Cell Tiss. Res. 174, 245-260 (1976)
Cell and Tissue Research 9 by Springer-Verlag 1976
Uitrastructural Localization of Prolactin, Growth Hormone and Luteinizing Hormone by Immunoeytochemical Techniques in the Bovine Pituitary F. Dacheux Laboratoire de Physiologie Compar~e, Facult6 des Sciences, Tours, France
M.P. Dubois I.N.R.A. - Station de Physiologic de la Reproduction, Nouzilly, France
Summary. The technique of ultrastructural immunocytochemistry involving the unlabeled antibody and the soluble peroxidase-antiperoxidase complex was used to identify and describe the prolactin (P) cells, somatotropic (STH) cells and luteinizing hormone (LH) cells in the bovine anterior pituitary gland. This method was used to localize the three hormones at the electron microscopic level. Staining of varying intensity was found on the secretory granules and on the small granules and vesicles within the Golgi complex. No stain was found in nuclei, on mitochondria or in the endoplasmic reticulum. Key words: Pituitary gland (bovine) - Prolactin - Growth hormone Luteinizing hormone - Immunocytochemistry, ultrastructural.
Introduction Sternberger et al. (1970) introduced the use of a soluble peroxidase-antiperoxidase complex (PAP) as an immunoenzymatic marker for the localization of antigenic sites in tissues at both the light and electron microscopic levels. This technique has been used successfully in rat pituitaries to localize adrenocorticotropic hormone (Moriarty and Halmi, 1972a, b; Moriarty et al., 1973), prolactin (Parsons and Erlandsen, 1974) and luteinizing hormone (Moriarty, 1975). In amphibians, Doerr-Schott and Dubois (1973) localized pituitary hormones at the ultrastructural level by the same method. The purpose of the present paper is to report findings on the application of this technique for the localization of prolactin hormone (P), luteinizing hormone (LH) and growth hormone (STH) in bovine pituitary cells at the electron microscopic level. This technique also permits an identification and ultrastructural characterization of these different cells. Send of/print requests to." M.P. Dubois, I.N.R.A. Physiologie Reproduction 37380 Nouzilly, France Abbreviations used in this article : P: Prolactin; STH: Growth Hormone; LH: Luteinizing Hormone;
A-oP: Antiserum to Ovine Prolactin; A-b LH: Antiserum to Bovine LH; A-pLt-I/~: Antiserum to Porcine LI.I/~ subunit; A-h STH: Antiserum to Human STH; HSA: Human Serum Albumin
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Material and Methods I. Ant&era Antigens used to prepare various antisera were: Ovine prolactin : NIH P S9 Bovine LH : NIH LH B 5 Human STH : KABI (Stockholm), 9 5 ~ monomeric growth hormone Porcine LH : fl subunit; prepared by Dr. Courte (France) Rabbit IgG : prepared by Dr. Levieux, I.N.R.A. (France) Anti O-Prolactin antisera was prepared by Dr. Gabrion (Facult6 de M6decine, Montpellier, France). The other antisera were prepared by one of the authors and their specificity checked as reported in previous publications (Dubois, 1969, 1971, 1972; Dubois and Dubois, 1974). The validity of A-h-STH as evidencing somatotrop cells was demonstrated by the identity of results using either A-b-STH (Dubois, 1969) or A-h-STH and by the ability of b-STH (NIH-STH B12) to inhibit the immunofluorescence reaction of somatotrop cells due to A-h-STH in the bovine pituitary.
II. Preparation of Tissues Bovine pituitaries from two nonpregnant cows and four oxen were studied. The tissue was fixed at 4~ either in 4 ~ glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, or in picric acid formaldehyde (PAF) for 8 h (Stefanini et al., 1967; Zamboni and De Martino, 1967). The pituitaries were then immersed 0.1 M phosphate buffer overnight at 4 ~C and post-fixed in 1~ OsO 4 for one h. After dehydration the pieces of tissues were embedded in Epon and polymerized at 60 ~C.
IIl. Immunocytological Reaction Ultrathin serial sections were cut on an ultramicrotome and mounted on gold grids. The technique adopted was that of Moriarty and Halmi (1972a). The sections were etched for 20 minutes with 10~o H202, washed in distilled deionized water and stained immunochemically for ten minutes in a drop of each bath. The baths consisted of: 1. normal sheep serum diluted 1/20; 2. antisera A-b LH, A-oP, A-pLHfl or A-h STH diluted 1/40 or 1/80; 3. antirabbit IgG sheep antiserum diluted 1/20; 4. PAP complex diluted 1/50 or 1/80 prepared by Dubois (M.P.) using the technique of Sternberger (1970). The staining solutions were diluted in tris buffered saline containing 0.25~ HSA except for normal sheep which was diluted in tris buffer. Between each bath, the grids were rinsed in tris buffered saline containing 0.25~ HSA. In all sections, peroxidase activity was revealed by 3-3'-diaminobenzidine and hydrogen peroxide (Graham and Karnovsky, 1966) or by 4 C 1-1 naphthol as substrate (Nakane, 1968, 1971). The reaction mixture was made up just before use. The solution was agitated during the staining procedure for five minutes in a beaker placed on a magnetic stirrer. After the reaction was completed, the grids were washed in distilled water and the reaction product was blackened with vapors of OsO 4 at 60 ~C. For immunohistochemical controls the specificity of the stain was tested by substitution of the following solutions for the antiserum: a) tris buffered saline, b) normal rabbit serum, c) absorbed antiserum. These saturated antisera were obtained by adding the antigen inhibitor in excess (LTH, STH, LH, LH/~) to the specific antiserum (100 or 300 gg antigen/ml antiserum). Additional controls involved the omission of one of the stain components: antirabbit IgG sheep antiserum, PAP or the substrate.
IV. Evidence for Cell Types: Comparison of Ultrastructural Morphology and Immunoeytology For this purpose, serial sections were observed on two different grids: on the first grid, the sections were stained with uranyl acetate and lead citrate for general morphology; the second grid was used for the immunocytochemical staining procedure. With this technique, the same cell could be observed for localization of antigen and for descriptive ultrastructural morphology.
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Results
A. Prolactin Cells (P cells) a) Ultrastructure. The cells containing prolactin are distributed throughout the pars distalis but are especially abundant in the peripheral area or "acidophilic zone". Unlike STH cells and LH cells which are scattered, P cells appear in clumps (Figs. 1-18). A marked polymorphism of prolactin cells is observed: some cells are large and oval in shape, while others are elongated with cytoplasmic processes (Fig. 18). A distinct polarization of organelles is also evident: the nucleus is eccentrically located in the cytoplasm, the well developed rough endoplasmic reticulum close to the plasma membrane is packed with regularly parallel cisternae at one pole of the cell (Fig. 7). However, enlargement of the cisternae is not uncommon, giving the appearance of irregularly outlined vacuoles (Fig. 18). The ribosomes are abundant and occur free in the cytoplasm or attached to the membrane of the endoplasmic reticulum (Fig. 7). The Golgi apparatus consists of circularly arranged stacks of cisternae with associated vesicles and a few larger vacuoles (Fig. 7). The mature secretory granules are located at the opposite pole of the cell. Evidence of granular extrusion by exocytosis is noted at the vascular pole of the cell where the granules are concentrated (Figs. 7, 8). The secretory granules, 350 nm to 500 nm in diameter, are spherical in shape and dense. Polymorphous granules as described by Smith and Farquhar (1966) in rat pituitary have not been observed. The mitochondria are well developed and usually elongated (Fig. 7). b) Immunocytochemical Staining. As illustrated in Figures 1, 2, 3, 4 and 8, prolactin cells in bovine pituitaries fixed with either glutaraldehyde or PAF are revealed by use of the unlabeled antibody-PAP complex technique. The reaction product associated with specific antibody is deposited over the round secretory granules of prolactin cells but not over the granules of other adjacent cell types (Figs. 1-4). Prolactin localization is not observed in association with the endoplasmic reticulum, mitochondria or nucleus (Fig. 8). However, similar to the larger mature granules, the small granules near the Golgi complex are stained
Figs. 1-3. Immunocytochemical localization of prolactin in PAF-fixed bovine pituitary (Figs. 1-2: cow pituitary; Fig. 3: ox pituitary). The positive staining prolactin cells (P) stand out in sharp contrast to the unstained secretory granules of somatotropic cells (S) and luteinizing hormone cells (L). 3-3' diaminobenzidine and H202 were used as substrate Fig. 4. Immunocytochemical localization of prolactin in cow pituitary. PAF fixation; substrate: C 1-1naphthol and H202. Unstained somatotropic cell (S) and clumps of stained prolactin cells (P) can be identified. Inset: Reaction product over the secretory granules Fig. 5. Immunocytochemical localization of somatotropic hormone in cow pituitary PAF fixation; substrate: 3-3'-diaminobenzidine and H202. The secretory granules are strongly stained (SG). Unstained nucleus (N) Fig. 6. High magnification of Figure 5. Reaction product over the secretory granules
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i m m u n o c y t o c h e m i c a l l y (Fig. 8). T h e stain, very dense over the granules, a p p e a r s as s p o t s o r c l u m p s o f v a r y i n g sizes at sites o f l o c a l i z a t i o n o f tissue a n t i g e n (Fig. 8, inset).
B. Somatotropic Cells ( S T H cells) a) Ultrastrueture. S T H cells are f o u n d t h r o u g h o u t the p a r s distalis a n d are d i s t r i b u t e d i n d e p e n d e n t l y a m o n g c l u m p s o f P cells. T h e y are r o u n d o r oval in shape, m e d i u m in size a n d c h a r a c t e r i z e d b y the presence o f a large a m o u n t o f s e c r e t o r y granules which are very dense, spherical in s h a p e a n d range f r o m 500 n m to 9 0 0 n m in d i a m e t e r (Figs. 17, 18). T h e e n d o p l a s m i c r e t i c u l u m a p p e a r s as series o f f l a t t e n e d sacs with a t t a c h e d ribosomes. O c c a s i o n a l l y , an e n l a r g e m e n t o f the sac occurs. T h e G o l g i a p p a r a t u s is well d e v e l o p e d a n d its l a m e l l a r structure is p r o m i n e n t (Figs. 17, 18) with a large n u m b e r o f small vesicles a n d secretory granules. T h e m i t o c h o n d r i a are u s u a l l y r o d - s h a p e d o r spherical a n d s o m e t i m e s t h e y m a y be elongated.
b) Immunocytoehemical Staining. E m p l o y i n g the P A P m e t h o d , secretory g r a n u l e s are specifically s t a i n e d i m m u n o c h e m i c a l l y for S T H (Figs. 5, 6). In c o n t r a s t , specific staining is n o t o b s e r v e d in a s s o c i a t i o n with the m e m b r a n e s o r cisternae o f the e n d o p l a s m i c reticulum. S o m e scattered P A P molecules are f o u n d a r o u n d the g r a n u l e s a n d in the c y t o p l a s m b u t in general such staining is n o t p r o m i n e n t (Fig. 6).
C. Gonadotropic Cells ( L H cells) a) Ultrastrueture. T h e g o n a d o t r o p i c cells in the b o v i n e p i t u i t a r y are d i s t r i b u t e d i n d e p e n d e n t l y in the p a r s distalis, being n u m e r o u s in the b a s o p h i l i c zone. The
Fig. 7. Prolactin cell in PAF-fixed cow pituitary. The rough endoplasmic reticulum (ER) is well developed close to the plasma membrane at one pole of the cell. The Golgi complex (G) near the nucleus (N) is associated with vesicles and small granules. The round secretory granules (RG)are located at the vascular pole, the mitochondria are often elongated (M). Note the granular extrusion by exocytosis
(arrow) Fig. 8. Same cell as in Figure 7 immunocytochemically stained with A-o P. Unstained endoplasmic reticulum (ER),mitochondria (M) and nucleus (N). Small granules (arrows)are positively stained near the Golgi complex (G). Inset: Reaction product over the secretory granules. The PAP complex molecules (whitearrows)can be identified over the granules as spots of varying sizes Fig. 9. Immunocytochemical localization of luteinizing hormone in gonadotropic cell of cow pituitary stained with A-p LHfl. Round secretory granules (RG) and some polymorphic granules (PG) show a strong staining. Globular bodies (GB)with clear vesiculation are negative Fig. 10. Gonadotropic cell stained with A-b LH. Unstained Golgi complex (G) and mitochondria (M). Round secretory granules (RG) and polymorphic granules (PG) are strongly stained. In the Golgi area, the saccules are negative (G) but the small positive secretory granules are numerous (arrows) Fig. 11. High magnification of Figure 10 with stained round granules (RG)and polymorphic granules (PG). Some PAP complex molecules around secretory granules or scattered in the cytoplasm (arrow) can be identified as flattened rings or pentagons
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Fig. 12. Gonadotropic cell (L) in glutaraldehyde-fixed pituitary stained with A-p LHfl. Secretory granules (SG) are heavily stained while those of an adjacent cell are negative. The saccules of Golgi complex are negative (G) Fig. 13. High magnification of Figure 12. There is variability in the intensity of the reaction on the granules. The stain is more intense on the round secretory granules (RG) and polymorphic granules (PG); the globular bodies (GB) show less reaction product
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cells are medium-sized, round or oval in shape, containing an eccentrically located large nucleus with a prominent nucleolus (Fig. 14). The mitochondria are spherical or elongated. The Golgi apparatus is moderately developed (Fig. 15). The cytoplasmic matrix sometimes has a high electron density. Most of the small granules, 250 nm to 400 nm in diameter are electron dense and regularly spherical (Figs. 14, 17). Accumulation of granules in the periphery of the cell is not uncommon; a clear zone without granules is frequently observed around the nucleus (Figs. 9, 12, 14, 15). Numerous large granules, irregular in shape and sometimes less dense than the smallest granules, are observed in the gonadotropic cells of all the adenohypophyses studied. The "polymorphic granules" are clearly membrane-enclosed and the globular content has an uniform granularity (Figs. 14, 17). In other granules or "globular bodies", the content is interrupted by occasional clear vesiculation (Fig. 14).
b) Immunocytochemical Staining. The subcellular binding sites of LH and LH fl are identical (Figs. 9, 10). The same cell type is positive for A-b LH and A-p LHfl (Figs. 9, 10). The small round secretory granules and some polymorphic granules show a strong staining (Figs. 9, 12, 13, 15, 16). The "globular bodies" often containing clear vesiculation show less reaction product (Fig. 13) or are negative (Figs. 9, 15). No staining is found in cisternae of endoplasmic reticulum, the mitochondria or the nucleus (Fig. 15). In the Golgi area, the saccules are negative (Figs. 10, 12, 15), but the small positive secretory granules are numerous (Figs. 10, 15). Diffuse staining is often found around the secretory granules and to a lesser extent scattered throughout the cytoplasm (Fig. 11). The PAP molecular complex can be identified as flattened rings or pentagons (Fig. 1l, arrows) which were originally described by Sternberger et al. (1970), Moriarty and Halmi (1972) and Moriarty (1973). The secretory granules of adjacent cells are completely devoid of immunologically localized reaction product (Figs. 9, 12).
D. Immunocytochemical Controls No staining is observed when antirabbit IgG sheep antiserum, PAP or the substrate are omitted. Substitution of normal serum or absorbed antiserum for the specific antiserum also prevents staining.
Fig. 14. Gonadotropic cell in PAF-fixed cow pituitary with eccentrically placed nucleus (N), elongated mitochondria (M), round secretory granules (RG), irregular polymorphic granules (PG)and some globular bodies with clear vesiculation (GB) Fig. 15. Immunocytochemical localization of luteinizing hormone in the same cell as in Figure 14, stained with A-p LH/L The small positive secretory granules (arrows)are present in the Golgi area (G); round mature secretory granules (RG) and polymorphic granules (PG) are strongly stained. N nucleus Fig. 16. High magnification of Figure 15. Reaction product over the round (RG) and polymorphic granules (PG)
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Discussion
Identification of Prolactin (P) Cells and STH Cells in the Bovine Adenohypophysis Mikami (1970) described "prolactin cells" in the peripheral area of the bovine adenohypophysis as being large in size, round or oval in shape and characterized by having the largest secretory granules (600 nm to 900 nm), spherical and very dense. With the unlabeled antibody and the peroxidase-antiperoxidase complex, these cells are identified as STH cells (Figs. 5, 6, 17). The cells described by Mikami (1970) as "STH cells" with secretory granules, 400 to 450nm in diameter, and cisternae of endoplasmic reticulum often dilated in the periphery of the cell correspond to P cells (Figs. 1, 2, 7, 8, 18).
Identification of Gonadotropic Cells Mikami (1970) and Heath (1970) described the tinctorial and ultrastructural characteristics of six types of glandular cells of the bovine pars distalis and identified two types of gonadotropic cells as LH and FSH cells. Contrary to these authors, we were not able to find a clear-cut ultrastructural difference speaking in favor of two types of gonadotropic cells. The ultrastructural immunocytology reveals only one type of gonadotropic cell staining with A-p LH fl and A-b LH and showing uniform morphology, according to light microscopic observations in this species (Dubois, 1972). The application of the c~ subunit-in order to inhibit the reaction obtained with antiserum to ovine FSH - produced, light microscopically, variable results showing in some cases persisting reactions of thyrotropic cells (cells positively marked by an antibody made specifically for TSH) and, in other cases, ofgonadotropic cells (Dubois, 1972). Under these circumstances it is not possible to draw positive conclusions with respect to the precise localization of FSH, in contrast to the light microscopic results obtained in man (Phifer et al., 1973) and in rat (Nakane, 1970; Tougard et al., 1971; Herbert, 1975).
Subcellular Localization of Prolactin, Growth Hormone and Lute&izing Hormone Secretory Granules. The secretory granules are always the main site of immunoreactivity for A-o P, A-b LH, A-p LH/~ and A-h STH. The polymorphic granules of LH cells are often strongly stained (Figs. 10, 11, 13, 16). Nevertheless, some polymorphic granules with vesiculation are negative (Figs. 9, 15). These results
Fig. 17. Bovine pituitary. PAF fixation. Note three somatotropic cells (S) with large, dense and round secretory granules (SG). The Golgi apparatus (G) is well developed and its lamellar structure is prominent. The gonadotropic cell (L) shows small secretory round granules (RG), globular bodies (GB) and elongated mitochondria (M) Fig. 18. Bovine pituitary. PAF fixation. Note the clump ofprolactin cells (P) with cytoplasmic processes, eccentrically located nucleus (N) and enlargement of the cisternae of endoplasmic reticulum (ER). Two STH cells (S) are seen with large dense secretory granules (SG)
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agree with those of Heath (1970) who demonstrated acid phosphatase activity in Golgi cisternae and very rarely in "globular bodies". These polymorphic bodies not staining with A-p LH/~ and A-b LH are probably lysosomes which correspond to vesiculation "globular bodies". The globular bodies of Heath (1970), negative for acid phosphatase, correspond to our large "polymorphic granules" strongly stained with antibodies A-p LH/~ and A-b LH. The secretory granules in prolactin cells of bovine pituitary are round and differ from those in rat pituitary which are often irregular and polymorphous (Smith and Farquhar, 1966; Nakane, 1970; Parsons and Erlandsen, 1974).
Golgi Complex and Endoplasmic Reticulum. No antigenicity is seen in the Golgi saccules, and the cisternae of the endoplasmic reticulum are generally negative. These results agree with the reports of Parsons and Erlandsen (1974) for prolactin cells and of Tougard et al. (1973) for LH cells: immunologic staining for prolactin cells and LH cells is not observed within cisternae of the endoplasmic reticulum or in the Golgi saccules. Contrary to this result, Nakane (1971) and Moriarty and Halmi (1972) for ACTH cells in rat pituitary localized the reaction product in the Golgi complex. In addition, these workers also reported occasional localization of prolactin and ACTH in the endoplasmic reticulum and its cisternae. Our immunocytochemical studies failed to reveal prolactin in dilated cisternae of the endoplasmic reticulum or in association with the membrane of the reticulum. The absence of intracisternal prolactin localization may mean that the concentration of prolactin in this site is below the level detectable by our procedure or that it may be due to alteration of antigenicity by fixation. However, since intense specific staining for prolactin was observed in small secretory granules within the Golgi area, our results may indicate, as Parsons and Erlandsen (1974) have suggested, that prolactin molecules are not immunologically recognizable until "packaging" and that tertiary structure is completed within the Golgi complex. In contrast to the results of Tougard et al. (1973), who found no positive secretory granules within the Golgi area in LH cells of rat pituitary, our results show small secretory granules stained with A-p LH/~ within the Golgi area. This difference may reflect differences between the immunohistochemical techniques used. In LH cells, cisternae of endoplasmic reticulum and Golgi saccules are negative. This is in agreement with the concept of the mechanism of glycoprotein biosynthesis in which the molecules synthetized on the ribosomes migrate into the endoplasmic reticulum, then into the Golgi complex where they are concentrated and completed by the addition of carbohydrate components. Scattered PAP molecules are found around the secretory granules or sometimes in the cytoplasm. However, in general, such cytoplasmic staining is not prominent. A cytoplasmic staining was reported for pituitary ACTH cells by Moriarty and Halmi (1972) and by Tougard et al. (1973) for LH cells with another immunohistochemical method. As the latter authors have suggested, this may represent LH molecules attached to ribosomes or polyribosomes or it may be due to LH stored free in the cytoplasm. In the present study, the specificity of the immunohistochemical method permits the identification of prolactin, STH and LH secreting cells in the bovine
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pituitary. It also provides findings concerning localization of LH fl and LH in the same gonadotroph cell. This does not exclude or include an affinity of these cells for anti-FSH antiserum.
References D oerr-Schott, J., Dubois, M.P.: Mise en 6vidence des hormones d'un Amphibien par la cyto-immunoenzymologie au microscope 61ectronique. C.R. Acad. Sci. (Paris) Ser. D 276, 2179-2182 (1973) Dubois, M.P.: Cytologic de l'hypophyse des bovins: s6paration des cellules somatotropes et des cellules prolactine par immunofluorescence. Identification des cellules LH darts la pars tuberalis et la pars intermedia. Bull. Ass. Anat. (Nancy) 145, 139-146 (1969) Dubois, M.P.: l~tude de l'apparition des secr6tions hormonales dans l'hypophyse foetale de bovin: mise en 6vidence par immunofluorescence des cellules somatotropes et des cellules ~t prolactine. C.R. Acad. Sci. (Paris) Ser. D 272, 433-435 (1971) Dubois, M.P.: Localisation par immunocytologie des hormones glycoprot6iques hypophysaires chez les Vert6br~s. Coll. ~Hormones glycoprotbiques hypophysaires~. I.N.S.E.R.M. 9, 27-47 (1972) Dubois, P.M., Dubois, M.P.: Mise en 6vidence par immunofluorescence de l'activit6 gonadotrope LH dans l'ant~hypophyse foetal humaine. Coll. ~Sexual Endocrinology of the perinatal period>~, Lyon (M.F. Forest & J. Bertrand, eds.). I.N.S.E.R.M. 32, 37-61 (1974) Graham, R.C., Jr., Karnovsky, M.: 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) Heath, E.: Cytology of the pars anterior of the bovine adenohypophysis. Amer. J. Anat. 127, 131-158 (1970) Herbert, D.C. : Localization of antisera to LH~ and FSHfl in the rat pituitary gland. Amer. J. Anat. 144, 379-385 (1975) Mikami, S. : Light and electron microscopic investigations of six types of glandular cells of the bovine adenohypophysis. Z. Zellforsch. 105, 45%482 (1970) Moriarty, G.C.: Adenohypophysis: ultrastructural cytochemistry. A review. J. Histochem. Cytochem. 21,855-894 (1973b) Moriarty, G.C.: Electron microscopic-immunocytochemical studies of rat pituitary gonadotrophs: a sc ~difference in morphology and cytochemistry of LH cells. Endocrinology 97, 1215-1225 (1975) 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, 590-603 (1972a) Moriarty, G.C., Halmi, N.S.: Adrenocorticotropin production by the intermediate lobe of the rat pituitary. An electron microscopic-immunohistochemical study. Z. Zellforsch. 132, 1-14 (1972b) Moriarty, G.C., Moriarty, C.M., Sternberger, L.A.: Ultrastructural immunocytochemistry with unlabeled antibodies and the peroxidase-antiperoxidase complex. A technique more sensitive than radioimmunoassay. J. Histochem. Cytochem. 21,825-833 (1973 a) Nakane, P.K.: Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat. J. Histochem. Cytochem. 16, 557-560 (1968) Nakane, P.K.: Classifications of anterior pituitary cell types with immunoenzyme histochemistry. J. Histochem. Cytochem. 18, 9-20 (1970) Nakane, P.K.: Application of peroxidase-labeled antibodies to the intracellular localization of hormones. Acta. endocr. (Kbh.), (Suppl.) 153, 190-202 (1971) Parsons, J.A., Erlandsen, S.L.: Ultrastructural immunocytochemical localization of prolactin in rat anterior pituitary by use of the unlabeled antibody enzyme method. J. Histochem, Cytochem. 22, 340-351 (1974) Phifer, R.F., Midgley, A.R., Spicer, S.S.: Immunohistologic and histologic evidence that folliclestimulating hormone and luteinizing hormone are present in the same cell type in the human pars distalis. J. clin. Endocr. 36, 125-141 (1973) Smith, R.E., Farquhar, M.G. : Lysosome function in the regulation of the secretory process in cells of the anterior pituitary gland. J. Cell Biol. 31, 319-347 (1966)
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Stefanini, M., Martino, C. de, Zamboni, L.: Fixation of ejaculated spermatozoa for electron microscopy. Nature (Lond.), 216, 173-174 (1967) Sternberger, L.A., Hardy, P.H., Jr., Cuculis, J.J., Meyer, H G : The unlabeled antibody enzyme method of immuno-histochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antiperoxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18, 315-333 (1970) Tougard, C., Kerdelhue, B., Tixier-Vidal, A., Justisz, M.: Localisation par cytoimmunoenzymologie de la LH, de ses sous-unit6s, ~ et/3 et de la FSH dans l'ad6nohypophyse de la Ratte castr6e. C.R. Acad. Sci. (Paris) Ser. D 273, 897-899 (1971) Tougard, C., Kerdelhue, B., Tixier-Vidal, A., Justisz, M.: 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. 58, 503-521 (1973) Zamboni, L., Martino, C. de: Buffered picric-acid formaldehyde: a new rapid fixation for electron microscopy. J. Cell Biol. 35, 148 A (1967)
Accepted July 12, 1976
Cell and Tissue Research 9 by Springer-Verlag 1976
Uitrastructural Localization of Prolactin, Growth Hormone and Luteinizing Hormone by Immunoeytochemical Techniques in the Bovine Pituitary F. Dacheux Laboratoire de Physiologie Compar~e, Facult6 des Sciences, Tours, France
M.P. Dubois I.N.R.A. - Station de Physiologic de la Reproduction, Nouzilly, France
Summary. The technique of ultrastructural immunocytochemistry involving the unlabeled antibody and the soluble peroxidase-antiperoxidase complex was used to identify and describe the prolactin (P) cells, somatotropic (STH) cells and luteinizing hormone (LH) cells in the bovine anterior pituitary gland. This method was used to localize the three hormones at the electron microscopic level. Staining of varying intensity was found on the secretory granules and on the small granules and vesicles within the Golgi complex. No stain was found in nuclei, on mitochondria or in the endoplasmic reticulum. Key words: Pituitary gland (bovine) - Prolactin - Growth hormone Luteinizing hormone - Immunocytochemistry, ultrastructural.
Introduction Sternberger et al. (1970) introduced the use of a soluble peroxidase-antiperoxidase complex (PAP) as an immunoenzymatic marker for the localization of antigenic sites in tissues at both the light and electron microscopic levels. This technique has been used successfully in rat pituitaries to localize adrenocorticotropic hormone (Moriarty and Halmi, 1972a, b; Moriarty et al., 1973), prolactin (Parsons and Erlandsen, 1974) and luteinizing hormone (Moriarty, 1975). In amphibians, Doerr-Schott and Dubois (1973) localized pituitary hormones at the ultrastructural level by the same method. The purpose of the present paper is to report findings on the application of this technique for the localization of prolactin hormone (P), luteinizing hormone (LH) and growth hormone (STH) in bovine pituitary cells at the electron microscopic level. This technique also permits an identification and ultrastructural characterization of these different cells. Send of/print requests to." M.P. Dubois, I.N.R.A. Physiologie Reproduction 37380 Nouzilly, France Abbreviations used in this article : P: Prolactin; STH: Growth Hormone; LH: Luteinizing Hormone;
A-oP: Antiserum to Ovine Prolactin; A-b LH: Antiserum to Bovine LH; A-pLt-I/~: Antiserum to Porcine LI.I/~ subunit; A-h STH: Antiserum to Human STH; HSA: Human Serum Albumin
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Material and Methods I. Ant&era Antigens used to prepare various antisera were: Ovine prolactin : NIH P S9 Bovine LH : NIH LH B 5 Human STH : KABI (Stockholm), 9 5 ~ monomeric growth hormone Porcine LH : fl subunit; prepared by Dr. Courte (France) Rabbit IgG : prepared by Dr. Levieux, I.N.R.A. (France) Anti O-Prolactin antisera was prepared by Dr. Gabrion (Facult6 de M6decine, Montpellier, France). The other antisera were prepared by one of the authors and their specificity checked as reported in previous publications (Dubois, 1969, 1971, 1972; Dubois and Dubois, 1974). The validity of A-h-STH as evidencing somatotrop cells was demonstrated by the identity of results using either A-b-STH (Dubois, 1969) or A-h-STH and by the ability of b-STH (NIH-STH B12) to inhibit the immunofluorescence reaction of somatotrop cells due to A-h-STH in the bovine pituitary.
II. Preparation of Tissues Bovine pituitaries from two nonpregnant cows and four oxen were studied. The tissue was fixed at 4~ either in 4 ~ glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, or in picric acid formaldehyde (PAF) for 8 h (Stefanini et al., 1967; Zamboni and De Martino, 1967). The pituitaries were then immersed 0.1 M phosphate buffer overnight at 4 ~C and post-fixed in 1~ OsO 4 for one h. After dehydration the pieces of tissues were embedded in Epon and polymerized at 60 ~C.
IIl. Immunocytological Reaction Ultrathin serial sections were cut on an ultramicrotome and mounted on gold grids. The technique adopted was that of Moriarty and Halmi (1972a). The sections were etched for 20 minutes with 10~o H202, washed in distilled deionized water and stained immunochemically for ten minutes in a drop of each bath. The baths consisted of: 1. normal sheep serum diluted 1/20; 2. antisera A-b LH, A-oP, A-pLHfl or A-h STH diluted 1/40 or 1/80; 3. antirabbit IgG sheep antiserum diluted 1/20; 4. PAP complex diluted 1/50 or 1/80 prepared by Dubois (M.P.) using the technique of Sternberger (1970). The staining solutions were diluted in tris buffered saline containing 0.25~ HSA except for normal sheep which was diluted in tris buffer. Between each bath, the grids were rinsed in tris buffered saline containing 0.25~ HSA. In all sections, peroxidase activity was revealed by 3-3'-diaminobenzidine and hydrogen peroxide (Graham and Karnovsky, 1966) or by 4 C 1-1 naphthol as substrate (Nakane, 1968, 1971). The reaction mixture was made up just before use. The solution was agitated during the staining procedure for five minutes in a beaker placed on a magnetic stirrer. After the reaction was completed, the grids were washed in distilled water and the reaction product was blackened with vapors of OsO 4 at 60 ~C. For immunohistochemical controls the specificity of the stain was tested by substitution of the following solutions for the antiserum: a) tris buffered saline, b) normal rabbit serum, c) absorbed antiserum. These saturated antisera were obtained by adding the antigen inhibitor in excess (LTH, STH, LH, LH/~) to the specific antiserum (100 or 300 gg antigen/ml antiserum). Additional controls involved the omission of one of the stain components: antirabbit IgG sheep antiserum, PAP or the substrate.
IV. Evidence for Cell Types: Comparison of Ultrastructural Morphology and Immunoeytology For this purpose, serial sections were observed on two different grids: on the first grid, the sections were stained with uranyl acetate and lead citrate for general morphology; the second grid was used for the immunocytochemical staining procedure. With this technique, the same cell could be observed for localization of antigen and for descriptive ultrastructural morphology.
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Results
A. Prolactin Cells (P cells) a) Ultrastructure. The cells containing prolactin are distributed throughout the pars distalis but are especially abundant in the peripheral area or "acidophilic zone". Unlike STH cells and LH cells which are scattered, P cells appear in clumps (Figs. 1-18). A marked polymorphism of prolactin cells is observed: some cells are large and oval in shape, while others are elongated with cytoplasmic processes (Fig. 18). A distinct polarization of organelles is also evident: the nucleus is eccentrically located in the cytoplasm, the well developed rough endoplasmic reticulum close to the plasma membrane is packed with regularly parallel cisternae at one pole of the cell (Fig. 7). However, enlargement of the cisternae is not uncommon, giving the appearance of irregularly outlined vacuoles (Fig. 18). The ribosomes are abundant and occur free in the cytoplasm or attached to the membrane of the endoplasmic reticulum (Fig. 7). The Golgi apparatus consists of circularly arranged stacks of cisternae with associated vesicles and a few larger vacuoles (Fig. 7). The mature secretory granules are located at the opposite pole of the cell. Evidence of granular extrusion by exocytosis is noted at the vascular pole of the cell where the granules are concentrated (Figs. 7, 8). The secretory granules, 350 nm to 500 nm in diameter, are spherical in shape and dense. Polymorphous granules as described by Smith and Farquhar (1966) in rat pituitary have not been observed. The mitochondria are well developed and usually elongated (Fig. 7). b) Immunocytochemical Staining. As illustrated in Figures 1, 2, 3, 4 and 8, prolactin cells in bovine pituitaries fixed with either glutaraldehyde or PAF are revealed by use of the unlabeled antibody-PAP complex technique. The reaction product associated with specific antibody is deposited over the round secretory granules of prolactin cells but not over the granules of other adjacent cell types (Figs. 1-4). Prolactin localization is not observed in association with the endoplasmic reticulum, mitochondria or nucleus (Fig. 8). However, similar to the larger mature granules, the small granules near the Golgi complex are stained
Figs. 1-3. Immunocytochemical localization of prolactin in PAF-fixed bovine pituitary (Figs. 1-2: cow pituitary; Fig. 3: ox pituitary). The positive staining prolactin cells (P) stand out in sharp contrast to the unstained secretory granules of somatotropic cells (S) and luteinizing hormone cells (L). 3-3' diaminobenzidine and H202 were used as substrate Fig. 4. Immunocytochemical localization of prolactin in cow pituitary. PAF fixation; substrate: C 1-1naphthol and H202. Unstained somatotropic cell (S) and clumps of stained prolactin cells (P) can be identified. Inset: Reaction product over the secretory granules Fig. 5. Immunocytochemical localization of somatotropic hormone in cow pituitary PAF fixation; substrate: 3-3'-diaminobenzidine and H202. The secretory granules are strongly stained (SG). Unstained nucleus (N) Fig. 6. High magnification of Figure 5. Reaction product over the secretory granules
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i m m u n o c y t o c h e m i c a l l y (Fig. 8). T h e stain, very dense over the granules, a p p e a r s as s p o t s o r c l u m p s o f v a r y i n g sizes at sites o f l o c a l i z a t i o n o f tissue a n t i g e n (Fig. 8, inset).
B. Somatotropic Cells ( S T H cells) a) Ultrastrueture. S T H cells are f o u n d t h r o u g h o u t the p a r s distalis a n d are d i s t r i b u t e d i n d e p e n d e n t l y a m o n g c l u m p s o f P cells. T h e y are r o u n d o r oval in shape, m e d i u m in size a n d c h a r a c t e r i z e d b y the presence o f a large a m o u n t o f s e c r e t o r y granules which are very dense, spherical in s h a p e a n d range f r o m 500 n m to 9 0 0 n m in d i a m e t e r (Figs. 17, 18). T h e e n d o p l a s m i c r e t i c u l u m a p p e a r s as series o f f l a t t e n e d sacs with a t t a c h e d ribosomes. O c c a s i o n a l l y , an e n l a r g e m e n t o f the sac occurs. T h e G o l g i a p p a r a t u s is well d e v e l o p e d a n d its l a m e l l a r structure is p r o m i n e n t (Figs. 17, 18) with a large n u m b e r o f small vesicles a n d secretory granules. T h e m i t o c h o n d r i a are u s u a l l y r o d - s h a p e d o r spherical a n d s o m e t i m e s t h e y m a y be elongated.
b) Immunocytoehemical Staining. E m p l o y i n g the P A P m e t h o d , secretory g r a n u l e s are specifically s t a i n e d i m m u n o c h e m i c a l l y for S T H (Figs. 5, 6). In c o n t r a s t , specific staining is n o t o b s e r v e d in a s s o c i a t i o n with the m e m b r a n e s o r cisternae o f the e n d o p l a s m i c reticulum. S o m e scattered P A P molecules are f o u n d a r o u n d the g r a n u l e s a n d in the c y t o p l a s m b u t in general such staining is n o t p r o m i n e n t (Fig. 6).
C. Gonadotropic Cells ( L H cells) a) Ultrastrueture. T h e g o n a d o t r o p i c cells in the b o v i n e p i t u i t a r y are d i s t r i b u t e d i n d e p e n d e n t l y in the p a r s distalis, being n u m e r o u s in the b a s o p h i l i c zone. The
Fig. 7. Prolactin cell in PAF-fixed cow pituitary. The rough endoplasmic reticulum (ER) is well developed close to the plasma membrane at one pole of the cell. The Golgi complex (G) near the nucleus (N) is associated with vesicles and small granules. The round secretory granules (RG)are located at the vascular pole, the mitochondria are often elongated (M). Note the granular extrusion by exocytosis
(arrow) Fig. 8. Same cell as in Figure 7 immunocytochemically stained with A-o P. Unstained endoplasmic reticulum (ER),mitochondria (M) and nucleus (N). Small granules (arrows)are positively stained near the Golgi complex (G). Inset: Reaction product over the secretory granules. The PAP complex molecules (whitearrows)can be identified over the granules as spots of varying sizes Fig. 9. Immunocytochemical localization of luteinizing hormone in gonadotropic cell of cow pituitary stained with A-p LHfl. Round secretory granules (RG) and some polymorphic granules (PG) show a strong staining. Globular bodies (GB)with clear vesiculation are negative Fig. 10. Gonadotropic cell stained with A-b LH. Unstained Golgi complex (G) and mitochondria (M). Round secretory granules (RG) and polymorphic granules (PG) are strongly stained. In the Golgi area, the saccules are negative (G) but the small positive secretory granules are numerous (arrows) Fig. 11. High magnification of Figure 10 with stained round granules (RG)and polymorphic granules (PG). Some PAP complex molecules around secretory granules or scattered in the cytoplasm (arrow) can be identified as flattened rings or pentagons
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Fig. 12. Gonadotropic cell (L) in glutaraldehyde-fixed pituitary stained with A-p LHfl. Secretory granules (SG) are heavily stained while those of an adjacent cell are negative. The saccules of Golgi complex are negative (G) Fig. 13. High magnification of Figure 12. There is variability in the intensity of the reaction on the granules. The stain is more intense on the round secretory granules (RG) and polymorphic granules (PG); the globular bodies (GB) show less reaction product
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cells are medium-sized, round or oval in shape, containing an eccentrically located large nucleus with a prominent nucleolus (Fig. 14). The mitochondria are spherical or elongated. The Golgi apparatus is moderately developed (Fig. 15). The cytoplasmic matrix sometimes has a high electron density. Most of the small granules, 250 nm to 400 nm in diameter are electron dense and regularly spherical (Figs. 14, 17). Accumulation of granules in the periphery of the cell is not uncommon; a clear zone without granules is frequently observed around the nucleus (Figs. 9, 12, 14, 15). Numerous large granules, irregular in shape and sometimes less dense than the smallest granules, are observed in the gonadotropic cells of all the adenohypophyses studied. The "polymorphic granules" are clearly membrane-enclosed and the globular content has an uniform granularity (Figs. 14, 17). In other granules or "globular bodies", the content is interrupted by occasional clear vesiculation (Fig. 14).
b) Immunocytochemical Staining. The subcellular binding sites of LH and LH fl are identical (Figs. 9, 10). The same cell type is positive for A-b LH and A-p LHfl (Figs. 9, 10). The small round secretory granules and some polymorphic granules show a strong staining (Figs. 9, 12, 13, 15, 16). The "globular bodies" often containing clear vesiculation show less reaction product (Fig. 13) or are negative (Figs. 9, 15). No staining is found in cisternae of endoplasmic reticulum, the mitochondria or the nucleus (Fig. 15). In the Golgi area, the saccules are negative (Figs. 10, 12, 15), but the small positive secretory granules are numerous (Figs. 10, 15). Diffuse staining is often found around the secretory granules and to a lesser extent scattered throughout the cytoplasm (Fig. 11). The PAP molecular complex can be identified as flattened rings or pentagons (Fig. 1l, arrows) which were originally described by Sternberger et al. (1970), Moriarty and Halmi (1972) and Moriarty (1973). The secretory granules of adjacent cells are completely devoid of immunologically localized reaction product (Figs. 9, 12).
D. Immunocytochemical Controls No staining is observed when antirabbit IgG sheep antiserum, PAP or the substrate are omitted. Substitution of normal serum or absorbed antiserum for the specific antiserum also prevents staining.
Fig. 14. Gonadotropic cell in PAF-fixed cow pituitary with eccentrically placed nucleus (N), elongated mitochondria (M), round secretory granules (RG), irregular polymorphic granules (PG)and some globular bodies with clear vesiculation (GB) Fig. 15. Immunocytochemical localization of luteinizing hormone in the same cell as in Figure 14, stained with A-p LH/L The small positive secretory granules (arrows)are present in the Golgi area (G); round mature secretory granules (RG) and polymorphic granules (PG) are strongly stained. N nucleus Fig. 16. High magnification of Figure 15. Reaction product over the round (RG) and polymorphic granules (PG)
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Discussion
Identification of Prolactin (P) Cells and STH Cells in the Bovine Adenohypophysis Mikami (1970) described "prolactin cells" in the peripheral area of the bovine adenohypophysis as being large in size, round or oval in shape and characterized by having the largest secretory granules (600 nm to 900 nm), spherical and very dense. With the unlabeled antibody and the peroxidase-antiperoxidase complex, these cells are identified as STH cells (Figs. 5, 6, 17). The cells described by Mikami (1970) as "STH cells" with secretory granules, 400 to 450nm in diameter, and cisternae of endoplasmic reticulum often dilated in the periphery of the cell correspond to P cells (Figs. 1, 2, 7, 8, 18).
Identification of Gonadotropic Cells Mikami (1970) and Heath (1970) described the tinctorial and ultrastructural characteristics of six types of glandular cells of the bovine pars distalis and identified two types of gonadotropic cells as LH and FSH cells. Contrary to these authors, we were not able to find a clear-cut ultrastructural difference speaking in favor of two types of gonadotropic cells. The ultrastructural immunocytology reveals only one type of gonadotropic cell staining with A-p LH fl and A-b LH and showing uniform morphology, according to light microscopic observations in this species (Dubois, 1972). The application of the c~ subunit-in order to inhibit the reaction obtained with antiserum to ovine FSH - produced, light microscopically, variable results showing in some cases persisting reactions of thyrotropic cells (cells positively marked by an antibody made specifically for TSH) and, in other cases, ofgonadotropic cells (Dubois, 1972). Under these circumstances it is not possible to draw positive conclusions with respect to the precise localization of FSH, in contrast to the light microscopic results obtained in man (Phifer et al., 1973) and in rat (Nakane, 1970; Tougard et al., 1971; Herbert, 1975).
Subcellular Localization of Prolactin, Growth Hormone and Lute&izing Hormone Secretory Granules. The secretory granules are always the main site of immunoreactivity for A-o P, A-b LH, A-p LH/~ and A-h STH. The polymorphic granules of LH cells are often strongly stained (Figs. 10, 11, 13, 16). Nevertheless, some polymorphic granules with vesiculation are negative (Figs. 9, 15). These results
Fig. 17. Bovine pituitary. PAF fixation. Note three somatotropic cells (S) with large, dense and round secretory granules (SG). The Golgi apparatus (G) is well developed and its lamellar structure is prominent. The gonadotropic cell (L) shows small secretory round granules (RG), globular bodies (GB) and elongated mitochondria (M) Fig. 18. Bovine pituitary. PAF fixation. Note the clump ofprolactin cells (P) with cytoplasmic processes, eccentrically located nucleus (N) and enlargement of the cisternae of endoplasmic reticulum (ER). Two STH cells (S) are seen with large dense secretory granules (SG)
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agree with those of Heath (1970) who demonstrated acid phosphatase activity in Golgi cisternae and very rarely in "globular bodies". These polymorphic bodies not staining with A-p LH/~ and A-b LH are probably lysosomes which correspond to vesiculation "globular bodies". The globular bodies of Heath (1970), negative for acid phosphatase, correspond to our large "polymorphic granules" strongly stained with antibodies A-p LH/~ and A-b LH. The secretory granules in prolactin cells of bovine pituitary are round and differ from those in rat pituitary which are often irregular and polymorphous (Smith and Farquhar, 1966; Nakane, 1970; Parsons and Erlandsen, 1974).
Golgi Complex and Endoplasmic Reticulum. No antigenicity is seen in the Golgi saccules, and the cisternae of the endoplasmic reticulum are generally negative. These results agree with the reports of Parsons and Erlandsen (1974) for prolactin cells and of Tougard et al. (1973) for LH cells: immunologic staining for prolactin cells and LH cells is not observed within cisternae of the endoplasmic reticulum or in the Golgi saccules. Contrary to this result, Nakane (1971) and Moriarty and Halmi (1972) for ACTH cells in rat pituitary localized the reaction product in the Golgi complex. In addition, these workers also reported occasional localization of prolactin and ACTH in the endoplasmic reticulum and its cisternae. Our immunocytochemical studies failed to reveal prolactin in dilated cisternae of the endoplasmic reticulum or in association with the membrane of the reticulum. The absence of intracisternal prolactin localization may mean that the concentration of prolactin in this site is below the level detectable by our procedure or that it may be due to alteration of antigenicity by fixation. However, since intense specific staining for prolactin was observed in small secretory granules within the Golgi area, our results may indicate, as Parsons and Erlandsen (1974) have suggested, that prolactin molecules are not immunologically recognizable until "packaging" and that tertiary structure is completed within the Golgi complex. In contrast to the results of Tougard et al. (1973), who found no positive secretory granules within the Golgi area in LH cells of rat pituitary, our results show small secretory granules stained with A-p LH/~ within the Golgi area. This difference may reflect differences between the immunohistochemical techniques used. In LH cells, cisternae of endoplasmic reticulum and Golgi saccules are negative. This is in agreement with the concept of the mechanism of glycoprotein biosynthesis in which the molecules synthetized on the ribosomes migrate into the endoplasmic reticulum, then into the Golgi complex where they are concentrated and completed by the addition of carbohydrate components. Scattered PAP molecules are found around the secretory granules or sometimes in the cytoplasm. However, in general, such cytoplasmic staining is not prominent. A cytoplasmic staining was reported for pituitary ACTH cells by Moriarty and Halmi (1972) and by Tougard et al. (1973) for LH cells with another immunohistochemical method. As the latter authors have suggested, this may represent LH molecules attached to ribosomes or polyribosomes or it may be due to LH stored free in the cytoplasm. In the present study, the specificity of the immunohistochemical method permits the identification of prolactin, STH and LH secreting cells in the bovine
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pituitary. It also provides findings concerning localization of LH fl and LH in the same gonadotroph cell. This does not exclude or include an affinity of these cells for anti-FSH antiserum.
References D oerr-Schott, J., Dubois, M.P.: Mise en 6vidence des hormones d'un Amphibien par la cyto-immunoenzymologie au microscope 61ectronique. C.R. Acad. Sci. (Paris) Ser. D 276, 2179-2182 (1973) Dubois, M.P.: Cytologic de l'hypophyse des bovins: s6paration des cellules somatotropes et des cellules prolactine par immunofluorescence. Identification des cellules LH darts la pars tuberalis et la pars intermedia. Bull. Ass. Anat. (Nancy) 145, 139-146 (1969) Dubois, M.P.: l~tude de l'apparition des secr6tions hormonales dans l'hypophyse foetale de bovin: mise en 6vidence par immunofluorescence des cellules somatotropes et des cellules ~t prolactine. C.R. Acad. Sci. (Paris) Ser. D 272, 433-435 (1971) Dubois, M.P.: Localisation par immunocytologie des hormones glycoprot6iques hypophysaires chez les Vert6br~s. Coll. ~Hormones glycoprotbiques hypophysaires~. I.N.S.E.R.M. 9, 27-47 (1972) Dubois, P.M., Dubois, M.P.: Mise en 6vidence par immunofluorescence de l'activit6 gonadotrope LH dans l'ant~hypophyse foetal humaine. Coll. ~Sexual Endocrinology of the perinatal period>~, Lyon (M.F. Forest & J. Bertrand, eds.). I.N.S.E.R.M. 32, 37-61 (1974) Graham, R.C., Jr., Karnovsky, M.: 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) Heath, E.: Cytology of the pars anterior of the bovine adenohypophysis. Amer. J. Anat. 127, 131-158 (1970) Herbert, D.C. : Localization of antisera to LH~ and FSHfl in the rat pituitary gland. Amer. J. Anat. 144, 379-385 (1975) Mikami, S. : Light and electron microscopic investigations of six types of glandular cells of the bovine adenohypophysis. Z. Zellforsch. 105, 45%482 (1970) Moriarty, G.C.: Adenohypophysis: ultrastructural cytochemistry. A review. J. Histochem. Cytochem. 21,855-894 (1973b) Moriarty, G.C.: Electron microscopic-immunocytochemical studies of rat pituitary gonadotrophs: a sc ~difference in morphology and cytochemistry of LH cells. Endocrinology 97, 1215-1225 (1975) 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, 590-603 (1972a) Moriarty, G.C., Halmi, N.S.: Adrenocorticotropin production by the intermediate lobe of the rat pituitary. An electron microscopic-immunohistochemical study. Z. Zellforsch. 132, 1-14 (1972b) Moriarty, G.C., Moriarty, C.M., Sternberger, L.A.: Ultrastructural immunocytochemistry with unlabeled antibodies and the peroxidase-antiperoxidase complex. A technique more sensitive than radioimmunoassay. J. Histochem. Cytochem. 21,825-833 (1973 a) Nakane, P.K.: Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat. J. Histochem. Cytochem. 16, 557-560 (1968) Nakane, P.K.: Classifications of anterior pituitary cell types with immunoenzyme histochemistry. J. Histochem. Cytochem. 18, 9-20 (1970) Nakane, P.K.: Application of peroxidase-labeled antibodies to the intracellular localization of hormones. Acta. endocr. (Kbh.), (Suppl.) 153, 190-202 (1971) Parsons, J.A., Erlandsen, S.L.: Ultrastructural immunocytochemical localization of prolactin in rat anterior pituitary by use of the unlabeled antibody enzyme method. J. Histochem, Cytochem. 22, 340-351 (1974) Phifer, R.F., Midgley, A.R., Spicer, S.S.: Immunohistologic and histologic evidence that folliclestimulating hormone and luteinizing hormone are present in the same cell type in the human pars distalis. J. clin. Endocr. 36, 125-141 (1973) Smith, R.E., Farquhar, M.G. : Lysosome function in the regulation of the secretory process in cells of the anterior pituitary gland. J. Cell Biol. 31, 319-347 (1966)
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Stefanini, M., Martino, C. de, Zamboni, L.: Fixation of ejaculated spermatozoa for electron microscopy. Nature (Lond.), 216, 173-174 (1967) Sternberger, L.A., Hardy, P.H., Jr., Cuculis, J.J., Meyer, H G : The unlabeled antibody enzyme method of immuno-histochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antiperoxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18, 315-333 (1970) Tougard, C., Kerdelhue, B., Tixier-Vidal, A., Justisz, M.: Localisation par cytoimmunoenzymologie de la LH, de ses sous-unit6s, ~ et/3 et de la FSH dans l'ad6nohypophyse de la Ratte castr6e. C.R. Acad. Sci. (Paris) Ser. D 273, 897-899 (1971) Tougard, C., Kerdelhue, B., Tixier-Vidal, A., Justisz, M.: 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. 58, 503-521 (1973) Zamboni, L., Martino, C. de: Buffered picric-acid formaldehyde: a new rapid fixation for electron microscopy. J. Cell Biol. 35, 148 A (1967)
Accepted July 12, 1976
