Cell Tiss. Res. 174, 233-244 (1976)
Cell and Tissue Research 9 by Springer-Verlag 1976
The Thyrotropic Cells of the Guinea Pig Pituitary Electron Microscopic Study after Characterization by ImmunocytochemicalMeans* J.-C. Beauvillain**, M. Mazzuca, M.P. Dubois Laboratory of Histology of the Faculty of Medicine, Lille Cedex, France Laboratory of the Physiologyof Reproduction INRA, CNRZ, Nouzilly, France
Summary. Three different immunocytoenzymatic techniques were used to identify and characterize the thyrotropic cells in the pituitary o f normal guinea pigs at the ultrastructural level (superimposition technique, i m m u n o cytochemical technique using P.A.P. and indirect immunohistoenzymatic method before embedding). These cells are characterized by a dark cytoplasm with granules ranging f r o m 1500 to 2000 A 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 T S H 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 o f the rough endoplasmic reticulum membranes. These results are discussed.
Key words: Anterior pituitary - Guinea pig - Thyrotropic cells - I m m u n o cytochemical techniques.
Introduction The characterization o f the thyrotropic cells, at the ultrastructural level by immunochemical means has only been done by N a k a n e (1970) in the pituitary of the rat. Because of the chemical similarities between the c~subunits of ovine L H and ovine TSH, Tougard et al. (1973), has also been able to describe T S H cells in the rat pituitary using anti-ovine LHe. But in most studies thyrotropic cells Send offprint requests to: J.-C. Beauvillain, Laboratoire d'Histologie et d'Embryologie, Place de
Verdun, F-59 - Lille, France * We thank D. Quief for technical assistance. This work was supported by a grant from U.E.R. III Lille ** Attach6 de Recherche INSERM
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were characterized and described by analogy to "thyroidectomy cells" (Farquhar and Rinehart, 1954; Barnes, 1962, 1963; Herlant, 1963; Kurosumi and Oota, 1966). However, much remains uncertain about these cells, especially their origin. Even if the thyroidectomy cells derive from the TSH cells, it is not always possible to characterize a cell type under physiological conditions by comparison with cells under experimental conditions especially for the thyrotropic cells which, according to Pelletier (1973), Pelletier and Puviani (1973), Cuerdo-Rocha and Zambrano (1974a, b) display differences in hormone storage depending on their secretory activity. In this study we have attempted to identify and to study the TSH cells of the guinea pig at the ultrastructural level by three methods: 1) by an immunocytochemical technique previously described by us (Beauvillain and Tramu, 1973; Tramu et al., 1974; Beauvillain et al., 1975). Immunocytochemical reactions are obtained on semi-thin sections and the same cells are then observed in adjacent ultrathin sections. 2) by using the complex peroxidase anti peroxidase (P.A.P.) of Sternberger according to Moriarty and Halmi (1972). 3) by a technique previously described by Nakane (1970) and modified by Mazzuca and Dubois (1974) and Mazzuca (1975), in which the tissues are frozen, exposed to antisera, and then embedded. The first technique permits a characterization of the cell type and the other two techniques allow a precise intracellular localization of the hormone. Material and Methods The studies were made on the pituitary glands of female guinea pigs.
Preparation and Study of Antibodies. The preparation of the Antisera to bovine TSH (NIH, TSH, B2), the immunisation method, the serological study and the specificity of these antisera have all been described previously (Dubois, 1971, 1972). The antiserum to bovine TSH was always absorbed with bovine LH (NIH, LH, Bs) before use. 1. Superimposition Technique Fixation, Embedding and Sectioning. Two fixatives were used. The first consists of a solution of 4 ~ glutaraldehyde and 2.2~o paraformaldehyde in 0.08 M cacodylate buffer adjusted to pH 7.4 (G.F. solution). The second fixative is a phosphate buffered picric-acid-formaldehyde solution (Stefanini et al., 1967) (P.A.F. solution). Pituitary glands were immersed for 2 h in the G.F. solution. Some of these were embedded after dehydration and the others were postfixed for two h in 1~o osmic acid in 0.1 M cacodylate buffer before embedding. Pituitaries were immersed for 4 h in the P.A.F. solution. After washing some of these were dehydrated and embedded and the others were postfixed in buffered osmic acid as above. For embedding Araldite medium was always used. Sections were cut on a Porter-Blum M T 1 ultramicrotome, m o u n t e d on grids and stained with lead citrate. Adjacent semi-thin sections were placed on glass slides. The first semi-thin section was used for the anti-bovine TSH reactions, the second for anti porcine LH/~ or anti c~17-39 A C T H antisera. Before these two reactions. Araldite was always removed with sodium methoxide according to Mayor et al. (1961). W h e n osmic acid postfixation was used, the semi-thin sections were treated with 10~o hydrogen peroxide for ten minutes.
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Fig. 1. Several cells immunocytochemically stained with anti-bovine TSH antiserum absorbed with bovine LH. These cells are often polyhedral and are sometimes grouped in clusters, but are not generally very numerous. • 550
Immunochemical Staining. As described by Nakane and Pierce (1967) the reaction used, is of the indirect type, using the rabbit antiserum containing the specific antibody in a first stage, a solution of sheepanti-rabbit gamma-globulin conjugated with horseradish peroxidase (Institut Pasteur, Paris) in a second stage. The horseradish peroxidase was localized histochemically with 3 3'-diaminobenzidine-tetrahydrochloride and fresh hydrogen peroxide according to the method of Graham and Karnovsky (1966). As controls, the anti b TSH was replaced by normal rabbit serum or omitted or compared with other antisera (antiporcine LH fl or anti ct 1%39 ACTH). 2. Immunohistochemical Staining Technique after Embedding Using P.A,P. The technique of Moriarty and Halmi (1972) was used. The pituitaries were fixed in a phosphate buffered picric-acid-formaldehyde solution for 4 h and embedded in Araldite. The different reactions were carried out on ultrathin sections as previously described (Moriarty and Halmi, 1972; Petrali et al., 1974; Beauvillain et al., 1975). As controls the anti b TSH antiserum was replaced by normal rabbit serum.
3. Immunohistochemieal Staining Technique before Embedding The pituitaries were fixed in the same picric acid-formaldehyde solution for 8 h. They were washed overnight with phosphate buffered saline. After impregnation with 10% glycerol for 1 h the tissues were frozen in liquid nitrogen at low pressure and were sectioned at about 20 ~tm. The sections were then exposed in succession to the normal sheep serum, anti b TSH, peroxidase labeled sheep anti-rabbit ~,-globulin, 3 3'-diaminobenzidine and osmium tetroxide. The details of this technique have already been described (Mazzuca and Dubois, 1974). The same controls were used as in the other methods.
Results With antiserum to b TSH, positive reactions were never observed following glutaraldehyde-formaldehyde fixation. However positive results were obtained w i t h a n t i p LH]~ a n d a n t i ~ 17-39 A C T H a n t i s e r a w i t h t h i s s a m e f i x a t i o n . P o s i t i v e
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Fig. 3. Higher magnification of the outlined part of the Figure 2 c. The granules show varying appearances; G golgi apparatus; E.R. rough endoplasmic reticulum, x 25,000
Fig. 2a-c. Superimposition between observations on semi-thin sections and ultrathin sections. On the first semi-thin section a anti-bovine TSH was used and a positive staining was obtained on one cell type; the thyropropic cells (7). On the second semi-thin section b anti-porcine LH/~ antiserum was used and a positive reaction was observable on an other cell type: the gonadotropin producing cells (G). The two cells are seen in the adjacent ultrathin section c. T thyrotropin cell; G gonadotropin producing cell. C capillary. • 7,500
Fig. 4 a and b. Different aspects of the thyrotropic cell. a Some of the granules (arrow) are homogeneously electron dense but others empty, with a small darker region. Between these two appearances m a n y intermediate aspects are seen (arrows) x 30,000. b In the neighbourhood of the Golgi complex (G), the granules are homogeneously electron dense, x 30,000
Fig. 5 a and b. Cells immunohistochemically stained with anti-bovine TSH antiserum (technique with P.A.P.)., All the granules show a positive reaction, a The rough endoplasmic reticulum consists of dilated cisternae (arrow); the concentration of the granules close to the blood capillary is well shown. x 11,000. Insert of a higher magnification shows the characteristic P.A.P. molecules (arrow). • 72,000. b The rough endoplasmic reticulum m a y also consists of flattened sacs (arrow). The entire granule is electron opaque, x 30,000
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results were obtained with picric-acid-formaldehyde fixation for all the antisera used (anti b TSH, anti pLHfi, anti ~ 17-39 ACTH immune sera).
1. Superimposition Technique a) Semi-thin Sections. A good immunochemical staining is obtained both with P.A.F. fixation alone and with P.A.F. osmic acid fixation. The immunochemical positive cells are not numerous and are located in the middle of the median and lateral parts of the pituitary. They sometimes appear in clusters (Fig. 1) larger than the neighbouring cells and commonly polyhedral in outline. Comparison of different sections shows that the cells stained with anti b TSH antiserum are not those stained using anti p LHfl or anti ~ 17-39 ACTH antisera. Furthermore, the number of positive cells following treatment with these two antibodies is much larger than with antiserum to b TSH. The thyrotropin producing cells like the gonadotropin producing cells are generally located near blood capillaries.
b) Thin Sections. All immunochemically stained cells share certain common characteristics though some variations occur. The cells are generally dark (Fig. 2 c) and the rough endoplasmic reticulum consists of few flattened sacs or sometimes small dilated vacuoles. The prominence of the Golgi complex varies from one cell to another but it never consists of dilated sacs. The mitochondria are small and not very numerous. Free ribosomes are scattered in the cytoplasm. The nucleus often contains a large nucleole. The ceils always have secretory granules which are from 1,500 to 2,200 A in diameter. Some of these granules display a marked electron density and a narrow space can be observed between the granule and the surrounding membrane. Granules of this type are not common and are usually concentrated close to the Golgi complex (Fig. 4 a). Most of the granules however are less electron dense and often have a darker region next to the surrounding membrane (Fig. 4 b). Empty vesicles of the same size as the granules are commonly found among the secretory granules (Fig. 4b). The granules appear in all parts of the cytoplasm but the majority are located close to the blood capillaries.
2. Immunocytochemical Technique after Embedding Using P.A.P. Staining is confined to secretory granules with diameters of 1,500 to 2,000A. Characteristic P.A.P. molecules can be seen at high magnification (Fig. 5). All the granules are homogeneously electron opaque and the area of greater electron
Fig. 6a---e. Immunocytochemical technique before embedding: a Only one cell type is stained; the rough endoplasmic reticulum consists of dilated cisternae; the positively stained granules are small; the nucleus and the mitochondria (m) are unstained, x 9,000. b The content of the cisternae is always negative but the membrane seems positive (arrow) and there is a diffuse staining in the cytoplasm. x 34,000. e No positive staining can be observed in the saccules of the Golgi complex (g). • 13,000
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density close to the membrane revealed with the first technique was not seen (Fig. 5). These positively stained cells, which are situated near blood capillaries contain a rough endoplasmic reticulum in the form of vacuoles (Fig. 5 a) or in some cases flattened sacs (Fig. 5b). The preferential localization of the granules close to capillaries is confirmed and is particularly clear because of the difference in electron density between these granules and the cytoplasm (Fig. 5 a, b).
3. Immunoeytochemical Technique before Embedding Positively stained cells displayed the same localization in the pituitary as with the previous techniques and no staining of other cell types was observed (Fig. 6 a). All the cells display similar characteristics. The rough endoplasmic reticulum is much more dilated than with the previous techniques, perhaps as a result of the freezing. The dense granule is small (around 800 A, in diameter) but is situated in a vesicle whose diameter ranges from 1,500 to 2,000 A (Fig. 6b). It seems that either there has been a concentration of the content of the granule or that the less dense part previously described, is no longer visible and only the densest part of the granule remains. No staining is observed in either the mitochondria or the nucleus. In the Golgi complex the saccules are always negative but a few granules surrounding the golgi zone are stained. A diffuse staining of the entire cytoplasm is observed in most cells and although the content of the dilated cisternae of rough endoplasmic reticulum is always negative, a positive reaction may be seen on the membranes of these cisternae.
Di~us~on
The use of P.A.F. fixative results in a positive immunochemical staining with anti-bovine b TSH in all three methods. The results agree with the study of Moriarty (1973). Furthermore, positive results with the superimposition technique were also obtained after osmium tetroxide post fixation which gives a slightly better preservation of the tissues. The localization and size of the stained cells in this study may be compared with results obtained at the light microscope level with different methods (thyroidectomy or alcian blue staining) for TSH cell localization in the same species (D'Angelo, 1953; Tramu, 1969) in the rat (Baker, 1971) and in the mouse (Messier, 1965). Such comparisons and our results with other immune sera lead us to conclude that the positive immunocytochemical staining is due to the anti-bovine TSH antisera. Unfortunately P.A.F. fixative is not so good as glutaraldehyde-formaldehydemixture to preserve morphological features at the ultrastructural level. Nevertheless the size of the granules and their localization close to blood capillaries are common characteristics obtained with both fixatives. These features are therefore important in characterizing the TSH cells whatever the fixative. The different appearances of the rough endoplasmic reticulum may represent stages of a secretory cycle in the same cell type, but as such differences cannot be seen using other fixatives it may be that these differences are fixative dependent.
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However, even though dilated cisternae may be due to P.A.F. fixative, they can also be observed in thyroidectomy cells with other fixatives: in the latter case they may represent particular stages of activity. The immunocytochemical technique after embedding using P.A.P. has been used to determine the intracellular localization of TSH. We obtained positive staining of the entire granule and were not able to observe a difference in concentration of TSH between the small dense granule and the part of the granule with a moderate electron density. With this approach, only the granules appear to be positive, but as it is sometimes difficult to see the Golgi complex and the rough endoplasmic reticulum, we used the immunocytochemical technique before embedding in order to determine whether TSH might be present in the rough endoplasmic reticulum or in the Golgi complex. Negative results of the content of dilated rough endoplasmic reticulum cisternae and saccules of the Golgi complex may be due to the absence of immunogenic TSH molecules or according to Nakane (1975) to the effect of fixation which destroy or differentially denatures the antigen sites in the Golgi complex, in the rough endoplasmic reticulum or in both. On the other hand most of the membranes of the rough endoplasmic reticulum appear positive. So even if TSH molecules are largely confined to the granules it may be that some are present in the cytoplasm and also next to the membranes (perhaps on ribosomes ?). In several types of hormone secreting cells in culture, Tixier-Vidal and Tougard (1975) have obtained analogous results. In conclusion, one cell type having the same characteristics has been revealed by three different immunocytochemical techniques. This study has permitted us to obtain some details of the ultrastructure of the thyreotropic cells under physiological conditions. The TSH molecules are essentially confined to the granules. References Barnes, B.G.: Electron microscope studies on the secretory cytology of the mouse anterior pituitary. Endocrinology 71, 618-628 (1962) Barnes, B.G.: The fine structure of the mouse adenohypophysis in various physiological states: In: Cytologic de l'ad6nohypophyse (J. Benoit and C. Da Lage, eds.). Paris: Editions du Centre National de la Recherche Scientifique 1963 Baker, B.L., Yu, Y.Y.: The thyrotropic cell of the rat hypophysis as studied with peroxidase labeled antibody. Amer. J. Anat. 131, 55 72 (1971) Baker, B.L., Yu, Y.Y.: Hypophyseal changes induced by thyroid deficiency and thyroxine administration as revealed by immunochemical staining. Endocrinology 89, 996-1004 (1971) Beauvillain, J.-C., Tramu, G.: Cellules fi activit~ corticotrope de l'hypophyse du L6rot (Eliomys quercinus): superposition des r6sultats de microscopic optique (immunofluorescence et colorations) et de microscopic ~lectronique. C.R. Acad. Sci. (Paris). 277, 1025-1028 (1973) Beauvillain, J.-C., Tramu, G., Dubois, M.P.: Characterization by different techniques of adrenocorticotropin and gonadotropin-producing cells in Lerot pituitary (Eliomys quercinus). Cell Tiss. Res. 158, 301-317 (1975) Cuerdo-Rocha, S., Zambrano, D.: The action of protein synthesis inhibitors and thyrotropin releasing factor on the ultrastructures of rat thyrotrophs. J. Ultrastruct. Res. 48, 1-17 (1974) Cuerdo-Rocha, S., Zambrano, D.: Thyrotrophs of the rat anterior pituitary after different periods of thyroidectomy. A conventional and histochemical electron microscope study. J. Ultrastruct. Res. 49, 312-321 (1974) D'Angelo, S.A.: Cytophysiologic aspects of thyrotrophic hormone secretion in the goitrous guinea pig. Endocrinology 52, 331-337 (1953) Dubois, M.P.: Les cellules g hormones glycoprotidiques du lobe ant~rieur de l'hypophyse: s6paration
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par immunofluorescence des cellules thyr6otropes et des cellules gonadotropes dans l'hypophyse des bovins, ovins, porcins. Ann. Rech. Veter. 2, 197~22 (1971) Dubois, M.P.: Localisation par immunocytologie des hormones glycoprotidiques hypophysaires chez les vert6br6s. In: Pituitary glycoprotein hormones (INSERM, Paris, ed.) 2 7 4 7 (1972) Farquhar, M.G., Rinehart, J.F.: Cytologic alterations in the anterior pituitary gland following thyroidectomy. An electron microscope study. Endocrinology 55, 85%876 (1954) Graham, R.C., Karnovsky, M.J.: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-302 (1966) Herlant, M.: Apport de la microscopic 61ectronique ~ l'6tude du lobe ant6rieur de l'hypophyse: In: Cytologic de l'ad6nohypophyse (J. Benoit and C. Da Lage eds.). Paris: Editions du Centre National de la Recherche Scientifique 1963 Kurosumi, K., Oota, Y. : Corticotrophs in the anterior pituitary gland of gonadectomized and thyroidectomized rats as revealed by electron microscopy. Endocrinology 79, 808-814 (1966) Mayor, H.D., Hampton, J.C., Rosario, B.: A simple method for removing the resin from epoxy embedded tissue. J. biophys, biochem. Cytol. 9, 909-910 (1961) Mazzuca, M.: Individualisation morphofonctionelle des terminaisons nerveuses p6rivasculaires de l'6minence m6diane (chez les Mammif6res) en microscopie 61ectronique. R6union de la D.G.R.S.T. "Biologic de la Reproduction et du D6veloppement", Paris, Mai 1975 Mazzuca, M., Dubois, M.P.: Detection of luteinizing hormone releasing hormone in the guinea pig median eminence with an immunohistoenzymatic technique. J. Histochem. Cytochem. 22, 993 996 (1974) Messier, B. : Number and distribution of thyrotropic cells in the mouse pituitary gland. Anat. Rec. 153, 343-348 (1965) Moriarty, G.C. : Adenohypophysis: Ultrastructural cytochemistry. A review. J. Histochem. Cytochem. 21,855 894 (1973) Moriarty, G.C., Halmi, N.S.: Electron microscopic study of the adrenocorticotropin producing cell with the use of unlabeled antibody and the soluble peroxidase-antiperoxidase complex. J. Histochem. Cytochem. 20, 590-604 (1972) Nakane, P.K.: Classifications of anterior pituitary cell types with immunoenzyme histochemistry. J. Histochem. Cytochem. 18, 9-20 (1970) Nakane, P.K.: Identification of anterior pituitary cells by immunoelectron microscopy: In: The anterior pituitary (A. Tixier-Vidal and M.G. Farquhar, eds.). New York: Acad. Press 1975 Nakane, P.K., Pierce, Jr. G.B.: Enzyme labeled antibodies for the light and electron microscopic localization of tissue antigen. J. Cell Biol. 33, 307-318 (1967) Pelletier, G.: Secretion and uptake of peroxidase by rat adenohypophyseal cells J. Ultrastruct. Res. 43, 445~459 (1973) Pelletier, G., Puviani, R.: Detection of glycoproteins and autoradiographic localization of (3H) fucose in the thyroidectomy cells of rat anterior pituitary gland. J. Cell Biol. 56, 600-605 (1973) Petrali, J.P., Hinton, D.M., Moriarty, G.C., Sternberger, L.A.: The unlabeled antibody enzyme method of immunocytochemistry. Quantitative comparison of sensitivities with and without peroxidaseantiperoxidase complex. J. Histochem. Cytochem. 22, 78~801 (1974) Stefanini, M., De Martino, C., Zamboni, L.: Fixation of ejaculated spermatozoa for electron microscopy. Nature (Lond.) 216, 173 (1967) Tixier-Vidal, A., Tougard, C. : Subcellular localization of several anterior pituitary and hypothalamic hormones as revealed by the peroxidase labeled antibody method. First International Symposium on Immunoenzymatic technique. INSERM, April 1975 (in press) Tougard, C., Kerdelhue, B., Tixier-Vidal, A.: Light and electron microscope localization of binding sites of antibodies against ovine luteinizing hormone and its two subunits in rat adenohypophysis using peroxidase labeled antibody technique. J. Cell Biol. 5g, 503 521 (1973) Tramu, G.: Les cellules thyr6otropes de l'ant6hypophyse du cobaye mille au cours de diverses circonstances exp6rimentales et physiologiques. C.R. Soc. Bio. (Lille) 163, 1560-1563 (1969) Tramu, G., Beauvillain, J.-C., Dubois, M.P.: Les cellules gonadotropes de l'hypophyse du L6rot (Eliomys quercinus): concordances des r6sultats de microscopic optique (immunofluorescence et coloration) et de microscopic 61ectronique. J. de Microscopic 21, 181 184 (1974)
Accepted May 25, 1976
Cell and Tissue Research 9 by Springer-Verlag 1976
The Thyrotropic Cells of the Guinea Pig Pituitary Electron Microscopic Study after Characterization by ImmunocytochemicalMeans* J.-C. Beauvillain**, M. Mazzuca, M.P. Dubois Laboratory of Histology of the Faculty of Medicine, Lille Cedex, France Laboratory of the Physiologyof Reproduction INRA, CNRZ, Nouzilly, France
Summary. Three different immunocytoenzymatic techniques were used to identify and characterize the thyrotropic cells in the pituitary o f normal guinea pigs at the ultrastructural level (superimposition technique, i m m u n o cytochemical technique using P.A.P. and indirect immunohistoenzymatic method before embedding). These cells are characterized by a dark cytoplasm with granules ranging f r o m 1500 to 2000 A 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 T S H 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 o f the rough endoplasmic reticulum membranes. These results are discussed.
Key words: Anterior pituitary - Guinea pig - Thyrotropic cells - I m m u n o cytochemical techniques.
Introduction The characterization o f the thyrotropic cells, at the ultrastructural level by immunochemical means has only been done by N a k a n e (1970) in the pituitary of the rat. Because of the chemical similarities between the c~subunits of ovine L H and ovine TSH, Tougard et al. (1973), has also been able to describe T S H cells in the rat pituitary using anti-ovine LHe. But in most studies thyrotropic cells Send offprint requests to: J.-C. Beauvillain, Laboratoire d'Histologie et d'Embryologie, Place de
Verdun, F-59 - Lille, France * We thank D. Quief for technical assistance. This work was supported by a grant from U.E.R. III Lille ** Attach6 de Recherche INSERM
234
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were characterized and described by analogy to "thyroidectomy cells" (Farquhar and Rinehart, 1954; Barnes, 1962, 1963; Herlant, 1963; Kurosumi and Oota, 1966). However, much remains uncertain about these cells, especially their origin. Even if the thyroidectomy cells derive from the TSH cells, it is not always possible to characterize a cell type under physiological conditions by comparison with cells under experimental conditions especially for the thyrotropic cells which, according to Pelletier (1973), Pelletier and Puviani (1973), Cuerdo-Rocha and Zambrano (1974a, b) display differences in hormone storage depending on their secretory activity. In this study we have attempted to identify and to study the TSH cells of the guinea pig at the ultrastructural level by three methods: 1) by an immunocytochemical technique previously described by us (Beauvillain and Tramu, 1973; Tramu et al., 1974; Beauvillain et al., 1975). Immunocytochemical reactions are obtained on semi-thin sections and the same cells are then observed in adjacent ultrathin sections. 2) by using the complex peroxidase anti peroxidase (P.A.P.) of Sternberger according to Moriarty and Halmi (1972). 3) by a technique previously described by Nakane (1970) and modified by Mazzuca and Dubois (1974) and Mazzuca (1975), in which the tissues are frozen, exposed to antisera, and then embedded. The first technique permits a characterization of the cell type and the other two techniques allow a precise intracellular localization of the hormone. Material and Methods The studies were made on the pituitary glands of female guinea pigs.
Preparation and Study of Antibodies. The preparation of the Antisera to bovine TSH (NIH, TSH, B2), the immunisation method, the serological study and the specificity of these antisera have all been described previously (Dubois, 1971, 1972). The antiserum to bovine TSH was always absorbed with bovine LH (NIH, LH, Bs) before use. 1. Superimposition Technique Fixation, Embedding and Sectioning. Two fixatives were used. The first consists of a solution of 4 ~ glutaraldehyde and 2.2~o paraformaldehyde in 0.08 M cacodylate buffer adjusted to pH 7.4 (G.F. solution). The second fixative is a phosphate buffered picric-acid-formaldehyde solution (Stefanini et al., 1967) (P.A.F. solution). Pituitary glands were immersed for 2 h in the G.F. solution. Some of these were embedded after dehydration and the others were postfixed for two h in 1~o osmic acid in 0.1 M cacodylate buffer before embedding. Pituitaries were immersed for 4 h in the P.A.F. solution. After washing some of these were dehydrated and embedded and the others were postfixed in buffered osmic acid as above. For embedding Araldite medium was always used. Sections were cut on a Porter-Blum M T 1 ultramicrotome, m o u n t e d on grids and stained with lead citrate. Adjacent semi-thin sections were placed on glass slides. The first semi-thin section was used for the anti-bovine TSH reactions, the second for anti porcine LH/~ or anti c~17-39 A C T H antisera. Before these two reactions. Araldite was always removed with sodium methoxide according to Mayor et al. (1961). W h e n osmic acid postfixation was used, the semi-thin sections were treated with 10~o hydrogen peroxide for ten minutes.
Characterization of Thyrotropic Cells
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Fig. 1. Several cells immunocytochemically stained with anti-bovine TSH antiserum absorbed with bovine LH. These cells are often polyhedral and are sometimes grouped in clusters, but are not generally very numerous. • 550
Immunochemical Staining. As described by Nakane and Pierce (1967) the reaction used, is of the indirect type, using the rabbit antiserum containing the specific antibody in a first stage, a solution of sheepanti-rabbit gamma-globulin conjugated with horseradish peroxidase (Institut Pasteur, Paris) in a second stage. The horseradish peroxidase was localized histochemically with 3 3'-diaminobenzidine-tetrahydrochloride and fresh hydrogen peroxide according to the method of Graham and Karnovsky (1966). As controls, the anti b TSH was replaced by normal rabbit serum or omitted or compared with other antisera (antiporcine LH fl or anti ct 1%39 ACTH). 2. Immunohistochemical Staining Technique after Embedding Using P.A,P. The technique of Moriarty and Halmi (1972) was used. The pituitaries were fixed in a phosphate buffered picric-acid-formaldehyde solution for 4 h and embedded in Araldite. The different reactions were carried out on ultrathin sections as previously described (Moriarty and Halmi, 1972; Petrali et al., 1974; Beauvillain et al., 1975). As controls the anti b TSH antiserum was replaced by normal rabbit serum.
3. Immunohistochemieal Staining Technique before Embedding The pituitaries were fixed in the same picric acid-formaldehyde solution for 8 h. They were washed overnight with phosphate buffered saline. After impregnation with 10% glycerol for 1 h the tissues were frozen in liquid nitrogen at low pressure and were sectioned at about 20 ~tm. The sections were then exposed in succession to the normal sheep serum, anti b TSH, peroxidase labeled sheep anti-rabbit ~,-globulin, 3 3'-diaminobenzidine and osmium tetroxide. The details of this technique have already been described (Mazzuca and Dubois, 1974). The same controls were used as in the other methods.
Results With antiserum to b TSH, positive reactions were never observed following glutaraldehyde-formaldehyde fixation. However positive results were obtained w i t h a n t i p LH]~ a n d a n t i ~ 17-39 A C T H a n t i s e r a w i t h t h i s s a m e f i x a t i o n . P o s i t i v e
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Characterization of Thyrotropic Cells
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Fig. 3. Higher magnification of the outlined part of the Figure 2 c. The granules show varying appearances; G golgi apparatus; E.R. rough endoplasmic reticulum, x 25,000
Fig. 2a-c. Superimposition between observations on semi-thin sections and ultrathin sections. On the first semi-thin section a anti-bovine TSH was used and a positive staining was obtained on one cell type; the thyropropic cells (7). On the second semi-thin section b anti-porcine LH/~ antiserum was used and a positive reaction was observable on an other cell type: the gonadotropin producing cells (G). The two cells are seen in the adjacent ultrathin section c. T thyrotropin cell; G gonadotropin producing cell. C capillary. • 7,500
Fig. 4 a and b. Different aspects of the thyrotropic cell. a Some of the granules (arrow) are homogeneously electron dense but others empty, with a small darker region. Between these two appearances m a n y intermediate aspects are seen (arrows) x 30,000. b In the neighbourhood of the Golgi complex (G), the granules are homogeneously electron dense, x 30,000
Fig. 5 a and b. Cells immunohistochemically stained with anti-bovine TSH antiserum (technique with P.A.P.)., All the granules show a positive reaction, a The rough endoplasmic reticulum consists of dilated cisternae (arrow); the concentration of the granules close to the blood capillary is well shown. x 11,000. Insert of a higher magnification shows the characteristic P.A.P. molecules (arrow). • 72,000. b The rough endoplasmic reticulum m a y also consists of flattened sacs (arrow). The entire granule is electron opaque, x 30,000
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results were obtained with picric-acid-formaldehyde fixation for all the antisera used (anti b TSH, anti pLHfi, anti ~ 17-39 ACTH immune sera).
1. Superimposition Technique a) Semi-thin Sections. A good immunochemical staining is obtained both with P.A.F. fixation alone and with P.A.F. osmic acid fixation. The immunochemical positive cells are not numerous and are located in the middle of the median and lateral parts of the pituitary. They sometimes appear in clusters (Fig. 1) larger than the neighbouring cells and commonly polyhedral in outline. Comparison of different sections shows that the cells stained with anti b TSH antiserum are not those stained using anti p LHfl or anti ~ 17-39 ACTH antisera. Furthermore, the number of positive cells following treatment with these two antibodies is much larger than with antiserum to b TSH. The thyrotropin producing cells like the gonadotropin producing cells are generally located near blood capillaries.
b) Thin Sections. All immunochemically stained cells share certain common characteristics though some variations occur. The cells are generally dark (Fig. 2 c) and the rough endoplasmic reticulum consists of few flattened sacs or sometimes small dilated vacuoles. The prominence of the Golgi complex varies from one cell to another but it never consists of dilated sacs. The mitochondria are small and not very numerous. Free ribosomes are scattered in the cytoplasm. The nucleus often contains a large nucleole. The ceils always have secretory granules which are from 1,500 to 2,200 A in diameter. Some of these granules display a marked electron density and a narrow space can be observed between the granule and the surrounding membrane. Granules of this type are not common and are usually concentrated close to the Golgi complex (Fig. 4 a). Most of the granules however are less electron dense and often have a darker region next to the surrounding membrane (Fig. 4 b). Empty vesicles of the same size as the granules are commonly found among the secretory granules (Fig. 4b). The granules appear in all parts of the cytoplasm but the majority are located close to the blood capillaries.
2. Immunocytochemical Technique after Embedding Using P.A.P. Staining is confined to secretory granules with diameters of 1,500 to 2,000A. Characteristic P.A.P. molecules can be seen at high magnification (Fig. 5). All the granules are homogeneously electron opaque and the area of greater electron
Fig. 6a---e. Immunocytochemical technique before embedding: a Only one cell type is stained; the rough endoplasmic reticulum consists of dilated cisternae; the positively stained granules are small; the nucleus and the mitochondria (m) are unstained, x 9,000. b The content of the cisternae is always negative but the membrane seems positive (arrow) and there is a diffuse staining in the cytoplasm. x 34,000. e No positive staining can be observed in the saccules of the Golgi complex (g). • 13,000
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density close to the membrane revealed with the first technique was not seen (Fig. 5). These positively stained cells, which are situated near blood capillaries contain a rough endoplasmic reticulum in the form of vacuoles (Fig. 5 a) or in some cases flattened sacs (Fig. 5b). The preferential localization of the granules close to capillaries is confirmed and is particularly clear because of the difference in electron density between these granules and the cytoplasm (Fig. 5 a, b).
3. Immunoeytochemical Technique before Embedding Positively stained cells displayed the same localization in the pituitary as with the previous techniques and no staining of other cell types was observed (Fig. 6 a). All the cells display similar characteristics. The rough endoplasmic reticulum is much more dilated than with the previous techniques, perhaps as a result of the freezing. The dense granule is small (around 800 A, in diameter) but is situated in a vesicle whose diameter ranges from 1,500 to 2,000 A (Fig. 6b). It seems that either there has been a concentration of the content of the granule or that the less dense part previously described, is no longer visible and only the densest part of the granule remains. No staining is observed in either the mitochondria or the nucleus. In the Golgi complex the saccules are always negative but a few granules surrounding the golgi zone are stained. A diffuse staining of the entire cytoplasm is observed in most cells and although the content of the dilated cisternae of rough endoplasmic reticulum is always negative, a positive reaction may be seen on the membranes of these cisternae.
Di~us~on
The use of P.A.F. fixative results in a positive immunochemical staining with anti-bovine b TSH in all three methods. The results agree with the study of Moriarty (1973). Furthermore, positive results with the superimposition technique were also obtained after osmium tetroxide post fixation which gives a slightly better preservation of the tissues. The localization and size of the stained cells in this study may be compared with results obtained at the light microscope level with different methods (thyroidectomy or alcian blue staining) for TSH cell localization in the same species (D'Angelo, 1953; Tramu, 1969) in the rat (Baker, 1971) and in the mouse (Messier, 1965). Such comparisons and our results with other immune sera lead us to conclude that the positive immunocytochemical staining is due to the anti-bovine TSH antisera. Unfortunately P.A.F. fixative is not so good as glutaraldehyde-formaldehydemixture to preserve morphological features at the ultrastructural level. Nevertheless the size of the granules and their localization close to blood capillaries are common characteristics obtained with both fixatives. These features are therefore important in characterizing the TSH cells whatever the fixative. The different appearances of the rough endoplasmic reticulum may represent stages of a secretory cycle in the same cell type, but as such differences cannot be seen using other fixatives it may be that these differences are fixative dependent.
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However, even though dilated cisternae may be due to P.A.F. fixative, they can also be observed in thyroidectomy cells with other fixatives: in the latter case they may represent particular stages of activity. The immunocytochemical technique after embedding using P.A.P. has been used to determine the intracellular localization of TSH. We obtained positive staining of the entire granule and were not able to observe a difference in concentration of TSH between the small dense granule and the part of the granule with a moderate electron density. With this approach, only the granules appear to be positive, but as it is sometimes difficult to see the Golgi complex and the rough endoplasmic reticulum, we used the immunocytochemical technique before embedding in order to determine whether TSH might be present in the rough endoplasmic reticulum or in the Golgi complex. Negative results of the content of dilated rough endoplasmic reticulum cisternae and saccules of the Golgi complex may be due to the absence of immunogenic TSH molecules or according to Nakane (1975) to the effect of fixation which destroy or differentially denatures the antigen sites in the Golgi complex, in the rough endoplasmic reticulum or in both. On the other hand most of the membranes of the rough endoplasmic reticulum appear positive. So even if TSH molecules are largely confined to the granules it may be that some are present in the cytoplasm and also next to the membranes (perhaps on ribosomes ?). In several types of hormone secreting cells in culture, Tixier-Vidal and Tougard (1975) have obtained analogous results. In conclusion, one cell type having the same characteristics has been revealed by three different immunocytochemical techniques. This study has permitted us to obtain some details of the ultrastructure of the thyreotropic cells under physiological conditions. The TSH molecules are essentially confined to the granules. References Barnes, B.G.: Electron microscope studies on the secretory cytology of the mouse anterior pituitary. Endocrinology 71, 618-628 (1962) Barnes, B.G.: The fine structure of the mouse adenohypophysis in various physiological states: In: Cytologic de l'ad6nohypophyse (J. Benoit and C. Da Lage, eds.). Paris: Editions du Centre National de la Recherche Scientifique 1963 Baker, B.L., Yu, Y.Y.: The thyrotropic cell of the rat hypophysis as studied with peroxidase labeled antibody. Amer. J. Anat. 131, 55 72 (1971) Baker, B.L., Yu, Y.Y.: Hypophyseal changes induced by thyroid deficiency and thyroxine administration as revealed by immunochemical staining. Endocrinology 89, 996-1004 (1971) Beauvillain, J.-C., Tramu, G.: Cellules fi activit~ corticotrope de l'hypophyse du L6rot (Eliomys quercinus): superposition des r6sultats de microscopic optique (immunofluorescence et colorations) et de microscopic ~lectronique. C.R. Acad. Sci. (Paris). 277, 1025-1028 (1973) Beauvillain, J.-C., Tramu, G., Dubois, M.P.: Characterization by different techniques of adrenocorticotropin and gonadotropin-producing cells in Lerot pituitary (Eliomys quercinus). Cell Tiss. Res. 158, 301-317 (1975) Cuerdo-Rocha, S., Zambrano, D.: The action of protein synthesis inhibitors and thyrotropin releasing factor on the ultrastructures of rat thyrotrophs. J. Ultrastruct. Res. 48, 1-17 (1974) Cuerdo-Rocha, S., Zambrano, D.: Thyrotrophs of the rat anterior pituitary after different periods of thyroidectomy. A conventional and histochemical electron microscope study. J. Ultrastruct. Res. 49, 312-321 (1974) D'Angelo, S.A.: Cytophysiologic aspects of thyrotrophic hormone secretion in the goitrous guinea pig. Endocrinology 52, 331-337 (1953) Dubois, M.P.: Les cellules g hormones glycoprotidiques du lobe ant~rieur de l'hypophyse: s6paration
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par immunofluorescence des cellules thyr6otropes et des cellules gonadotropes dans l'hypophyse des bovins, ovins, porcins. Ann. Rech. Veter. 2, 197~22 (1971) Dubois, M.P.: Localisation par immunocytologie des hormones glycoprotidiques hypophysaires chez les vert6br6s. In: Pituitary glycoprotein hormones (INSERM, Paris, ed.) 2 7 4 7 (1972) Farquhar, M.G., Rinehart, J.F.: Cytologic alterations in the anterior pituitary gland following thyroidectomy. An electron microscope study. Endocrinology 55, 85%876 (1954) Graham, R.C., Karnovsky, M.J.: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-302 (1966) Herlant, M.: Apport de la microscopic 61ectronique ~ l'6tude du lobe ant6rieur de l'hypophyse: In: Cytologic de l'ad6nohypophyse (J. Benoit and C. Da Lage eds.). Paris: Editions du Centre National de la Recherche Scientifique 1963 Kurosumi, K., Oota, Y. : Corticotrophs in the anterior pituitary gland of gonadectomized and thyroidectomized rats as revealed by electron microscopy. Endocrinology 79, 808-814 (1966) Mayor, H.D., Hampton, J.C., Rosario, B.: A simple method for removing the resin from epoxy embedded tissue. J. biophys, biochem. Cytol. 9, 909-910 (1961) Mazzuca, M.: Individualisation morphofonctionelle des terminaisons nerveuses p6rivasculaires de l'6minence m6diane (chez les Mammif6res) en microscopie 61ectronique. R6union de la D.G.R.S.T. "Biologic de la Reproduction et du D6veloppement", Paris, Mai 1975 Mazzuca, M., Dubois, M.P.: Detection of luteinizing hormone releasing hormone in the guinea pig median eminence with an immunohistoenzymatic technique. J. Histochem. Cytochem. 22, 993 996 (1974) Messier, B. : Number and distribution of thyrotropic cells in the mouse pituitary gland. Anat. Rec. 153, 343-348 (1965) Moriarty, G.C. : Adenohypophysis: Ultrastructural cytochemistry. A review. J. Histochem. Cytochem. 21,855 894 (1973) Moriarty, G.C., Halmi, N.S.: Electron microscopic study of the adrenocorticotropin producing cell with the use of unlabeled antibody and the soluble peroxidase-antiperoxidase complex. J. Histochem. Cytochem. 20, 590-604 (1972) Nakane, P.K.: Classifications of anterior pituitary cell types with immunoenzyme histochemistry. J. Histochem. Cytochem. 18, 9-20 (1970) Nakane, P.K.: Identification of anterior pituitary cells by immunoelectron microscopy: In: The anterior pituitary (A. Tixier-Vidal and M.G. Farquhar, eds.). New York: Acad. Press 1975 Nakane, P.K., Pierce, Jr. G.B.: Enzyme labeled antibodies for the light and electron microscopic localization of tissue antigen. J. Cell Biol. 33, 307-318 (1967) Pelletier, G.: Secretion and uptake of peroxidase by rat adenohypophyseal cells J. Ultrastruct. Res. 43, 445~459 (1973) Pelletier, G., Puviani, R.: Detection of glycoproteins and autoradiographic localization of (3H) fucose in the thyroidectomy cells of rat anterior pituitary gland. J. Cell Biol. 56, 600-605 (1973) Petrali, J.P., Hinton, D.M., Moriarty, G.C., Sternberger, L.A.: The unlabeled antibody enzyme method of immunocytochemistry. Quantitative comparison of sensitivities with and without peroxidaseantiperoxidase complex. J. Histochem. Cytochem. 22, 78~801 (1974) Stefanini, M., De Martino, C., Zamboni, L.: Fixation of ejaculated spermatozoa for electron microscopy. Nature (Lond.) 216, 173 (1967) Tixier-Vidal, A., Tougard, C. : Subcellular localization of several anterior pituitary and hypothalamic hormones as revealed by the peroxidase labeled antibody method. First International Symposium on Immunoenzymatic technique. INSERM, April 1975 (in press) Tougard, C., Kerdelhue, B., Tixier-Vidal, A.: Light and electron microscope localization of binding sites of antibodies against ovine luteinizing hormone and its two subunits in rat adenohypophysis using peroxidase labeled antibody technique. J. Cell Biol. 5g, 503 521 (1973) Tramu, G.: Les cellules thyr6otropes de l'ant6hypophyse du cobaye mille au cours de diverses circonstances exp6rimentales et physiologiques. C.R. Soc. Bio. (Lille) 163, 1560-1563 (1969) Tramu, G., Beauvillain, J.-C., Dubois, M.P.: Les cellules gonadotropes de l'hypophyse du L6rot (Eliomys quercinus): concordances des r6sultats de microscopic optique (immunofluorescence et coloration) et de microscopic 61ectronique. J. de Microscopic 21, 181 184 (1974)
Accepted May 25, 1976
