (Knutton et al., '75) and causes dramatic changes in cellmembrane morphology (Shay and Clark, '77; Furcht et al., '76). To our knowledge, no studies have attempted to establish a correlation between changes in secretion and cell-surface morphology after colchicine treatment. Moreover, the effects of colchicine on anterior pituitary cells in monolayer culture have not yet been investigated. In previous studies (Antakly et al., '78, '791, we have shown a good correlation between surface morphology and secretory activity of anterior pituitary cells in primary culture. In the present report, we have used the same system to investigate the modifications induced by colchicine on the secretion, ultrastructure, surface morphology and immunocytochemical properties of anterior pituitary cells. MATERIALS AND METHODS
Materials Except as otherwise indicated, chemicals were purchased from Fisher Scientific Company. Colchicine was obtained from Sigma (St. Louis, Missouri) whereas the media for cultures were from Grand Island Biologicals Co., Grand Island, New York. Glutaraldehyde (special grade) was purchased from Ladd. Preparation of dispersed anterior pituitary cells Adult female Sprague-Dawley rats a t random stages of the estrous cycle, and weighing between 200 and 400 g (purchased from Canadian Breeding Farms, St. Constant, Quebec), were used for preparation of primary cultures of anterior pituitary cells as described previously (Labrie et al., '73b). Briefly, the anterior pituitary glands (30-50 per experiment) were cut into small fragments and digested a t room temperature either under constant mechanical agitation or in a rotary agitator, in 10 ml of 0.1% hyaluronidase (Sigma), 0.35% collagenase (Worthington) and 3% bovine serum albumin (BSA) in HEPES buffer (25 mM HEPES, 137 mM NaCl, 5 m M KCl, 0.7 mM Na2HP0,, 10 mM glucose, pH 7.2). Digestion was continued for approximately 90 minutes, or until the suspension became finely granular and opaque. This suspension was then centrifuged for 7 minutes a t 50 x g a t room temperature and the pellet was resuspended in 10 ml of 0.25% Viokase in HEPES buffer before continuing digestion (25-40 min)
until the tissue became dispersed into predominantly single cells. The cells were then washed by centrifugation through a layer of 4% BSA in HEPES buffer. The cells were further washed 4 times in HEPES buffer and plated in Corning polystyrene Petri dishes (35 x 10 mm) in 1.5 ml of Dulbecco's Modified Eagle's Medium (DMEM) containing 2.5% fetal calf serum and 10% horse serum (adsorbed with dextran-coated charcoal), non-essential amino acids, penicillin (50 U,'ml) and streptomycin (50pg/ml). Each Petri dish was seeded with 8.5 x lo5 cells. The cells were maintained at 37°C in a saturated atmosphere of 95% O 2 -5% CO, up to 6 days in culture. Incubation procedure with colchicine In time-course experiments, the cells were M coltreated a t 12-hour intervals with chicine for 24, 48 or 72 hours. In other experiments, the cells were treated once with M colchicine for 4, 8 or 16 hours. The culture media were substituted by fresh medium 4 hours before the end of each experiment, and prolactin release was measured only in the last 4 hrs of incubation. After incubation, the cells were washed 4 times with sterile serumfree DMEM and centrifuged at 100 x g for 7 minutes at 4"C, while the culture medium was stored a t -20°C until assayed. At t h e time of tissue fixation or prolactin assay, all cells had been in culture for the same time (6 days). In order to determine t h e prolactin content, pituitary cells were incubated overnight a t 4°C with 1ml of 50 mM Na,CO,, 2 mM EDTA (pH 8.3) to induce cell lysis. Subsequently, t h e Petri dishes were rinsed with 1ml acidified DMEM and the pooled suspensions (pH 7.0) were frozen, thawed, and centrifuged at 1,000 x g for 7 minutes a t 4°C. The supernatant was stored a t -20°C until assayed. Transmission (TEM) and scanning &EM) electron microscopy For TEM, cells were washed 3 times in 0.15 M sodium-cacodylate buffer, pH 7.4, and fixed at room temperature for 1hour in 0.15 M sodium-cacodylate buffer, pH 7.4, containing 1%glutaraldehyde. After fixation, the cells were gently scraped off the bottom of the Petri dishes with a rubber policeman and the suspension was centrifuged into a pellet. The fixative was then removed and the pellets were rinsed overnight in 0.131 sodium-cacodylate buffer, pH 7.4, containing 5% sucrose (rinse buffer). The pellets were divided into
COLCHICINE AND CULT13RED PITUITARY CELLS
two groups: the first group was post-fixed in 1%OsO, in 0.1 M Na cacodylate, pH 7.4, for 1 hour, rinsed in distilled water and stained “en bloc” with 1%uranyl acetate, whereas the second group was not post-fixed. Both groups were dehydrated conventionally and embedded in Araldite, and ultrathin sections were cut using a Reichert ultramicrotome. Some sections were stained with lead citrate. Observations were performed with a Siemens Elmiskop 102 electron microscope operated a t 60 kv. For SEM, the cells were rinsed 3 times in rinse buffer pre-warmed to 37OC, before fixation in situ a t 37OC for 1hour in 1%glutaraldehyde in 0.15 M sodium-cacodylatebuffer, pH 7.4 (390 mosm/liter), and storage overnight in rinse buffer a t 4°C. Cells were subsequently post-fixed in 1%Oso, in 0.1 M cacodylate buffer (pH 7.4) for 1hour and dehydrated with increasing concentrations of ethanol before critical-point-drying in a Samdri PVT3 apparatus using liquid COP They were finally coated with approximately 20 nm of gold-palladium alloy in a Hummer I1 sputter-coater (Technics Co.). Observations were made with the use of an ETEC Autoscan U1 scanning electron microscope at 30 kv, accelerating voltage, and sometimes a JEOL F-15 scanning microscope fitted with a cold-field emission gun. To eliminate the possibility of biased observations, samples were, in most cases, coded and the observer was not aware of treatments.
Immunocytochemistry and cytochemistry Cells were rinsed 3 times in 0.05 M Tris-HC1 buffer, pH 7.6, containing 0.9% NaCl (Trisbuffered saline) and fixed in Petri dishes with Bouin’s fixative for a period of 20 hours. Following fixation, Petri dishes were rinsed 4 times with Tris-buffered saline. Immunocytochemical reactions were performed with the unlabeled antibody method of Sternberger (‘74). Antisera to r a t FSH, LH, TSH, prolactin and GH, supplied by Dr. A. F. Parlow for the National Institute of Arthritis, Metabolic and Digestive Diseases (NIAMDD), Rat Pituitary Hormone Program, and anti-sera to porcine ACTH (Pelletier et al., ’77) were used a t a dilution of 1:1,000to 1:3,000.These antisera were from the same lot as those used for the radioimmunoassays. Although antibodies used were shown to be specific by radioimmunoassays, control reactions were also performed to test the specificity of the immunocytochemical reactions in two ways: absorp-
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tion of the antibodies with excess antigens prior to the incubation, or substitution of the specific antiserum with serum obtained from non-immunized rabbits. The specificity of t h e cytochemical reaction of peroxidase was checked by omitting H 2 0 2or the hydrogen donor (diaminobenzidine)from the incubation mixture. The relative intensity of the immunocytochemical reaction was evaluated by 3 different observers. Acid phosphatase, a known marker enzyme for lysosomes, was localized as described by Leduc e t al. (’671, using a mixture of 10 mg of a-glycerophosphate and 10 mg of P-glycophosphate per 10 ml of medium as substrates.
Hormone assays and calculations Prolactin was measured in duplicate by double-antibody radioimmunoassay (Labrie e t al., ’73b) using rat prolactin 1-2 and rabbit antisera (anti-rat prolactin-S-3) kindly supplied by Dr. A. F. Parlow from the National Institute of Arthritis and Metabolic Diseases, Rat Pituitary Hormone Program. Radioimmunoassay data were analyzed with a HewlettPackard calculator, model 9830A, according to a program based on model I1 of Rodbard and Lewald (’70).All data are presented as means t SEM of duplicate measurements of triplicate Petri dishes. Statistical significance was measured according to the multiple-range test of Duncan-Kramer (Kramer, ’56). RESULTS
Effect of colchicine on prolactin secretion Short-term treatment with colchicine reduced basal prolactin release, a 40% decrease in hormone release being found after four hours (p < 0.01). The colchicine-induced inhibition of prolactin secretion persisted and even increased a t longer time intervals (fig. 1).Moreover, as compared to non-treated controls, the cell prolactin content increased by 24% following exposure to colchicine for 16 hours (p < 0.05). At longer time intervals (2496 hours), the inhibition of prolactin secretion was accompanied by a marked decrease in the cell content (p < 0.01 a t 4 days, fig. 2). Light microscopic immunocytochemistry In previous experiments (Antakly et al., ’79) the relative numbers of cells immunoreactive for prolactin, TSH, ACTH, FSH, LH and GH were determined. Prolactin-positive cells do, in fact, represent about 70% of the total glan-
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ANTAKLY, PELLETIER, ZEYTINOGLU AND LABRIE
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RELEASE0 CELL CONTENl
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HOURS OF INCUBATION
Fig. 1 Time-course of the effect of 10-6Mcolchicine (up to 16 hours), on prolactin release and cell content cell content), in rat anterior pituitary cells in primary culture. as well as on total hormone (released Prolactin release was measured during the last four hours of incubation. Note the progressive decrease of prolactin release with a corresponding accumulation of intracellular hormone, leading t o no significant change of total prolactin for up to 16 hours of incubation.
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RELEASED CELL CONTENT TOTAL
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DAYS OF INCUBATION M colchicine (up to 4 days) on prolactin release and cell content, Fig. 2 Time-course of the effect of as well as total hormone (released cell content), in r a t anterior pituitary cells in primary culture. Note the progressive decrease of prolactin release up to t h e last time interval studied, while cell content increased for up to 24 hours in the presence of colchicine, and decreased thereafter. These results are taken from another experiment than those shown in figure 1.
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COLCHICINE AND CULT1JRED PITUITARY CELLS
dular cell population. Although we could also identify LH-, FSH-, ACTH-, growth hormoneand TSH-positive cells, we focused our attention on prolactin cells. This choice was made not only because mammotrophs are most numerous (70%of glandular cell population) but also because, in parallel experiments, we have obtained detailed information about the interactions between hypothalamic and peripheral hormones on the control of this pituitary hormone. The prolactin-positive cells seemed to be almost completely filled with reaction products showing a fine granular staining in the cytoplasm, whereas the nuclei remained negative (fig. 3a). After one hour of incubation with colchicine, the intensity of the reaction for prolactin was already increased. After four hours in the presence of colchicine, the reaction became clearly more intense. In fact, although the intensity of the reaction clearly increased during the same period (fig. 3b), the percentage of prolactin-positive cells did not seem to show any increase up to 48 hours; however, between 48 hours and 4 days of treatment the percentage was decreased by 20%. This was estimated by counting about 800 cells under the microscope and determining the percentage of positive ones. The type of staining in the positive cells also changed, the reaction becoming coarsely granular and even “patchy.” After two and four days of treatment, a gradual decrease in the intensity of the reaction was observed, however. Scanning electron microscopy The SEM morphology of untreated cells in culture for six days has already been described briefly (Antakly e t al., ’78, ’79). Cells were mostly rounded up and measured about 8-12 pm in diameter. High-resolution SEM (fig. 4) showed numerous protrusions and projections from the cell surface consisting of microvilli of about 0.1 p m in diameter, blebs 0.4-0.8 p m in diameter and, sometimes, filamentous extensions (filopodia) which seemed to attach the cells to the surface of the Petri dish or to other cells. Fibroblasts could also be clearly seen underlying the glandular cells. After four hours of treatment with colchicine, noticeable changes in cellular shape could be observed, the cells becoming less spherical (fig. 51, with the rare presence of microvilli. However, in some cells, the number of blebs, as well as their size, increased (fig. 51, whereas in other cells, the cell surface became only more folded than in controls. After eight hours of treat-
357
ment, the effects mentioned above were more pronounced, and a t 16 hours almost all cells were clearly affected (fig. 6). The cell diameter decreased to 5-8 pm and the blebs were observed to cover preferentially the lower part of the cells, and even to dissociate from them (fig. 7). Only rarely could microvilli then be seen. Not infrequently large blebs of 1-3 p m were observed bulging from the cells, resulting in a characteristic deformed cell shape. This bulging was even more dramatic a t 48 or 96 hours of treatment, when the cell surface became covered with numerous folds. The generally uniform appearance of the cells suggests that the effects of colchicine are found on all pituitary cell types. Transmission electron microscopy (TEM) The ultrastructure of glandular cells in control cultures strongly resembled that of pituitary cells fixed in vivo. Prolactin-secreting cells could be easily identified. Prolactin granules exhibited their characteristic irregular shapes (fig. 8 ) and showed a positive reaction when stained for the immunocytochemical localization of prolactin. Microtubules were routinely observed (fig. 8b), while microvilli were found in most cells. Fibroblasts were easily identified in all the sections. After four hours in the presence of colchicine, no cytoplasmic microtubule could be seen, while accumulation of secretory granules was apparent in most cells a t eight hours and up to two days (fig. 9). In some cells, the cisternae of both the Golgi complex and the endoplasmic reticulum appeared to contain electron-opaque material. After 8, 16 and 24 hours of treatment, more lysosomes, which showed acid-phosphatase reaction, and vacuoles were observed in the prolactin cells (fig. 91, and some of these vacuoles were autophagic. Microfilaments could be seen in some cells. At both long or short time intervals, we noticed a change in the shape of the cells: in the presence of colchicine, the cells became flattened and microvilli were rarely seen. However, blebs of varying size and structure were frequently observed. In some cases, these blebs were seen to contain cell organelles such as endoplasmic reticulum, mitochondria or even secretory granules. Long-term treatment of the cells with colchicine (48 and 96 hours) resulted in a marked decrease in the number of secretory granules, as well as an increase in densely osmiophilic bodies which could probably be considered
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ANTAKLY, PELLETIER, ZE:YTINOGLU AND LABRIE
lysosomes (fig. 10).Autophagic vacuoles could also be seen (fig. 10) empty vacuoles were observed in some instances. Although some growth-hormone cells were filled with granules in the presence of colchicine, the majority of the cells were significantly degranulated as compared to controls. Golgi elements were absent from most sections (fig. 10). Prolonged exposure to colchicine induced dramatic changes in the morphology of the nuclei of most prolactin as well as other cells. Some nuclei were multilobed and/or fragmented (fig. 10).Microfilaments, rarely seen a t short timeintervals, became a common feature in cells after 96 hours of treatment (fig. 101, although their prominence varied from one cell to another. The presence of these microfilaments seemed to be closely associated with the presence of phagocytic vacuoles and the disappearance of granules. DISCUSSION
The present data show for the first time that colchicine leads to a rapid (less than 4 hours) and progressive inhibition of prolactin release in rat anterior pituitary cells in primary culture. These findings are in agreement with our previous report showing that vincristine can inhibit growth hormone and prolactin secretion in hemi-pituitaries incubated in vitro (Labrie et al., '73a). The inhibited prolactin release is accompanied by markedly increased cellular hormone content, for up to 24 hours in the presence of colchicine. This temporary accumulation of intracellular prolactin is followed by a progressive decrease, which indicates increased prolactin degradation and/or decreased prolactin synthesis. The present measurements of prolactin secretion are consistent with the immunocyto- 7 chemical and ultrastructural observations. In fact, for up to 24 hours of colchicine treatment, more prolactin was detected by immunocytochemistry and an increased number of prolactin granules and Golgi vacuoles was observed by TEM. The crowding of secretory granules into the cells is probably an indication of a decrease in hormone release. On the other hand, the appearance of numerous phagocytic vacuoles and lysosome-like bodies may account, as mentioned earlier, for the decrease in cell content after prolonged treatment with colchicine. Prolactin originally contained in the granules is probably being hydrolyzed by the lysosomes, a phenomenon already well described in prolactin cells after
cessation of the stimulus of lactation (Farquhar, '69). Moreover, the synthesis of pro-~ lactin is probably being decreased, since the rough endoplasmic reticulum was less prominent in colchicine-treated cells than in controls; in addition, Golgi elements were absent from most sections of cells treated for 96 hours with colchicine. Colchicine has already been shown to inhibit protein synthesis in the liver (Redman et al., '78) and to result in the dispersion of the Golgi apparatus (Moskalewski et al., '76; Thyberg et al., '77). The secretory cell nuclei, which indirectly control protein synthesis, were also affected by long-term treatment with colchicine. It is not excluded, however, t h a t colchicine might have a toxic effect on these cells. The present study confirms previous transmission electron microscopic reports suggesting that microtubules are responsible for t h e maintenance of the cell cytoskeleton. In fact, the colchicine-treated cells lose their spherical configuration, flatten and seem to collapse. Moreover, as shown in figures 5-7, the cell surface becomes somewhat irregular and folded, suggesting that intracellular supporting elements have been lost. We also observed a retraction of the cells (from 8-12 p m in untreated to 5-8 p m in colchicine-treated cells) which might be explained either by decreased synthesis of the structural material, loss of cell material or membrane folding. This might also account the decrease in the number and prom>.for inence of microvilli following colchicine treatment, since microvilli have been considered as a reserve of cellular membranes (Knutton et al., '75). On the other hand, the disappearance of microvilli seems to be directly related to a decrease in cell secretion, since in a previous study we have shown that a n increase in secretory activity of the same cell type is accompanied by an increase in the number and prominence of microvilli activity (Antakly et al., '78, '79). Transformed and tumor cells are richer in microvilli than their normal counterparts (Knutton et al., '75; Porter and Fonte, '73) and it is well known t h a t these cells have a high secretory activity. In the studies mentioned above, as well as in ours, t h e diameter of the microvilli was the same, about 0.1 pm. The role of the blebs, which increase in number and prominence after colchicine treatment, is less well understood. Several studies (Bruni et al., '77; Antakly e t al., '78, '79; Antakly et al., in preparation) show that they
COLCHICINE AND CULTURED PITUITARY CELLS
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also increase in number and size as a response to the stimulus of secretion. In the present study, however, the cells exposed to colchicine showed inhibited secretion, which seems a contradiction. It is suggested t h a t there might be two functionally different types of blebs (or bleb-like structures). One type is promoted after stimulation, as noted after long-term exposure of anterior pituitary cells to estradiol (Antakly e t al., '78, '791, in cells of the ependyma1 cells lining the third ventricle of the rabbit (Bruni e t al., '77) or in cells transformed in vitro (Malick and Langenbach, '76; Porter et al., '73). This type of bleb seems to be related to increased cellular activity. The second type of bleb would be a result of cellular transformation due to the loss of microtubules, and would represent material dissociating from the cells. This is supported by the observation that, in most treated cells, blebs were mostly seen a t the base of the cells or even slightly remote from them (figs. 5-7), as though they were dissociating from the cells. Moreover, the size of these blebs (1-3 pm in diameter) is larger than that of untreated cells (fig. 41,or of cells which have been stimulated with estradiol (Antakly et al., '78, '79). It is already known that colchicine and cytochalasin can induce cytoplasmic extrusions and even complete enucleation of mammalian cells (Ege et al., '74; Shay and Clark, '77). In addition, i t is possible that these effects on the cell surface could be a direct action of colchicine on the cell membrane. ACKNOWLEDGMENTS
T. A. is a recipient of an award from the National Council for Scientific Research, Lebanon. LITERATURE CITED Antakly, T., G. Pelletier, F. Zeytinoglu and F. Labrie 1979 Scanning electron microscopy of rat pituitary cells in monolayer culture. In: Scanning Electron Microscopy. 0. Johari, ed. SEM Inc. AMF OHare, Il., Vol. 3, pp. 389-397, i n press. Antakly, T., F. Zeytinoglu, G. Pelletier and F. Labrie 1978 Surface morphology and secretory activity of rat anterior pituitary cells in primary culture. Electron Microscopy, 1978. J. M. Sturgess, ed. Papers presentedat t he Ninth Int. Congress on Electron Microscopy, Toronto, VOl. 11, pp. 584-585. Bruni, J. E., D. G. Montemurro and R. E. Clattenburg 1977 Morphology of the ependymal lining of t he rabbit third ventricle following intraventricular administration of synthetic luteinizing hormone-releasing hormone (LHRH): a scanning electron microacopic investigation. Am. J. Anat., 150: 411-426. Ege, T., H. Hamberg, U. Krondahl, J. Ericsson and N. R. Ringertz 1974 Characterization of minicells (nuclei) ob-
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tained by cytochalasin enucleation. Exp. Cell Res., 87: 365-377. Farquhar, M. G. 1969 Lysosome function in regulating secretion. Disposal of secretory granules in cells of the anterior pituitary gland. In: Lysosomes in Biology and Pathology. J. T. Dingle and H. B. Fell, 4 s . North-Holland Publishers, Amsterdam, Vol. 2, pp. 462-541. Feldmann, G., M. Maurice, C. Sapin and J. P. Benhamou 1975 Inhibition by colchicine of fibrinogen translocation in hepatocytes. J. Cell. Biol., 67: 237-243. Furcht, L. T., R. E. Scott and P. B. Maercklein 1976 Independent alterations in cell shape and intramembranous particle topography induced by cytochalasin B and colchicine in normal and transformed cells. Cancer Res., 36: 4584-4589. Gillespie, E., R. J. Levine and S. E. Malawista 1968 Histamine release from ra t peritoneal mast cells: inhibition by colchicine and potentiation by denterium oxide. J. Pharmacol. Exp. Ther., 264: 158-165. Knutton, S., M. C. B. Summer and C. A. Pasternak 1975 Role of microvilli in surface changes of synchronized P8154 mastocytoma cells. J. Cell Biol., 66: 568-576. Kramer, C. Y. 1956 Extension of multiple-range test to group means with unequal numbers of replications. Biometrics, 12: 307-310. Labrie, F., M. Gauthier, G. Pelletier, P. Borgeat, A. Lemay and J. J. Gouge 1973a Role of microtubules in basal and stimulated release of growth hormone and prolactin in the ra t adenohypophysis in uitro. Endocrinology, 93: 903-914. Labrie, F., G. Pelletier, A. Lemay, P. Borgeat, N. Barden, A. Dupont, M. Savary, J. C6te and R. Boucher 1973b Control of protein synthesis in anterior pituitary gland. In: Karolinska Symposium on Research Methods in Reproductive Endocrinology. E. Diczfalusy, ed. Geneva, pp. 301-340. Lacy, P. E., S. R. Howell, D. A. Young and C. J. Fink 1968 New hypothesis of insulin secretion. Nature, 219: 1177-1179. Leduc, E. H., W. Bernhard, S. J. Holt and J. P. Tranzer 1967 Ultrathin frozen section. 11. Demonstration of enzymic activity. J. Cell Biol., 34: 773-786. Malaisse, W. J., V. Leclerq-Meyer, E. Van Obberghen, G. Somers, G. Devis, M. Ravazzola, F. Malaisse-Lagae and L. Orci 1973 The role of microtubular-microfilamentous system in insulin and glucagon release by the endocrine pancreas. In: Microtubule and Microtubule Inhibitors. M. Borgers and M. de Brafander, eds. North-Holland, Amsterdam, pp. 143-153. Malick, L. E., and R. Langenbach 1976 Scanning electron microscopy of in vitro chemically transformed mouse embryo cells. J. Cell Biol., 68: 654-664. Moskalewski, S., J. Thyberg and U. Friberg 1976 In uitro influence of colchicine on the Golgi complex in A- and Bcells of guinea pig pancreatic islets. J. Ultrastruct. Res., 54: 304-317. Pelletier, G., and M. B. Bornstein 1971 Effect of colchicine on rat anterior pituitary gland in tissue culture. Exptl. Cell. Res., 70: 221-223. Pelletier, G., R. Leclerc, F. Labrie, J. C6te, M. Chretien and M. Lis 1977 Immunohistochemical localization of @ l i p tropic hormone in the pituitary gland. Endocrinology, 100: 770-776. Porter, K, and V. Fonte 1973 Observations on the topography of normal and cancer cells. IITRUScanning Electron Microscopy 1973, pp. 683-688. Porter, K., D. Prescott and J. Frye 1973 Changes in surface morphology of Chinese hamster ovary cells during the cell cycle. J. Cell Biol., 57: 815-836. Poisner, A. M., and J. Bernstein 1971 A possible role for
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microtubules in catecholamine release from adrenal medulla: effect of colchicine vinca alkaloids and deuterium oxide. J. Pharmacol. Exp. Ther., 177: 102-108. Redman, C. M., D. Banerjee, K. Howell and G . E. Palade 1975 The step a t which colchicine blocks the secretion of plasma protein by r a t liver. Ann. N.Y. Acad. Sci., 253: 780-788. Redman, C. M., D. Banerjee, C. Manning, C. Y. Huang and K. Green 1978 In uiuo effect of colchicine on hepatic protein synthesis and on t h e conversion of pro-albumin to serum albumin. J. Cell. Biol., 77: 400-416. Rodbard, D., and J. E. Lewald 1970 Computer analysis of radioligand assay and radioimmunoassay data. In: Second Karolinska Symposium on Research Methods in Reproductive Endocrinology. E. Diczfalusy, ed. Stockholm, pp. 79-103.
Shay, J. W., and M. A. Clark 1977 Morphological studies on the enucleation of colchicine-treated L-929cells. J. Ultrastruct. Res., 58: 155-161. Sternberger, L. A. 1974 Immunocytochemistry. PrenticeHall, E n g l e w d s Cliffs, New Jersey, pp. 129-171. Thyberg, J., S. Nilaon, S. Moskalewski and A. Hinck 1977 Effects of colchicine on t h e Golgi complex and lysosomal system of chondrocytes in monolayer culture. An electron microscopic study. Cytobiology, 15: 175-191. Williams, J. A,, and J. Wolff 1970 Possible role of microtubules in thyroid setion. Proc. Natl. Acad. Sci. (U.S.A.),67: 1901-1908. Wolff, J., a n d B. Bhattacharya 1975 Microtubules and thyroid hormone mobilization. Ann. N.Y. Acad. Sci., 253: 763-770.
PLATE 1 EXPLANATION OF FIGURES
3 Immunocytochemical localization of prolactin in anterior pituitary cells in culture. Bars = 2 0 p m . x 1,000.
a Control. Note granular type of stain in most positive control cells. A few negative cells are also seen. b Twenty-four-hour colchicine-treated cells show increased immunoreactivity of the
positive cells, which are almost completely covered with reaction product. The staining becomes coarsely granular (patchy) in these cells. Note also a retraction of most of the cells incubated in the presence of colchicine.
COLCHICINE AND CULTURED PITUITARY CELLS Antakly, Pelletier, Zeytinoglu and Labrie
PLATE 1
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Figs. 4-7 Scanning electron microscopy (SEM) of the cultured cells. All bars in the SEM micrographs equal 1 km. All cells shown are representative of each group.
PLATE 2 EXPLANATION OF FIGURES
4
SEM of untreated cell (control)
a
Whole cell showing surface protrusions. x 4,000. Note t h a t the untreated cell is usually more or less spherical.
b
Details of the cell shown in (a). Microvilli (double-headed arrow) as well as small blebs (arrow) can be observed. x 20,000.
5 SEM of cells treated for four hours with colchicine a
Note smooth cell surface and cell deformation. Large blebs are seen. x 6,000.
h
Details of t h e cell shown in (a). The cell surface IS now folded and microvilli have disappeared. A large bleb is seen (arrow). X 20,000.
COLCHICINE AND CULTURED PITUITARY CELLS Antakly, Pelletier, Zeytinoglu and Labrie
PLATE 2
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PLATE 3 EXPLANATION OF FIGURES
6 SEM of cells treated with colchicine for 16 hours. a
Note the decreased cell size and t h e appearance of numerous blebs. Material seen a t the lower right side of the cell seems to be dissociating from the cell. X 5,000.
b At high resolution, no microvilli are seen, but blebs of various sizes cover most of the cell surface. x 20,000. 7
SEM of cells treated with colchicine for 24 hours.
a
Note a clear decrease in cell size as compared with untreated (control) cells. The blebs seem to cover preferentially the lower part of the cell (closest to the surface of the Petri dish on which cells are growing). X 5,000.
b Details of the same cell, showing that the part of t h e cell which is not covered with blebs is smoother than in the case of a 4-hour-treated cell (fig. 3B). X 20,000.
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COLCHICINE AND CULTURED PITUITARY CELLS Antakly, Pelletier, Zeytinoglu and Labrie
PLATE 3
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Figs. 8-10 Transmission electron microscopy (TEM) of rat anterior pituitary cells in primary culture. All specimens were fixed and processed under identical conditions.
PLATE 4 EXPLANATION OF FIGURES
8 Details of control cells.
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a
Section passing through Golgi elements which are well developed (Go). Scattered secretory granules can also be recognized (arrows). x 30,000.
b
High-resolution picture showing microtubules (arrows). Their frequency varies from one section to another, but they are usually not numerous. x 60,000.
COLCHICINE AND CULTURED PITUITARY CELLS Antakly, Pelletier, Zeytinoglu and Labrie
PLATE 4
PLATE 5 EXPLANATION OF FIGURE
9 Prolactin cell treated for 24 hours with colchicine. The cells were processed for the cytochemical localization of acid phosphatase. Note accumulation of secretory granules ( G ) in t h e cytoplasm. Some granules show lead deposits (arrows), resulting from the acid-phosphatase reaction. Such granules have probably fused with lysosomes. Other more characteristic lysosomes are also seen (Ly). x 30,000.
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COLCHICINE AND CULTURED PITUITARY CELLS Antakly, Pelletier, Zeytinoglu and Labrie
PLATE 5
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PLATE 6 EXPLANATION OF FIGURES
10 Cell treated for 96 hours colchicine
a
Very few granules (G) of irregular shape can be observed a t this stage and most of the cells are degranulated. Numerous dense bodies which are most probably of lysosomal origin (Ly), can be seen, as well a s an autophagic vacuole. Note indented nucleus (N). x 30,000.
b Details from a cell showing two such dense lysosomal bodies and an autophagic vacuole (V). Note large bundles of microfilaments (F). X 60,000.
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COLCHICINE AND CULTURED PITUlTARY CELLS Antakly, Pelletier, Zeytinoglu and Labrie
PLATE 6