Electron Microscopy and Histochemical Correlation of Human Anterior Pituitary Cells

Carlos Paiz, MD and Gordon R. Hennigar, MD

THE PARS ANrERIOR of human adenohypophysis has been studied extensively by histochemical and tinctorial methods."-8 Multiple nomenclatures have been developed to describe the various cell types, but the ultimate goal is to adopt a terminology based solely on function. Electron microscopy has served to clarify in part the fine structure of the adenohypophysis, and its contribution to date has been the attempted correlation of granular size and shape with specific hormonal secretion.9-" Similar studies have been made in animal species other than man.12-'7 In several human cell types, the granules are so similar that it is fre- quently difficult if not impossible to distinguish the cells on morphologic grounds alone. The use of thick-thin section correlation for light and electron microscopy can, in part, clarify this problem, since histochemical properties at the light level may be correlated with fine structural differences within the same cell at the electron microscopic level. Such correlations have served three purposes: (1) We have been able to relate certain serous, mucoid, and seemingly chromophobe cells of light microscopy with the corresponding electron microscopic equivalents. (2) In so doing, we have demonstrated that cells having the same or similar granule morphology with electron microscopy are strik- ingly different with light microscopy histochemistry. (3) The thick-thin section method of comparison has provided us with the opportunity to relate fine structural differences within pituitary cells with the corresponding variable dye binding seen in a single cell with light microscopy. Materials and Methods Adenohypophyses were obtained from 11 cases of necropsy and in 1 instance following surgical hypophysectomy (Table 1). The adenohypophysis obtained at surgery was received in small fragments so From the Departments of Pathology of the State University of New York Downstate Medical Center, Brooklyn, NY, and Veterans Administration Hospital, Medical University of South Carolina, Charleston, SC. Supported by NIH Grants CA06081 and AM10956. Accepted for publication Oct 14, 1969. Address for reprint requests: Dr. Gordon R. Hennigar, Department of Pathology, Med- ical University of South Carolina, 80 Barre St, Charleston, SC 29403. 43 44 PAIZ AND HENNIGAR American Journal of Pathology

Table 1. Survey of Cases of Human Adenohypophysis Interval between death No. Age Sex Race and fixation Diagnosis Cause of death 1 40 M W 6hr 50min Diabetes mellitus Heart failure 2 52 M W 10hr 00min Diabetes mellitus Acute myocardial infarction 3 87 F W 3hr 45min Bronchopneumonia Bronchopneumonia 4 51 M PR lhr 00min Laennec's cirrhosis Acute abdominal hemorrhage 5 37 M N 8hr 15min Alcoholism Bronchopneumonia 6 33 F N 6hr 00min Pregnancy Abortion; Uterine rupture 7 60 F W 4hr 30min Obesity Bronchopneumonia 8 53 M W 2hr 10min Laennec's cirrhosis Postoperative shock 9 29 F N 32hr 00min Suicide 10 35 M N 9hr 15min Homicide 11 22 M W 16hr 00min Suicide 12 62 F W Ohr 10min Ca of breast Surgical specimen; alive that it was impossible to determine the anatomic site of origin of the cells. On the other hand, necropsy specimens could be oriented as to anatomic site. The glands were first cut in their greatest horizontal diameter. A 1-mm slice of tissue following the original plane of section was then made and the tissue was placed on a sheet of dental wax and covered with several drops of glutaraldehyde. Tissue excised for electron microscopy was obtained from three different zonal sources labeled A, B, and C (Text-fig 1). Five 1-cu mm tissue blocks were obtained from each zone of the horizontal sections and fixed 1 hr at 6°C in Sabatini's glutaraldehyde.18 The tissue was then washed in three changes of phosphate buffer (30 min each) and then postosmicated (1 hr) in 1% osmium tetroxide in Palade's phosphate buffer (pH 7.4) 19 containing sucrose. The blocks were rapidly dehydrated through graded alcohol solutions and embedded in Epon.20 Thin sections were cut with a Huxley ultramicrotome and stained with either lead hydroxide or uranyl acetate.21 The adjacent thick section was treated with a solution of 3% NaOH in 80% ethyl alcohol for 5 min. This solution dissolved the embedding media and facilitated the staining of the thick section by PAS-Orange G sequence. Magnifications in the electron microscope were determined against a carbon grating replica calibration standard (No. 115) with 28,800 lines/linear in. (Ern- est F. Fullam, Inc., Schenectady, NY). The time elapsed between the death of the individual and fixation of the pituitary varied from 30 min to 32 hr. The surgical specimen was fixed within 10 min of its removal. This relatively wide range provided us with some preliminary basis for the evaluation of pituitary cell preservation following various postmortem in- tervals. Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 45 April 1970 ZONAL SOURCE ANT. OF SAMPLE

TEXT-FIG 1. Ant = anterior, P = posterior, pars nervosa, A = serous wing, B = mucoid wedge, C = postenror mucoid wedge. Tissue excised for electron microscopy obtained from three different zonal sources labelled A, B, and C. Zone A corresponds to lateral wings of adenohypophysis, and is heavily populated by "serous or acidophil cells." Zone B consisted of anteromedial portion of gland which has a predominantly mucoid cell population. Zone C was the area equivalent to the pars intermedia in other than human animal species.

One surgical specimen (Case 12) and one necropsy specimen (Case 6) were correlated by the adjacent thick-thin section method. The same cell field in electron micrographs was compared to the adjacent section stained by PAS-Orange G.22,23 Results Although postmortem autolysis is fairly rapid, necropsy material may in part be suitable for limited electron microscopic investigation. How- ever, in cases of sudden death, glands obtained up to 8 hr postmortem revealed better preservation than those obtained at a shorter postmortem interval from people with severe illnesses. It was observed that shock and anoxia play an important role in the acceleration of cytolysis. The McManus PAS procedure22 followed by an Orange G counter- stain enables differentiation of pituitary cells into 3 large groups: the orangeophils or serous cells; the PAS-positive reacting group or mucoid cells; 23 and a large group of colorless cells or chromophobes. Electron microscopically, each group may be subdivided according to the cytoplasmic characteristics of the different cell types. The serous cells comprise a heterogeneous and large population which predominates in the lateral wings of the pars anterior (Text-fig 1). For practical purposes they are subdivided into cells with large or small secretory granules. The acidophils with large granules are the most common. Their secre- 46 PAIZ AND HENNIGAR American Journal of Pathology tory granules are uniformly dense, varying in size from 250 m,u to 500 mp with a mean diameter of 350 mu (Fig 1 and 4). The smallest granulations are round and the largest somewhat irregular, varying in shape from round to oval to piriform. The ergastoplasm is prominent, composed of flattened cisternae which are sometimes arranged in parallel rows or whirlpools. In some acidophils with large granules a faint PAS reaction was observed about the centrosphere (Fig 2 and 6). At higher magnification this PAS-positive area (Fig 7) is seen to contain a lamellar and tubular Golgi complex and a few seemingly immature or early granules. The second type of acidophil with smaller granules is less common. The smaller granules within it have a clear area between the granular core and its plasma membrane. The larger granules which measured from 200 to 300 ma are more electron dense and there is no distinct perigranular clear zone (Fig 8). The ergastoplasm is poorly developed and composed of minute vesicles and short tortuous cisternae. The hyaloplasm is abundant, with a moderate amount of free ribosomes and microtubules. The mitochondria are scarce (Fig 5). A section 2 ,M thick usually fails to demonstrate the serous nature of the cell. However, if the section thickness is increased to 4 or 6 u the cell acidophilia is demonstrable, provided sufficient numbers of granules are present. The PAS-positive or mucoid cells are also subdivided into cells charac. terized by large and small granules. The most common form is the cell with small granules. The secretory characteristics are manifested by a very electron-dense area and are ovoid to pear-shaped. The granular dimensions in the maximally granulated cell range from 300 to 800 mp with a mean of 450 m,&. The ergastoplasm is well developed and composed of flat cisternae. These cells are seen more commonly topograph- ically positioned in the middle and anterior portion of pars distalis. On occasion it is impossible to differentiate this cell from the serous cell with 350 mu diameter granules since their smaller granulations overlap considerably and the ergastoplasm is similar (Fig 3 and 9). The second, less common type of PAS-positive mucoid cell, which has large granules, is usually found in tissue blocks from the most anterior portion of pars distalis. This mucoid cell is readily identified electron microscopically due to the peculiar characteristics of their secretory granules which are packed together during the storage phase. Furthermore, the secretory granules are of variable density with the smaller sizes being more electron dense and the larger less electron Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 47 April 1970 dense. Both types of secretory granules measure from 350 to 1100 mu. Occasionally we have observed cells with low-density granules up to 2000 mu in diameter. This may either represent a variation in secretory function, or perhaps another closely related cell type (Fig 14). The appearance of the small granules overlaps with those in the most common type of mucoid cell. The larger granules are very distinct and their matrix tends to lose electron density proportionately as the granule size increases. This peculiar combination of secretory granules makes this cell easily identifiable solely on a morphologic basis. The endoplasmic reticulum is not usually prominent, but it sometimes is expanded into large irregular cisternae. By application of the thin-thick correlation technique we have studied a large pool of cells that on light microscopy were devoid of color affinity. These chromophobe cells comprise a heterogeneous population of cells. We classify them into 3 main groups: degranulated pituitary cells, serous (?) or mucoid (?), follicular cells, and oncocytic cells. The degranulated cells might correspond to either serous or mucoid series depicted in an earlier or late state of its secretory function. Cell type identification is extremely difficult because the secretory granules are scarce, of variable size, and always smaller than those which occur in the fully granulated cell. The follicular cells, originally described by Farquhar and Rinehart,1'24 are attached to one another by desmosomes and form either potential intercellular spaces lined by microvilli, or are bound to an acinar lumen containing an amorphous dense material. The cytoplasm in general lacks secretory granules and is essentially occupied only by microtubules of smooth endoplasmic reticulum and clusters of free ribosomes. At the apical border a few small vesicles containing a less electron-dense material are seen (Fig 10). The amorphous material in the acinar lumen is seen to be strongly PAS-positive when thick-thin correlations are performed. By electron microscopy, one is sometimes able to demonstrate secretory cells in continuity with the colloid although they are usually separated from it by a follicular cell. Since both acidophils and mucoid cells surround the colloid, it is possible that this luminal material has a dual origin and comes from both types of cells. The apical portion of the follicular cell resembles somewhat the apical portion of the thyroid follicular cell, portions of which are also lined by microvilli and contain small vesicles about its free zone.24 Also, we have classed as chromophobes those cells which occasionally contain secretory granules and whose cytoplasmic space is almost com- 48 PAIZ AND HENNIGAR American Journal of Pathology pletely occupied by mitochondria. The numerous closely packed mitochondria seem to be unusually large and have abundant cristae and numerous large intramitochondrial granules (Fig 13). The cell bound- aries are smooth and regular. Because of the rare occurrence of this particular cell type, we consider the finding of possible limited signifi- cance. The material examined revealed its presence in only 5 instances. The ultrastructure is similar to that described in oncocytomas of several organs and to our knowledge it is the first time this particular cell has been described in electron microscopic studies of human adenohypophyses. Discussion There is some agreement on the development of pituitary cells from less differentiated elements by a process of progressive maturation. Definite secretory cycles occur constantly in adenohypophyseal cells and the stage of a given cycle may be characterized by nuclear changes, metamorphosis of the ergastoplasm, changes in the Golgi, and alteration of granule morphology.2529 In general, the terms serous and mucoid are applied to a cellular state in which enough appropriate cytoplasmic material is demonstrable by histochemical techniques. Therefore, many chromophobes at the light microscopy level probably correspond to degranulated serous or mucoid cells in either early or very late stages of their secretory cycle. Some of the most active somatotrophic hormone- producing (STH) adenomas in humans reveal a scantily granulated cytoplasm that is chromophobic by light microscopic criteria.26 The 350- m,u serous cell of our study is comparable to that described as an STH cell in humans 9,30 and in other animal species.'31'32 Occasionally some STH cells contain secretory granules which measure up to 800 mA. In such cells, the ergastoplasm becomes prominent and vesicular. Possibly this peculiar appearance of the ergastoplasm represents a different cell type (prolactin) or merely a stage in the secretory cycle. The STH cells and possible prolactin cells predominate in the horizontal sampling in the lateral and anteromedial portions of pars distalis. On the other hand, the finely granulated serous (175 m,) cell is found randomly scattered throughout the gland. These secretory granules sometimes fail to demonstrate their acidophilic properties. This cell might be responsible for ACTH production since adenohypophyseal tumors inducing Cushing's disease have been shown to be composed of finely granulated "acidophils," 25 similar to the small acidophil of this study. A morphologically identical cell has been demonstrated in ACTH- Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 49 April 1970

producing adenomas 33 experimentally induced in the mouse. Electron microscopically, Foncin11 and Foncin and LeBeau 30 have described the corticotroph as having fine secretory granules 90-130 m,u in average diameter. The amphophil cells have been discussed at length.27 The term amphophil indicates a blending of acidophilic and basophilic secretory granules. The existence of such hybrid cells has never been proven.26 However, we have demonstrated that the Golgi zone of acidophils sometimes exhibit an area that is PAS-positive in thick-thin section correlations. We believe that this phenomenon is not necessarily related to the secretory granule but to the material within the centrosphere (Golgi apparatus, immature granulations, and ground substance). Occasionally lysosomes are demonstrable in serous as well as in mucoid cells. At the electron microscopic level they are seen as dense osmophilic bodies containing a lipid-like substance. In the thick-thin correlation these structures are extremely PAS-positive. These lysosomes impart to acidophils the amphophilic properties which are not necessarily attributed to the secretory granules. The mucoid cells with granules averaging 450 mnu are the most numerous of the PAS-positive cells and they are predominant in the most medial portion of pars anterior. They might represent one of the cells incorporated in the beta group of Greek nomenclatures, which are thought to possibly secrete either MSH, TSH, and/or ACTH.6 The other mucoid cell with large granules might represent one or both of the gonadotrophs. Large numbers of this cell type were seen in a block examined from the most anteromedial portion of pars anterior. The stage or form with less differences in electron density of its two granule types bears some resemblance to Foncin's FSH cell. This mucoid cell sometimes acquires greater cytoplasmic proportions and becomes filled by large numbers of secretory granules which are markedly less electron dense. These granules give the cytoplasm a vesicular appearance at the light microscope level. This cell, in our correlative study, appeared sometimes as a chromophobe since only the small dense granules stained with PAS. It is not known whether the vesicular form is a separate cell or whether it merely represents a stage of secretion. Appar- ently, the vesicular form shows affinity for dialyzed iron and has been tentatively labeled a gonadotroph32 and possibly the source of LH. There are extensive observations regarding the FSH and LH gonadotrophs in several animal species 28 which do not correlate with our observations about this particular cell. Mast cells were rarely seen in our electron microscopic survey. They 50 PAIZ AND HENNIGAR American Journal of Pathology were found in the interstitial connective tissue, and contain in their cytoplasm dense secretory granules with an average diameter of 450 mIy, corresponding to the description of other investigators.29 The chromophobes comprise a large pool of cells which deserve further study. This correlative study was able to add little new informa- tion with regard to these cells. Some chromophobe cells contain abundant ribosomes and endoplasmic reticulum (dark cells) while others are seemingly free of these protein-synthesizing units (Fig 12), indi- cating possible stages in its secretory function. The follicular cell deserves some attention, since very little has been recorded concerning its physiologic role. Examination of the micrographs discloses that these cells are quite frequent. The lumen they form is not always occupied by colloid material. Our observations demonstrate that the stored colloid is derived from both mucoid and serous cells. We suggest that the follicular cell might use its numerous microvilli to absorb a hormone or hormones for immediate use (ACTH?) from the colloid. Rennels 31 exposed rats to severe stress by scaling and has demonstrated accumulation of material in the follicular cells under such conditions. In our study the follicular cells did not always separate secretory cells from the luminal colloid. Other workers have reported that the secretory cells are always separated from the colloid by a portion of a follicular cell.34'35

Summary Two types of mucoid cells, 2 serous cells, and 1 type of chromophobe all were differentiated by PAS-Orange G staining of Epon-embedded thick sections of human adenohypophysis and correlated with their electron microscopic equivalents in adjacent thin sections. One mucoid cell apparently corresponded to the light microscopic beta cell and contained granules averaging 450 m/A in diameter. The second mucoid cell, possibly a gonadotroph, contained 2 granule types: 1 granule was relatively electron dense and averaged 250-500 mu in diameter; the other was less electron dense and ranged from 500 to 1000 mu in diameter. One serous cell, the growth hormone cell, contained dense granules averaging 350 mu in diameter. The other serous form, presumably the adrenocorticotroph, contained granules averaging 150 m,u. The chromophobe type studied was the follicular cell. It is suggested that this cell may absorb one or more specific proteins from the colloid. Both mucoid and serous cells were found to contribute to the formation of colloid. Occasional mast cells and oncocytes were identified. Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 51 April 1970

References 1. RoMEIS, B. Hypophyse. In Handbuch der mikroskopischen Anatomie des Menschen. Hrsg. von. W.v. Mollendorff, Bd. 6.3, Teil, J. Springer, Berlin, 1940. 2. HALMI, N. S., DE GROOTE, J. W. Remarks concerning two types of basophil cells in the human adenohypophysis. J Clin Endocr 21:732-735, 1961. 3. HERLANT, M. Les cellules basophiles du lobe anterieur de l'hypophyse chez l'homme. Arch Biol (Liege) 67:539-553, 1956. 4. EZRIN, C., MURRAY, S. The cells of the human adenohypophysis in pregnancy, thyroid disease and adrenal cortical disorders. Centre National de la Rech-erche Scientifique: Cytologie de L'Adenohypophyse 128:183-201, 1963. 5. PEARSE, A. G. E., VAN NOORDEN, S. The functional cytology of the human adenohypophysis. Canad Med Ass J 88:462-471, 1968. 6. PURVES, H. D. "Cytology of the Adenohypophysis." In The Pituitary Gland (Vol. 1, Chap. 4), Harris, G. W. and Donovan, B. T., Eds. University of California Press, Berkeley and Los Angeles, 1966. 7. PHIFER, R. F., SPICER, S. S., HENNIGAR, G. R. Human adenohypophyseal mucoid cell staining (abst). Amer J Path 55:72A, 1969. 8. PHIFER, R. F. Cell type correlation classification and glycoprotein char- acterization in human adenohypophyseal mucoid cells (abst). Texas Rep Biol Med 26:3, 1968. 9. SCHELIN, U. Chromophobe and acidophil adenoma of the human pituitary gland. A light and electron microscopic study. Acta Path Microbiol Scand 158 (Suppl.):80, 1962. 10 PAIZ, C., HENNIGAR, G. R. Electron microscopy and histochemical correlation of human anterior pituitary cells (abst). Fed Proc 25:536, 1966. 11. FONCINc, J. F. Etudes sur l'hypophyse humaine au microscope electronique. Extrait de Pathologie Biologie 14:893-902, 1966. 12. BARNES, B. G. The fine structure of the mouse adenohypophysis in various physiological states. Centre National de la Recherche Scientifique: Cytologie de L'Adenohypophyse 128:91-110,1963. 13. FARQUHAR, M. G., RINEHART, J. F. Electronmicroscopy studies of the anterior pituitary gland of castrate rats. Endocrinology, 54:516-541, 1954. 14. FARQUHAR, M. G., RINEHART, J. F. Cytologic alterations in the anterior pituitary gland following thyroidectomy. An electron microscope study. Endocrinology 55:857-876, 1954. 15. HERLANT, M. Apport de la microscopie electronique a l'etude du lobe anterieur de l'hypophyse. Centre National de la Recherche Scicntifique: Cytologie de radenohypophyse. 128:73-90, 1963. 16. RINEHART, J. F., FARQUHAR, M. G. Electron microscopic studies of the anterior pituitary gland. J Histochem Cytochem 1:93-113, 1953. 17. BARNES, B. G. Electron microscopic studies on the secretory cytology of the mouse anterior pituitary. Endocrinology 71:618-628, 1962. 18. SABATINI, D. D., BENSCH, K., BARNETT, R. J. Cytochemistry and electronmicroscopy. I Cell Biol 17:19-58, 1963. 19. PALADE, G. E. A study of fixation for electron microscopy. J. Exp Med 95:285-297, 1952. 52 PAIZ AND HENNIGAR American Journal of Pathology

20. LUFT, J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 9:409-414, 1961. 21. WATSON, M. L. Staining of tissue sections for electron microscopy witl heavy metals. I Biophys Biochem Cytol 4:475-481, 1958. 22. MCMANUS, J. F. A. Histological demonstration of mucin after periodic acid. Nature (London) 158:202-218, 1946. 23. PEARCE, A. G. E. Observations on the localization, nature, and chemical constitution of some components of the anterior hypophysis. J Path Bact 64:791-809, 1952. 24. FARQUHAR, M. G. Corticotrophs of the rat adenohypophysis as revealed by electron microscopy (abst). Anat Rec 127:291, 1957. 25. HERLANT, M., KLASTERSKY, J. Etude au microscope electronique des cellules corticotropes de l'hypophyse. C R Acad Sci (Paris) 256:2709-2711, 1963. 26. HALMI, N. S. Some unsolved problems of anterior pituitary histophysiology. Centre National de la Recherche Scientifique: Cytologie de radenohypophyse 128:19-31, 1963. 27. MELLGREN, J. Anterior pituitary in hyperfunction of adrenal cortex; ana- tomical study with special reference to Syndroma Morgagni and notes on prostatic hypertrophy. Acta Path Microbiol Scand 22: (Suppl. 60):1-177, 1945. 28. GIROD, C., DUBOIS, P. Etude ultrastructurale des cellules gonadotropes an- tehypophysaires, chez le hamster dore. I Ultrastruct Res 13:212-232, 1965. 29. THIERY, J. P. Etude au microscope electronique de la maturation et de l'excretion des granules des mastocytes. I Micro-scopie 2:549-556, 1963. 30. FONCIN, J. F., LEBEAU, J. Identification au microscope electronique des cellules adrenocorticotropes de l'hypophyse humaine. C R Soc Biol (Paris) 158:2276-2279, 1964. 31. RENNELS, E. G. Electron microscopic alterations in the rat hypophysis after scalding. Amer I Anat 114:71-76, 1964. 32. PHIFER, R. F., SPICER, S. S. Ultrastructural localization of acid mucosub- stances in human adenohypophyseal cells (abst). Program of the His- tochemical Society. Twentieth Annual Meeting, April, 1969. 33. FURTH, J., CLIFTON, K. H. "Experimental Pituitary Tumors." In Ciba Foun- dation Colloquia on Endocrinology (Vol. 12). Wolstenholme, G. E. W., and O'Connor, M., Eds. Little, Boston, 1958, pp 3-21. 34. MAzzocm, G. Sulle modalita di liberazione dei prodotti ormonali da parte della cellule adenoipofisarie con particolare riguardo alla frue morfologia di follicoli e spazi interstiziali esisterite dell'adenoipofisi del maiale. Arch Ital Anat Embriol 72:351-357, 1967. 35. BERGLAND, R. M. Follicular cells of the human pituitary. (Unpublished).

The authors wish to thank Dr. H. Rawling Pratt-Thomas, Mr. Robert F. Phifer, and Miss Comelia Campbell for assistance in the preparation of the manuscript, Mr. Allen Lieberman for his technical assistance, and Mr. Jon R. Benthal for the preparation of photomicrographs. Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 53 April 1970

[Illustratiomn follow] 54 PAIZ AND HENNIGAR American Journal of Pathology

Legends for Figures Fig 1. Stained with PAS-Orange G, to be correlated with electron micrograph, Fig 4. For orientation, note blood vessel (bv) in lower right corner. Hemiacinus is composed of two cell types; serous cells and few chromophobes. Serous cell labelled (1) is deeply orangeophilic. One small weakly orangeophilic cell (2) and a group of colorless cells (3) are also seen. X 900. Fig 2. Reproduction from thick section stained with PAS-Orange G sequence; correlate with Fig 6. Mucoid cells are stained red-purple; the serous cell orange. Mucoid cells (1 and 2) and the serous cells (3 and 4) will be correlated in the corresponding thin section illustrated in Fig. 6. PAS-Orange G. X 900. Fig 3. Encompasses area containing those cells seen in adjacent thin section (Fig 9). For orientation, note hourglass-shaped follicle (fi) which contains a PAS-positive material. Note 3 mucoid cells (m) and 3 serous cells (s) usually separated from follicle by colorless follicular cell (Fc). PAS-Orange G. X 900. I

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3 Fig 4. Electron micrograph taken from the corresponding adjacent thin section; correlate with Fig 1. Three cells correspond to serous cells with large granules averaging 350 mra (1). One small serous cell with granules averaging 75 mrt (2) and group of colorless cells (3). Blood vessel (bv) in lower right corner. Area encompassed by rectangle is illustrated at higher magnification in Fig 5. (Fig 4 X 3,400). Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 57 April 1970

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Fig 5. Secretory granules of the two types of serous cells are illustrated in area encompassed by rectangle in Fig 4. Cell on left has round, regular electron-dense secretory granules 350 mti average diam, and ergastoplasm is flat. Cell on right has a vesicular ergastoplasm and its secretory granules are sometimes pear-shaped and average 350 my. This structural difference may represent phases of the secretory cycle and not necessarily 2 different cell types. At bottom, the second type of acidophil with granules averaging 75 mz is seen. Lead citrate. X 14,000. Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 59 April 1970

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Fig 6. Low power electron micrograph taken from adjacent thin section illustrating the 2 types of mucoid cells; correlate with Fig 2. Cell 1 corresponds with granules averaging 450 mgt. Cell 2 represents a heavily granulated cell with granules up to 800 mR diam. Serous cell (3) illustrates amphophilic properties in that its centrosphere was faintly stained PAS-positive. However, its secretory granules (350 mg) are strongly orangeophilic. High power electron micrograph of area in rectangle is seen in Fig 7. Serous cell (4) has granules with mean diam of 150 m,t and is illustrated at higher magnification in Fig 8. Lead citrate. X 3,400. Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 61 April 1970

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Fig 7. High power magnification of area in rectangle in Fig 6. This portion of cytoplasm contains intricate tubular and vesicular system of Golgi apparatus as well as a centriole and a few small immature granules. This area was stained faintly PAS- positive in thin section. Cell below is mucoid cell (2) depicted in Fig 6. Notice variable size and density of secretory granules and prominent vesiculated ergastoplasm. Sec- retory granules are closely packed together. Lead citrate. X 24,800. Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 63 April 1970

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Fig 9. Electron micrograph encompasses area containing those cells seen in adjacent thick section (Fig 3). Note hourglass-shaped follicle (Fl), 3 mucoid cells (M), and 3 serous cells (S). Follicular cell labeled (Fc). Rectangles enclose areas illustrated at higher magnification in Fig 10 and 11. Lead citrate. X 3,400. Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 67 April 1970

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Fig 10. (upper) Higher power magnification of area in rectangle containing mucoid cell in Fig. 9. Follicular space (Fl) contains an amorphous dense material. Small portion of mucoid cell cytoplasm containing a few secretory granules is separated from follicle by follicular cells (Fc) which are in contact with colloid and project microvilli into it. Lead citrate. X 24,800. Fig 11. (lower) Higher magnification of area enclosed by rectangle in Fig 9 containing serous cell. Note portion of free plasma membrane of serous cell lining follicular space. Secretory granules in proximity with plasma membrane (arrow) may be about to be secreted. Smooth and rough endoplasmic reticulum of this cell is particularly abundant in this area. Lead citrate. X 24,800. Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 69 April 1970

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Fig 12. (upper) These 2 cells were chromophobes in correlative thick section stained with PAS-Orange G. They are not agranular since a few secretory particles do appear along cell periphery. Chromophobe cell in center contains abundant endoplasmic reticulum while the others are seemingly free of it. Lead citrate. X 5,400. Fig 13. (lower) Chromophobe cell whose cytoplasm is virtually occupied by mitochondria, some of which are large with a prominent intramitochondrial granule. This cell has been referred to in the literature as an oncocyte. Lead citrate. X 6,000. Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 71 April 1970

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Fig 14. Possible alternate form of mucoid cell containing two types of granules. Smaller granules, 350 m,u in average diameter, were extremely PAS-positive in thick-thin correlation. Large nonelectron-dense granules were neither PAS-positive nor acidophilic and measure up to 3200 m,u. Lead citrate. X 3400. Vol. 59, No. 1 ANTERIOR PITUITARY CELLS 73 April 1970

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[End of Article]