MICROSCOPY RESEARCH AND TECHNIQUE 20:162-176 (1992)

Effect of Drugs on Pituitary Ultrastructure W. SAEGER Department of Pathology, Marienkrankenhaus Hamburg, 0-2000 Hamburg 76, Federal Republic of Germany

KEY WORDS

Drug effects, Anterior pituitary, Light and electron microscopic structure

ABSTRACT Various drugs and hormones influence the light microscopic and especially the electron microscopic structure of the anterior pituitary and its tumors. Many structural effects are known only from animal experiments since specimens from human pituitaries are mostly not available. The structure of growth hormone (GH) cells is relatively stable. A massive GH cell hyperplasia is known only in rare cases with growth hormone releasing factor (GRF) excess from tumors. Prolactin cells can be stimulated by drugs, neurotransmitters, and hormones which decrease the dopamine inhibition. Adrenocorticotropic hormone (ACTH) cells are stimulated by stress, some hormones, loss of adrenals, and drugs which activate the al-and P-receptors or inhibit the a,-receptors. They are suppressed and changed into Crooke’s cells by treatment with glucocorticoids. Thyroid-stimulating hormone (TSH) cells increase in number and size in states of overstimulation especially by thyrotropin releasing hormone (TRH). A decrease results from hyperthyroidism and possibly from somatostatin, L-dopa, and dopamine. Gonadotroph cells transform into castration cells in strongly hyperactive states (gonadectomy, antiandrogens, gonadotropin releasing hormone [Gn-RHlagonists, aminoglutethimide). Special types of pituitary adenomas can be treated with drugs which suppress hormone production and proliferation. Dopamine agonists and somatostatin reduce the tumor size of varying proportions of GH secreting adenomas in acromegaly. Ultrastructurally, a decrease of cytoplasmic and nuclear volume and an increase of lysosomes are found. Bromocriptine and other dopamine agonists are established in the treatment of prolactin secreting adenomas. They induce a shrinkage in many cases. Ultrastructurally, a reduction of cellular and nuclear size, an increase in number of secretory granules and of lysosomes, and a reduction of rough endoplasmic reticulum can be demonstrated. INTRODUCTION The different cell types of the anterior pituitary can show various alterations after treatment with special drugs which can stimulate or suppress the hormone secretion or lead to a proliferation of cell types or inhibit their proliferation. In many conditions the stimulating effect is combined with an increased proliferation whereas a suppressing effect is often associated with a cytostatic action. Growth hormone (GH) cells show few or no morphologic alterations under many influences. Prolactin cells can react very fast upon stimulation (better: upon decreased or lacking inhibitions), transforming into socalled pregnancy cells. In cases of increased inhibition they shrink. Corticotroph cells transform into Crooke’s cells in states of hyperglucocorticism. Thyroid-stimulating hormone (TSH) cells shrink due to inhibition and enlarge due to stimulation. Gonadotroph cells transform into castration cells after excessive stimulation by drugs or hormones. Pituitary adenomas can react to a drug in the same way as the cell types from which they develop or can show alterations different from those of their non-tumorous counterparts. Some effects are visible a t the level of light microscope, others require a careful ultrastructural examination. Many experiments have been performed on isolated

0 1992 WILEY-LISS.INC.

cells in suspension or monolayer culture or in tissue culture as they have advantages either due to the possibilities for demonstrating the direct effect of a drug, or due to the absence of influences of the hypothalamus or the hormones of peripheral endocrine glands. This review discusses the influence of drugs and hormones on the morphology, especially on the ultrastructure, of the different pituitary cell types and their adenomas. Ultrastructural studies of the normal human non-tumorous hypophysis are rare for many reasons (Horvath and Kovacs, 1988; Pelletier et al., 1978; Saeger, 1977; Uei and Kanzaki, 1984; Von Lawzewitsch et al., 1972): 1) fresh specimens for electron microscopy are scarce since surgical hypophysectomies are only rarely performed for palliative treatment of patients with metastatic prostatic or breast cancer and autopsy material is not suitable for electron microscopic studies; 2) any tissues of patients with metastatic cancers are not normal because of preoperative treatments; 3) peritumorous pituitary tissue obtained from adenoma resections cannot be considered normal; 4) the ultra-

Received August 9, 1990; accepted in revised form September 7, 1990. Address reprint requests to Wolfgang Saeger, M.D., Department of Pathology, Marienkrankenhaus Hamburg, Alfredstrak 9, D-2000 Hamburg 76, Federal Republic of Germany.

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structure of the adenohypophysis may be altered by underlying diseases, especially those of the endocrine target organs as well as by the sex and age of patients. The normal ultrastructure is therefore hard to define. We know from many animal experiments that pituitary ultrastructure can be changed by drugs and hormones. It would thus seem logical that human pituitaries react in similar ways and show comparable structural alterations, especially if blood hormone levels are changed in the expected manner. In our review we have to refer to experimental data for comparison with supposed alterations of the human pituitaries because, with very few exceptions, ultrastructural studies on the human pituitary under treatment with drugs or hormones cannot be performed due to the reasons mentioned above. In the second part of our review we describe the druginduced changes in pituitary adenomas. These alterations can be better studied as many adenomas are removed surgically after short- or long-term treatment with special drugs. Hence untreated adenomas of one type can be compared with treated adenomas of the same type in order to find specific and significant effects of the drug or hormone. In particular morphometry is a valuable method in this field.

NORMAL PITUITARY GH Cells About 50% of the pituitary cell population is composed of GH cells which are localized mostly in the lateral wings. GH cells, in contrast to all other pituitary cell types, are very stable in number, granule content, and ultrastructure. Allthough GH is most important in the period of growth, the structure of GH cells does not change from childhood to old age. GH cells are acidophil, medium-sized or large, showing spherical shapes and spherical nuclei. They stain with eosin and orange G. The ultrastructure (Asa et al., 1984; Horvath and Kovacs, 1988; Saeger, 1977) demonstrated parallel areas of rough endoplasmic reticulum, globular Golgi apparatus, and many dense spherical granules with diameters mostly between 350 and 500 nm. Lysosomes vary in number and size, but large complexes are lacking. Many conditions are known (Tables l a , lb) which elevate or reduce GH levels in blood but structural alterations are not known. In rats, injections of growth hormone releasing factor (GRF) increase the number of extrusions of secretory granules from GH cells after 2.5 minutes (Couch et al., 1969). GH injections in rats suppress GH cells as demonstrated by morphometry which show decrease of cell area and diameter and numbers of secretory granules (Nakayama and Nickerson, 1973). Propylthioracil (PTU) treatment in rats depletes the number of large secretory granules in some GH cells and induces the enlargement of the cell as well as extensive proliferation of the endoplasmic reticulum (Horvath and Kovacs, 1988; Yoshimura et al., 1973). From other studies (Wood et al., 1987) we know that expression of the GH gene is strongly reduced in rats with PTU-induced hypothyroidism. T, replacement reverses this effect so

TABLE l a . GH stimulating drugs and hormones' Druglhormone GH-RH Glucocorticoid' Triiodothyroninez TRH3 Ldopa Dopamine agonists' (bromocriptine) Metoclopramide Amphetamine

Mechanism of action Direct stimulation of GH cells Increase of number of pituitary GH-RH receptors Increase of action of GH-RH Stimulation of adenoma cells Increase of catecholamine synthesis Stimulation of dopamine receptors ?

?

'For review see Muller and Nistico, 1989. 'In vitro studies. 30nly in acromegaly, not in healthy persons. 4Antagonisticeffect in pathologic hypersecretion (acromegaly).

TABLE Ib. GH suppressing drugs and hormones' Drudhormone Somatostatin SMS 201-995 Phentolamine ~

Mechanism of action I n h i b i t i o n of G H x e a s e from GH cells Inhibition of GH release from GH cells Blockade of a,- and a,-receptors

'For review see Miiller and Nistico, 1989.

TABLE l c . Drugs in treatment of acromegaly' Dopamine agonists Ldopa Dopamine Apomorphine Piribedil Bromocriptine Lisuride Pergolide Carhergoline 5-hydroxytryptamineantagonists Metergoline Cyproheptadine Somatostatin analogue Octreotide 'For review see Muller and Nistico. 1989.

the GH m-RNA values normalize. The marked hyperplasia of TSH cells which is demonstrable in hypothyroid rats is not accompanied by increased mitosic rate or thymidine labeling (Corenblum et al., 1977) SO that one can suppose the GH cells may transform into TSH producing cells during development of hypothyroidism in rats (Horvath and Kovacs, 19881, but further studies have to be performed for confirmation or exclusion of such a cell transdifferentiation. In human pathology there is only one condition which may alter the number and possibly the structure of GH cells: the excess of growth hormone releasing hormone (GH-RH)by rare tumors of the pancreatic islets, the gut, the lung, or the hypothalamus. A massive GH cell hyperplasia was demonstrated (Thorner et al., 1980) but a significant alteration of the single cells was not found with the exception of an enlargement of the Golgi apparatus and a slight increase in the average diameter of the secretory granules. Therefore, although these cells are hyperactive resulting in mark-

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W. SAEGER

Fig. 1. Prolactin cell hypevlasia adjacent to GH secreting adenoma: elongated prolactin cells with lobated nuclei, strongly developed rough endoplasmic reticulum, and Very sparse secretory C a n ules. x 4,000.

edly elevated GH levels in blood their ultrastructure is nearly stable. The strongly increased stimulation of hormone production apparently leads to an increase of GH cell number.

Prolactin Cells Between 10% and 30%of pituitary cells are prolactin cells. Their number depends on age, sex, and, in females, on the number of pregnancies. Men have the lowest numbers and multiparous women the highest. In the normally functional state prolactin cells are acidophil and indistinguishable from GH cells by conventional stains. The electron microscope (Asa et al., 1984; Horvath and Kovacs, 1988; Saeger, 1977) reveals elongated cell bodies, moderately developed rough endoplasmic reticulum, and many spherical or irregularlyshaped granules with diameters of about 600 nm. A second prolactin cell type with signs of higher secretory activity can be distinguished. It contains a well developed rough endoplasmic reticulum with formation of “nebenkernen,” a prominent Golgi apparatus, and fewer and smaller granules which measure between 150 and 300 nm. Exocytoses are frequently found. In the period of advanced pregnancy and lactation, a maximal physiologic stimulation for prolactin cells, they transform into pregnancy cells which are characterized by their large elongated size and the sparse granulation (Asa et al., 1984; Horvath and Kovacs, 1988; Saeger, 1977).They comprehend about 50% of all pituitary cells. Prolactin cells are stimulated by different drugs, neurotransmitters, and hormones (Table 2a; Fig. 1). The stimulation is mostly the effect of a decreased dopamine inhibition, as the direct stimulation by increased secretion of prolactin releasing factor (PRF) or of thyrotropin releasing hormone (TRH) into the portal blood plays only a minor role in the regulation of prolactin secretion Won Werder, 1988). On the other hand, in cultured rat pituitary cells a direct stimulating effect of estradiol on prolactin gene

Fig. 2 . Estrogen-induced prolactin cell hyperplasia of the rat: large cells with large h l g i areas, strongly increased rough endoplasmic reticulum, and varying secretory granules. X 3,500.

transcription is documented (Shull et al., 1987). Longterm treatment of rats with estrogens induces prolactin cell hyperplasia (Saeger, 1977) (Fig. 2) which develops into prolactin cell adenomas (Saeger, 1977) (Fig. 3). The stimulated prolactin cell (due to decreased suppression) has an ultrastructure similar to that of the pregnancy cell. The cytoplasmic volume is markedly increased. The cells become elongated, The nuclei may be slightly lobated and contain more than one nucleolus but the heterochromatin is not increased. The rough endoplasmic reticulum is extremely developed showing highly organized parallel membranes with many ribosomes. The Golgi apparatus is distinctly enlarged with slightly dilated sacculi and increased numbers of immature granules. In contrast, the mature secretory granules are very sparse, slightly pleomorphic, and relatively small (up to 250 nm). Lysosomes are scanty, small, and rich in pigment. Mitochondria are also sparse, oval, or elongated. It is not known whether the markedly stimulated prolactin cells (pregnancy cell type) of the human pituitary react upon short-term suppressions as in animals. It has been shown that removal of the suckling young results in the accumulation of secretory granules in the prolactin cells of lactating rats (Smith and Farquhar, 1966). Then the secretory granules become incorporated into dense lysosomal bodies, which are subsequently degraded to yield vacuolated bodies. Finally, these form free lipid droplets and small dense bodies. Such investigations are not possible in humans. In normal persons or in intact animals most prolactin cells are suppressed (Table 2b) by inhibiting hypothalamic control so that a further suppression by dopamine agonists does not induce significant morphologic alterations. But in states with decreased inhibition the number and size of prolactin cells increase (Fig. 1).Dopamine agonist treatment of those animals normalizes the number and structure of prolactin cells (McComb et al., 1986). Bromocriptine also blocks the mitotic activity of those prolactin cells (Takahashi and Kawashima, 1987). In diabetic rats prolactin cells are found to be atrophic (Yamauchi and Shiino, 1986).

TABLE 2a. Prolactin stimulating drugs and hormones' Drupdhormones

Mechanism of action

Antihypertensives a-methyldopa Reserpine Phenoxybenzamine Phentolamine Antiemetics Metoclopramide Thiethylperazine H, receptor antagonists Cimetidine Ranitidine Antidepressants/Neuroleptics' Imipramine Amitriptiline Chlorpromazine Haloperidol Domperidone Su 1piride

Interference in dopamine biosynthesis ? Inhibition of norepinephrine uptake Blockade of pituitary dopamine receptors Dopamine receptor antagonists Dopamine receptor antagonists Suppression of prolactin inhibiting factor (?) Suppression of prolactin inhibiting factor (?) Inhibition of norepinephrine uptake Inhibition of norepinephrine uptake Dopamine receptor antagonism Dopamine receptor antagonism Dopamine receptor antagonism Dopamine receptor antagonism

hormone^^.^

Stimulation of prolactin secretion Dopamine antagonism

TRH Estrogens Substance P Enkeuhalin B-endorDhin ~~

7

? ? ~

~

'For review see Dericks-Tan, 1988; Fliickiger et al., 1982; Miiller and Nistico, 1989. 'Green and Brown, 1988. 3Serri et al., 1989. 4Stimulatory actions in vitro: calcitonin, cholecystokinin (CCK), vasoactive intestinal peptide (VIP), metenkephalin, Gn-RH.

TABLE 2b. Prolactin suppressing drugs and hormones' Drugdhormones

Mechanism of action

Dopamine agonists Phenylethylamines 2-Amino-tretralines Apomorphine Ergolines Methergoline Methysergide Bromocriptine Lisuride Buspirone' Pyrrolo-ethylamines Piperazines Indirectly acting drugs pamphetamine Amantadine Nomifensine Methylphenidate Peptides and derivates Prolactin inhibiting factor (dopamine) y-aminobutyric acid (GABA) Neurotensin Calcitonin a-MSH Pyroglutamic acid Others De~amethasone~

Stimulation of dopamine stimulation of dopamine Stimulation of dopamine Stimulation of dopamine

receptors receptors receptors receptors

Stimulation of dopamine receptors Stimulation of dopamine receptors Enhancement of dopaminergic neurotransmission Enhancement of dopaminergic neurotransmission Enhancement of dopaminergic neurotransmission Enhancement of dopaminergic neurotransmission Direct inhibition of hormone secretion in prolactin cells ? ? ? ? ? ?

'For review see Dericks-Tan, 1988; Fliickiger et al., 1982; Muller and Nistico, 1989; Von Werder, 1988. 'Seppala et al., 1987. 3Rupprechtet al., 1987.

TABLE 2c. Drugs in the treatment of

hwerurolactinemia'

Dopamine agonists Bromocriptine Pergolide Lisuride Metergoline Terguride Mesulergin 'For review see Muller and Nistico, 1989.

W. SAEGER

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Fig. 3. Estrogen-induced prolactin cell adenoma of the rat: medium-sized cells with well developed Golgi fields and rough endoplasmic reticulum and sparse varying secretory granules and lysosomes. x 4,200.

Injections of insulin to these rats seem to stimulate the activity of prolactin cells but granule ex trusions are reduced (Yamauchi and Shiino, 1986). Castration of rats decreases prolactin levels, which can be corrected by estrogen administration leading to a reduction of cell size and accumulation of secretory granules (Osamura et al., 1982). Somatostatin induces an increase of lysosomes of prolactin cells in estrogen-pretreated rats (Saunders et al., 1983). Suppressed human prolactin cells can be found in the paraadenomatous glands of patients with prolactin secreting adenomas treated with dopaminergic drugs. These prolactin cells show a shrinkage of the nuclear and cytoplasmic volume. The nuclei are slightly irregular and more heterochromatic. The cytoplasm is small showing few rough endoplasmic reticulum membranes and very small Golgi apparatus, but secretory granules seem to be increased in number which is obviously the effect of the shrinkage of the cytoplasm. Exocytoses may be observed despite the inactive state of these cells (Horvath and Kovacs, 1988). Other suppressed prolactin cells may possess only very sparse secretory granules so that they resemble small chromophobe cells which are designated as null cells by Kovacs et al. (1980).

ACTH Cells Adrenocorticotropic hormone (ACTH) is part of the large proopiomelanocortin molecule which contains melanocyte-stimulating hormone (MSH), lipotropin (LPH), and endorphins as well. The ACTH cells can therefore be immunoreactive for other subunits of the large molecule. On the other hand, cells with immunohistologic content of MSH, LPH, or endorphins may be immunonegative for ACTH (Saeger et al., 1990b). Especially the ACTH cells in the pars intermedia and pars posterior possess more subunits than the ACTH cells of the pars anterior (Celio et al., 1980). Due to the problems in the immunohistologic demonstration of ACTH cells, many different antibodies should be applied for identification of all parts of the

molecule. The exact frequency of ACTH cells is not known. They may represent approximately 15-25% of pituitary cells. The number can increase in senescence. A reduced number of ACTH cells is abnormal. No differences exist between the two sexes (Asa et al., 1984; Kovacs et al., 1981). The main localization of the ACTH cells is the central “mucoid” wedge of the pituitary where they comprise the majority of parenchymal cells. The ACTH cells are medium-sized or large and oval with round nuclei and small nucleoli (Asa et al., 1984; Kovacs et al., 1981; Saeger, 1977). In the paranuclear region of many ACTH cells a large irregular vacuolar unstained body is demonstrable, the so-called enigmatic body (Asa et al., 1984; Horvath and Kovacs, 1988) which is characteristic but not specific for ACTH cells. Since acid phosphatase was demonstrated in these bodies they could be identified as lysosomes (Saeger, 1977). ACTH cells are strongly stained with periodic acid-Schiff (PAS), lead hematoxylin, and aniline blue, and are argyrophil (Saeger, 1977). By ultrastructural examination (Asa et al., 1984; Horvath and Kovacs, 1988; Saeger, 1977) the ACTH cells are ovoid or have angular outlines facing the capillaries. The nucleus lies eccentrically harboring a nucleolus in the vacinity of the nuclear membrane. In the relatively electron-opaque cytoplasm a moderately well developed and conspicuous Golgi complex with often dilated sacculi is found. The immature secretory granules in the Golgi areas are mostly haloed. The mature secretory granules are usually numerous, spherical, oval or slightly irregular, and varying in electron density. They measure 200-400 nm. Cytokeratin filaments of the type I are apparent mainly in the paranuclear region forming bundles of different thicknesses. They are reliable markers of the ACTH cells. The enigmatic bodies are of different sizes. They show a dense peripheral part and a varying central part with lipid vacuoles and represent autophagic lysosomes for the uptake and probably recycling of undischarged secretory material. A hyperfunction of ACTH cells (Table 4) can be induced by stress, stimulating hormones, loss of the adrenals, and drugs (Table 3a). The hyperactive ACTH cells are enlarged with euchromatic large nuclei and decreased size and number of enigmatic bodies. These cells are called Crooke-Russell cells (Crooke and Russell, 1935). Electron microscopic studies of these hyperactive cells have not been reported but in Cushing’s disease some paraadenomatous ACTH cells can also show signs of hyperactivity whereas others are in the state of suppression (Saeger, 1974a; Saeger et al., 1988). The stimulation is indicated by an increase of rough endoplasmic reticulum and Golgi areas and a decrease of the large lysosomes or enigmatic bodies. Animal experiments with adrenalectomy or treatment with adrenostatic drugs (metyrapone, aminoglutethimide) in the rat demonstrate by morphometric method a numerical increase of ACTH cells and an enlargement of rough endoplasmic reticulum and Golgi areas whereas mature secretory granules and lysosomes remain unchanged (Saeger and Caselitz, 1974). Zak et al. (1985) found increased diameters of secretory granules. A significant dose-related increase in volume

EFFECT OF DRUGS ON PITUITARY ULTRASTRUCTURE

167

TABLE 3a. ACTH stimulating drugs and hormones’

Drugsihormones CRF Vasopressin Oxytocin Epinephrinelnorepinephrine Methoxamine Ldopa Yohimbine Desmethylimipramine Metyrapone Aminoglutethimide

Mechanism of action Stimulation of ACTH release from ACTH cells Stimulation of ACTH release Stimulation of ACTH release Activation of a,-and preceptors a,-adrenoreceptor agonism Increase of dopamine, epinephrine, and norepinephrine Inhibition of a,-receptors Blockade of norepinephrine reuptake Blockade of steroid hormone synthesis Blockade of steroid hormone synthesis ~~

‘For review see Muller and Nistico, 1989 Sehulte, 1989.

TABLE 3b. ACTH suppressing drugs and hormones’

Drugs/hormones Dexamethasone Cortisol a-helical CRF-9-41 Clonidine Meclastine Baclofen y-vinyl-GABA

Mechanism of action Antagonism of CRF stimulated secretion Inhibition of H, stimulated accumulation of cyclic adenosine monophosphate (CAMP) Suppression of ACTH increase in ether stress Stimulation of a,-receptors (in primary effective disorders) Blockade of HI-receptor Inhibition of GABA transferase Inhibition of GABA transferase

‘For review see Miiller and Nistico, 1989.

TABLE 3c. Drugs in treatment of Morbus Cushing’

Drugs Cyproheptadine Bromocriptine Sodium valproate

Mechanism of action Serotonin antagonism, blockade of Caz+ entry into tumor cells (?) Dopamine agonism in cases with deficiency of tubohypophyseal domaninergic tract Reduction of aspertate Increase of GABA activitv

‘For review see Muller and Nistico, 1989.

density and in mitotic activity of rat ACTH cells has been shown after corticotropin releasing factor (CRF) (McNicol et al., 1988). Granule content is also increased (Kubba and McNicol, 1987). The addition of vasopressin to the higher dose of CRF induces a further increase in cell area and a reduction in granule content. A low dose of CRF was accompanied by an increase in mean granule diameter whereas high doses result in a decrease. Similar results have been obtained in the rat intermediate lobe which demonstrate that the proopiomelanotropic cells in this location change their ultrastructure under stimulation by adrenalectomy or adrenostatic drugs (John, 1983). A chronic suppression of ACTH cells by sustained elevation of cortisol plasma levels (Tables 3b, 4) induces in humans highly characteristic alterations of the ACTH cells which have been known as Crooke’s cells (Crooke, 1935) (Fig. 4). In the light microscope, they show a diminished heterochromatic oval nucleus in peripheral location and an enlarged cytoplasm exhibiting a broad hyalin ring, very large enigmatic bodies in the paranuclear region, and sparse secretory granules surrounding the large lysosomes or located underneath the cell membranes. The immunohistologic hormone content does not change (Saeger et al.,

1990b). The hyalin ring is immunohistologically positive for 55-57 KD-Keratin but negative for 68 KDKeratin, vimentin, desmin, neurofilament, and glial fibrillary acid protein (GFAP) (Uei, 1988). By electron microscopy the hyaline material consists of marked accumulation of type I microfilaments (KOvacs et al., 1970). Rough endoplasmic reticulum and Golgi areas are strongly diminished. The mostly enlarged secretory granules are displaced t o the cell periphery or the paranuclear region. The prominent enigmatic bodies are often surrounded by secretory granules which seem to fuse with the vacuoles (De Cicco et al., 1972; Saeger, 1977; WBgermark and Wersall, 1968). The amount of filament bundles in Crooke’s cells or in cells which seem to be developing into Crooke’s cells (so-called pre-Crooke’s cells) shows an inverse relationship to glucocorticoid plasma levels (Horvath and Kovacs, 1988). In rodents and most other animals, Crooke’s changes cannot be induced by application of glucocorticoidsbut the number of ACTH cells decreases after long-term treatment (Fig. 5). Volume densities of organelles also decrease. Accumulation of cytofilaments cannot be observed (Caselitz and Saeger, 1979).Somatostatin given intracisternally to rats leads to a significant decrease of immature granules, rough endoplasmic reticulum,

168

W. SAEGER TABLE 4. Causes of hypercortisolism ~

~~~~

~~~

I. Hypothalamic-hypophyseal disorder with elevated ACTH

Fig. 4. Crooke’s cells in Morbus Cushing: two cells adjacent to ACTH secreting adenoma with circular densely arranged microfilaments, paranuclear secretory granules, and lysosomes. x 5,200.

and lysosomes in ACTH cells (Caselitz and Saeger, 1979).

TSH Cells TSH cells are angular, polygonal cells of medium or large size. They are located in the medial and anterior parts of the adenohypophysis and mostly close to the capillaries (Asa et al., 1984; Saeger, 1977). Constituting less than 10% of pituitary cells, they are the least common cell type of the adenohypophysis. The cytoplasm is stainable with hematoxylin, PAS, alcian blue, aldehyde fuchsin, aldehyde thionin, and lead hematoxylin (Saeger, 1977). Their ultrastructure (Asa et al., 1984; Horvath and Kovacs, 1988; Saeger, 1977) is characterized by the angular cell shape, the round euchromatic nucleus with very small or lacking nucleolus, and the relatively high amount of organelles in the cytoplasm. The rough endoplasmic retimlum is developed to a medium degree showing medium long, in part slightly dilated arrays. The globoid Golgi apparatus contains several vesicles and flattened sacculi. The secretory granules measure 80-250 nm and are variably electron dense and spherical. They are scattered throughout the cytoplasm or arranged in a single layer underneath the cell membrane. Lysosomal bodies are sparse. Microtubules may be numerous. In states of stimulation, especially by TRH oversecretion due to thyroid hypofunction (Table 5a), the TSH cells increase in number and size. TSH hyperplasia can be accompanied by prolactin cell hyperplasia and lead to an increase in weight and size of the pituitary and the sella (Khalil et al., 1984; Siebenmann et al., 1971). The enlarged TSH cells in hypothyroidism are called thyroidectomy cells. They are characterized by a vacuolated slightly PAS positive cytoplasm harboring also large globules strongly positive for PAS. The electron microscope (Horvath and Kovacs, 1988; Kovacs and Horvath, 1986) reveals a strongly increased and dilated rough endoplasmic reticulum as being responsible for the slight vacuolation seen at the light microscope level. The Golgi complexes are also markedly

Pituitary ACTH secreting pituitary adenoma ACTH cell hyperplasia Hyperstimulated ACTH cells Adrenals Bilateral hyperplasia 11. Ectopic ACTH and/or CRF secretion Pituitary ACTH cell hyperplasia Hyperstimulated ACTH cells Adrenals Bilateral hyperplasia 111. Glucocorticoid secreting adrenal tumors Pituitary Crooke’s cells Adrenals Aden oma Carcinoma Adenomatosis IV. Side effect of drugs: ACTH treatment Pituitary Crooke’s cells Adrenals Bilateral hyperplasia V. Side effects of drugs: Glucocorticoid treatment Pituitary Crooke’s cells Adrenals Bilateral atrouhv

enlarged but the sacculi are not very dilated. The secretory granules vary in size and number, for the most part being slightly decreased. The alterations are reversible. After treatment of hypothyroidism the sella and the hypophysis return to normal size and thyroidectomy cells disappear probably by reversing into normally active TSH cells. In tissue culture of rat pituitaries, thyroidectomy cells can be induced by TRH (Shiino et al., 1973). After short-term treatment with thyroxine, secretory granules accumulate in these cells (Shiino et al., 1973). Functional suppression of TSH cells can be induced by hyperthyroidism and other conditions (Table 5b). It results in a decrease of size and number of TSH cells (Murray and Ezrin, 1966; Racadot and Peillon, 1966). Whereas ultrastructural studies in suppressed human TSH cells are not known to us, animal experiments show an accumulation and enlargement of the secretory granules and lysosomes (Kaul and Vollrath, 1974).

FSH/LH Cells The gonadotroph cells comprise approximately 1020% of anterior pituitary cells. They are scattered throughout the entire adenohypophysis, mostly with contact to capillaries and often adjacent to prolactin cells (Asa et al., 1984; Horvath and Kovacs, 1988; Kovacs et al., 1981). Whereas the number of gonadotroph cells is low before puberty, it increases in the climacterium (Kovacs et al., 1981, 1988). They are mediumsized, oval, or slightly irregular. The mostly oval nuclei are often eccentrically located. The cytoplasm stains with hematoxylin, PAS, alcian blue, aldehyde fuchsin, aldehyde thionin, and lead hematoxylin (Saeger, 1977).

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EFFECT OF DRUGS ON PITUITARY ULTRASTRUCTURE

TABLE 5a. TSH stimulating drugs and hormones' Drugsihormones

Mechanism of action

TRH Propanolol Metoclopramide Sulpiride Domperidone

Stimulation of TSH release Blockade of P-receptor Blockade of dopamine receptor Blockade of dopamine receptor Blockade of dopamine receptor

'For review see Miiller and Nistico, 1989.

TABLE 5b. TSH suppressing drugs and hormones' Drugdhormones

Mechanism of action

Somatostatin Triiodothyronine

Inhibition of TSH secretion Inhibition of 5-hydroxytryptamine turnover and TSH release Increase of dopamine, norepinephrine, and epinephrine Stimulation of dopamine receptors

L-dopa Dopamine

'For review see Miiller and Nistico. 1989.

Fig. 5. ACTH cell of the rat after methylprednisolone treatment: angular cell contacting the basement membrane with regular structure. No accumulation of cytofilaments or lysosomes. x 4,250.

Immunohistology identifies follicle-stimulating hormone (FSH) and luteinizing hormone (LH) mostly in the same cells (Kovacs et al., 1981) but some cells show only FSH or LH positivity. Probably structural differences between the bihormonal and monohormonal gonadotrophs do not exist. An intermittently varying production of those gonadotroph hormones, as demonstrated in rats (Childs et al., 1987), seems feasible in humans too. By electron microscopy (Asa et al., 1984; Horvath and Kovacs, 1988)FSH/LH cells are medium-sized harboring spherical and eccentric euchromatic nuclei and abundant cytoplasm. The rough endoplasmic reticulum is well developed with short, often slightly dilated profiles. The Golgi apparatus is prominent with numerous sacculi and vesicles and includes many immature secretory granules. The mature secretory granules vary considerably in size, structure, and number. Two types of secretory granules exist: one type measures 150-250 nm, the other 350-600 nm. In women, most secretory granules are of the larger type. Light bodies with spherical shape, granular content, and dense core seem to be characteristic for gonadotroph cells (Holck et al., 1987). There are many conditions in which gonadotroph cells are stimulated and hyperactive (Table 6a). In those states the gonadotroph cells increase in number and slightly in size showing densely arranged vacuoles and variably diminished granules. Since these alterations are demonstrated after removal of the gonads, these cells have been called castration cells or gonadectomy cells. Electron microscopically (Foncin and Le Beau, 1966; Kovacs and Horvath, 1975; Saeger et al., 1983) the vacuoles are identified as strongly dilated rough endoplasmic reticulum. There seems t o be a progression from smaller to larger vacuoles. The Golgi apparatus is enlarged and the number of secretory granules decreased.

In animals, similar alterations can be observed after treatment with antiandrogens (cyproterone) (Fig. 6) or with the steroidogenesis inhibitor aminoglutethimide (Fig. 7) (Saeger, 1974b) or with luteinizing hormone releasing hormone (LH-RH) (Shiino, 1979) or gonadotropin releasing hormone (Gn-RH) agonist (Yi-bin et al., 1987). Suppression of gonadotrophs can be expected in many states (Table 6b) but morphologic and especially ultrastructural findings are not reported in humans. From animal experiments we suppose that estrogens may lead to involution of gonadotrophs since a decrease in number and size was reported (Asa et al., 1984).

PITUITARY ADENOMAS GH Producing Adenomas in Acromegaly There are three main adenoma types in acromegaly (Table 7): the differentiated GH cell adenoma (or densely granulated GH cell adenoma and in part mammosomatotroph cell adenoma according to the classification of Kovacs and Horvath [19861), the undifferentiated adenoma (or sparsely granulated GH cell adenoma including the acidophil stem cell adenoma in the classification of Kovacs and Horvath [19861), or the mixed GH cell/prolactin cell adenoma. The ultrastructure of these adenomas is extensively described and characterized (Horvath and Kovacs, 1988; Kovacs and Horvath, 1986,1987; Saeger, 1977,1981).The electron microscopic differences between the main types have been morphometrically analyzed and found to be significant (Saeger et al., 1987b). In the treatment of patients with acromegaly (Table lc) two drugs have been introduced: dopamine agonists and the somatostatin analogue SMS 201-995. Both drugs can lower GH plasma levels, reduce tumor size, and improve the clinical symptoms of acromegaly in varying proportions of cases in the different reports published in the literature (Bassetti et al., 1988; Besser et al., 1976; Lamberts et al., 1985; Nilson, 1978; Schatz et al., 1988). The effect of shrinkage is generally minimal in comparison with the shrinkage of cells in pro-

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W.SAEGER

TABLE 6a. Gonadotropin stimulating drugs and hormones’ Drugdhormones LH-RH Buserelin* Naloxone Metuclopramide Histamine Estrogen Progesterone Cyproterone3

Mechanism of action Stimulation of LH and FSH release LH-RH analog Stimulation of LH release Stimulation of LH release in mid-luteal phase Potentiation of LH-RH-induced LH rise Increase of responsiveness to LH-RH in the second part of cycle Initial augmentation of responsiveness to LH-RH Testosterone antagonism

‘For review see MBller and Nistico, 1989.

‘No effect in gonadotropin secreting adenomas (Sassolas et al., 1988). 3V0n Berswordt-Wallrabe and Neumann (1968).

TABLE 6b. Gonadotropin suppressing drugs and hormones‘ Drugdhormones Inhibin Testosterone Estrogen Progesterone Endogenous opoid peptides GABA

Mechanism of action Negative feedback Negative feedback Decrease of responsiveness in the first part of cycle Suppression of responsiveness to LH-RH after initial augmentation In mid-luteal phase

Fig. 6. Gonadotropic cell of rat after cyproterone treatment: strongly dilated rough endoplasmic reticulum. x 7,800.

Inhibition of LH-RH-induced LH rise

‘For review see Miiller and Nistico, 1989.

lactin secreting adenoma after bromocriptine treatment. Clinical improvement following bromocriptine in acromegaly does not always imply tumor shrinkage (Besser et al., 1976; Nilson, 1978). Electron microscopy reveals in a small collection of bromocriptine- and TRH-treated adenomas a dense granulation, a dilated rough endoplasmic reticulum, and frequent misplaced exocytoses. From these facts the authors (Fanghanel-S. et al., 1978) concluded that bromocriptine inhibits the spontaneous release of GH but does not interfere with the abnormal GH response to TRH. In GH producing adenomas treated with a somaFig. 7. Gonadotropic cell of rat after aminoglutethimide treattostatin analogue SMS 201-995 (octreotide) a decrease ment: cystic dilation of rough endoplasmic reticulum (“castration of volume of cytoplasm and nuclei and an increase of cell”). x 5,300. lysosomes were found by ultrastructural morphometric analysis of one case in comparison with untreated adenomas (George et al., 1987). Others (Garcia Garcia et al., 1989)found a decrease of lysosomal and mitochon- show stimulation of GH by TRH in culture when there drial volume densities. Perivascular spaces were wid- is the same effect in vivo and no TRH effect in vitro ened containing cell debris. More tumors should be in- when GH does not respond in vivo (Marcovitz et al., vestigated by morphometry to clarify the mechanism of 1982). Apomorphine and somatostatin suppress GH somatostatin effect in vivo. and prolactin secretion in cultured adenoma tissue In vitro studies demonstrate dopamine receptors in (Marcovitz et al., 1982). Cortisol was found to increase GH secreting adenomas (Bression et al., 1982) and a GH secretion and simultaneously decrease prolactin significant inhibition of GH release by dopamine (Bres- secretion in long-term tissue cultures of GH producing sion et al., 1982). Electron microscopic studies (Garcia adenomas (Adams et al., 1981; Loras et al., 1988). EsGarcia et al., 1989) (Fig. 8A,B) show an increase in tradiol stimulates prolactin from a mixed adenoma number and a decrease in diameter of secretory gran- (Adams et al., 1981). Insulin has no effect, T, and T4 ules after bromocriptine. together suppressed GH and prolactin (Adams et al., Functional in vitro studies of GH producing ade- 1981). GRF has been found to stimulate GH secretion nomas not accompanied by morphometric analysis in 5 or 13 cultured adenomas (Westphal et al., 1987).

EFFECT OF DRUGS ON PITUITARY ULTRASTRUCTURE

171

TABLE 7. Adenoma types in acromegaly Classification of Kovacs and Horvath (1986) Densely granulated HG cell adenoma' Sparsely granulated GH cell adenoma* Mixed GWprolactin cell adenoma5 Acidophil stem cell adenoma5 Mammosomatotroph cell adenoma5

Classification of Saeger ( 1981) Highly differentiated GH cell adenoma3 Undifferentiated acidophil adenoma4 Unclassified adenoma Chromophobe adenomas Oncocytic adenoma

N' 74 209 12 13

2 310

(%)

(23.9) (67.4) (3.9) (4.2)

(0.6) 100.0

'Collective of tumors in acromegaly (Saeger et al., 1987a). 2Mostly isolated GH hyperfunction. 3Correspondingto densely granulated GH cells adenoma. 4Correspondingto sparsely granulated GH cell adenoma or to acidophil stem cell adenoma. 'Mostly acromegaly and hyperprolactinernia.

Fig. 8. A: Untreated cell suspension of an undifferentiated acidophi: adenoma in acromegaly: well developed Golgi area and rough endoplasmic reticulum, sparse secretory granules. x 9,270. B: Cell suspension of the same adenoma (like Fig. 8A) after treatment with bromocriptine (90 minutes): lobated nuclei, accumulation of secretory granules, increased lysosomes. x 4,440.

Prolactin Producing Adenomas Prolactin secreting adenomas in patients with isolated hyperprolactinemia are classified into large cell chromophobe adenomas (respectively sparsely granulated prolactin cell adenomas in the classification of Kovacs and Horvath 119861) or densely granulated prolactin cell adenomas. However, other adenoma types (especially small cell chromophobe adenomas) can also

Fig. 9. A Untreated cell suspension of prolactin cell adenoma in hyperprolactinemia: elongated cells with slightly lobated nuclei, large Golgi fields, medium amounts of rough endoplasmic reticulum, and pleomorphic secretory granules. x 5,320.B: Cell suspension of the same adenoma (like Fig. 9A) after treatment with bromocriptine (90 minutes): decreased cytoplasmic volume, condensed nuclear chromatin, dilated rough endoplasmic reticulum. x 5,460.

produce mild to moderate hyperprolactinemia (Riedel et al., 1986) (Table 8). In the therapy of prolactinomas (Table 2c) bromocriptine and other dopamine agonists have been well established for many years. They reduce prolactin plasma levels and tumor size and improve endocrine, visual, and neurologic disturbances in many cases (Chiodini et al., 1981; Fluckiger et al., 1982; Molitch et al., 1985; Nissim et al., 1982; Thorner et al., 1980).

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W.SAEGER TABLE 8. A d e n o m types in hyperprolactinemia Classification of Kovacs and Horvath (1986) Densely granulated prolactin Containing adenoma' Sparsely granulated prolactin containing adenoma2 Mixed GH celllprolactin cell adenoma5 Acidophil stem cell adenoma5 Mammosomatotroph cell adenoma'

Classification of Saeger (1981)

N'

c/o

Highly differentiated prolactin cell aden~ma',~ Large cell chromophobe aden~ma'.~ Undifferentiated acidophil adenomas

6 62 8

4.7 48.8 6.3

Small cell chromophobe adenoma Oncocytic adenoma

44

34.7 5.5 100.0

7 127

~

'Collective of tumors with isolated hyperprolactinernia (Riedel et al., 19861. 21solated hyperprolactinemia. 3Correspondingto densely granulated prolactin containing adenoma. 4Corresponding to sparsely granulated prolactin containing adenoma. 51n combination with acromegaly.

Morphofunctional studies at the light microscopic level show a distinct reduction of cytoplasmic volume. In our collection (Hamester et al., 1987) we confirmed this effect by morphometric analysis. The nuclei also seemed to be smaller after treatment but the differences were not significant. The shrinkage of the cells results in transformation of large cell chromophobe adenomas into small cell chromophobe adenomas. Studies on the question whether dopamine agonist treatment induces necrosis of adenoma cells have led to controversial results. Several authors (Barrow et al., 1984; Nissim et al., 1982) concluded that there are no signs of increased necrosis in treated prolactinomas, whereas others (Gen et al., 1983; Ludecke et al., 1983; Mori et al., 1985) reported conspicuous necrobiotic alterations. Morphometric studies in our collection of treated adenomas show (Hallenga et al., 1988) that the rate of cell necrosis was significantly higher after treatment in comparison with preoperatively untreated prolactinomas and inactive adenomas. At the ultrastructural level many studies have been undertaken on dopamine agonist-treated adenomas (Fig. 9A,B) (Anniko and Wersall, 1981; Barrow et al., 1984; Bassetti et al., 1984; Landolt, 1984; Larraza et al., 1980; Mori et al., 1985; Nissim et al., 1982; Rengachary et al., 1982; Saitoh et al., 1986b; Schottke et al., 1986; Tindall et al., 1982). Morphometry reveals a reduction of cellular and nuclear size, an increase in number but a reduction in size of secretory granules, an increase in lysosomes, and a reduction of rough endoplasmic reticulum (Figs. 10A,B, llA,B) (Bassetti et al., 1984; Landolt et al., 1985; Nissim et al., 1982; Saitoh et al., 1986b; Schottke et al., 1986). The individual tumor cell volume is reduced by approximately 40% within 7 days (Landolt et al., 1985; Saitoh et al., 1986b). The number of secretory granules discharged into the intercellular space increases after short-term treatment (Landolt et al., 1985; Saitoh et al., 1986b). The perivascular fibrous tissue accumulates after treatment for more than 3 months (Landolt, 1984; Mori et al., 1985). Morphometric results in tissue culture are similar to those obtained in surgical biopsies taken after shortterm administration (Duffy et al., 1988) indicating a direct effect of bromocriptine. Incubation with testosterone can inhibit prolactin in adenomas and can be accompanied by a development of myelin figures and

Fig. 10. A Untreated cell suspension of large cell chromophobe adenoma in hyperprolactinemia: lobated nucleus, well developed rough endoplasmic reticulum, sparse small secretory granules, microvilli at the membranes. x 3,980. B: Cell suspension of the same adenoma (like Fig. 10A): lobated nuclei, decreased cytoplasmic volume, increased secretory granules and lysosomes. x 4,120.

accumulation of amorphous dense inclusion bodies in cytoplasm (Anniko et al., 1983). In vitro studies reveal a decrease in number of exocytoses and an increase in the volume density of lysosomes 60 minutes after treatment of adenoma cell suspensions (Fig. 9A,B) (Saeger et al., 1985). Estrogen-induced prolactin secreting adenomas of rat (Fig. 3) and their hormone secretion can be reduced by bromocriptine (Niwa et al., 1987; Saitoh et al.,

EFFECT OF DRUGS ON PITUITARY ULTRASTRUCTURE

Fig. 11. A Large cell chromophobe adenoma in hyperprolactinemia without treatment with dopamine agonists: large cells with well developed rough endoplasmic reticulum and secretory granules. x 5,200. B Chromophobe adenoma in hyperprolactinemia after treatment with dopamine agonists: lobated nucleus, decreased cytoplasmic volume, increased secretory granules. x 5,200.

1986a). The development of those experimental adenomas but also of spontaneous adenomas can be prevented by dopomine agonists (Dall’Ara et al., 1988; El Etreby et al., 1983) and by the antiestrogens clomiphene (Sumi et al., 1984) and tamoxifen (De Quijada et al., 1980).

173

Fig. 12. A Untreated cell suspension of ACTH secreting adenoma in Cushing’s disease: medium-sized cells with well developed Golgi fields and rough endoplasmic reticulum, many secretory granules. x 4,210. B Cell suspension of same adenoma (like Fig. 12A) after treatment with lysine-vasopressin (15 minutes): sparse secretory granules, increased rough endoplasmic reticulum. x 4,880.

shape, size, and electron opacity measuring 200-600 nm in diameter. Rough endoplasmic reticulum is distributed throughout the cytoplasm and is partly dilated. Ribosomes are relatively numerous. Medical treatment of pituitary adenomas in Cushing’s disease (Table 3c) or Nelson’s syndrome may be useful with cyproheptadine, bromocriptine, or sodium ACTH Producing Adenomas valproate due to their possible ACTH lowering effect Corticotroph adenomas are subdivided into well dif- (Muller and Nistico, 1989). Adenoma shrinkage by ferentiated ACTH cell adenomas (73% of all adenomas such treatment has been documented only in very rare in Cushing’s disease and Nelson’s syndrome) (Table 4) cases: chronic administration of sodium valproate re(Saeger et al., 1988) and undifferentiated mucoid cell duces a macroadenoma in Nelson’s syndrome (Loli et adenomas (27%)(classification of Saeger [19771).Light al., 1988) but morphologic studies were not performed. microscopy shows distinct similarities with the normal The mechanism by which sodium valproate affects the ACTH cells in the highly differentiated type which are size of the tumor is unknown. In vitro studies show that mostly well granulated. The undifferentiated type con- ACTH secretion from isolated adenoma cells in supertains often only sparse PAS positive secretory gran- fusion chambers is not or only partially suppressed by ules. cortisol, which not only fails to suppress vasopressinThe ultrastructure of ACTH secreting adenomas is induced ACTH secretion but also seems to enhance vadescribed in detail elsewhere (Asa et al., 1984; Horvath sopressin stimulation in some experiments (Ludecke et and Kovacs, 1980; Kovacs and Horvath, 1986; Saeger, al., 1980). This finding suggests a missing or inverse 1973, 1974a, 1977, 1981). Type I microfilaments local- feedback action of glucocorticoids in adenoma cells. ized in the perinuclear region are typical in cases of Electron microscopic morphometry of these isolated adCushing’s disease. Secretory granules are varying in enoma cells reveals different results (Heemcke et al.,

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W. SAEGER

1987).In one case hormone synthesizing organelles are significantly decreased after cortisol treatment (Fig. 12A,B),in a second case volumes of secretory granules and mitochondria are increased. Further studies seem to be necessary to clarify the obviously complicated mechanism. Other Endocrine Active Adenoma Types and Inactive Tumors There are only very few reports on the effect of treatment upon gonadotropin secreting adenomas (Asa et al., 1988) or on inactive adenomas. Gn-RH administration in patients with gonadotropin secreting pituitary adenomas shows different effects: persistent stimulation of LH in one patient with LH secreting adenoma, reduction of LH and FSH secretion and tumor size in a patient with a FSH and LH secreting adenoma, and no effects in other patients (for review: Sassolas et al., 1988). Treatment with the Gn-RH agonist buserelin failed to reduce gonadotropin release by the tumors of further two patients and had no antitumoral effect (Sassolas et al., 1988). Morphologic alterations by this treatment are not known. The medical treatment of adenomas, which are clinically and immunohistologically inactive, is ineffective (Zarate et al., 1985).Bromocriptine does not reduce the size of those tumors and the structural features remain unchanged (Zarate et al., 1985). The question of whether those adenomas which are clinically inactive but contain prolactin immunohistologically (Saeger et al., 1990) react to dopamine agonist medication is unresolved and requires further studies. Clarification of this problem could be helpful in selecting appropriate postoperative treatment for patients with residual tumors. REFERENCES Adams, E.F., Brajkovich, I.E., and Mashiter, K. (1981) Growth hormone and prolactin secretion by dispersed cell cultures of human pituitary adenomas. Long term effects of hydrocortisone, estradiol, insulin, 3,5,3'-triiodothyronine and thyroxine. J . Clin. Endocrinol. Metab., 53:381-386. Anniko, M., Werner, S., and Eneroth, P. (1983) Oestroaen and testosteron effects on hormone secretion and cell morphology of human pituitary tumours. Virchows Arch. (A), 399:149-162. Anniko, M., and Wersall J . (1981) Clinical and morphological findings in two cases of bromocriptine-treated prolactinoms. Acta Pathol. Microbiol. Scand. Sect. A, 89:41-47. Asa, S.L., Gerrie, B.M., Kovacs, K., Horvath, E., Singer, W., Killinger, D.W., and Smyth, H. (1988) Structure-function correlations of human pituitary gonadotroph adenomas in vitro. Lab. Invest., 58: 403-410. Asa, S.L., Horvath, E., Kovacs, K., and Ezrin, C. (1984) Cytology of the normal pituitary and pituitary tumours. In: Pituitary tumors. D. Ode11 and D.H. Nelson, eds. Futura Publishing Company Inc., Mount Kisco, New York, pp. 71-143. Barrow, D.L., Tindall, G.T., Kovacs, K., Thorner, M.O., Horvath, E., and Hoffmann, J.C. (1984) Clinical and pathological effects of bromocriptine on prolactin secreting and other pituitary tumours. J . Neurosurg., 6O:l-7. Bassetti, M., Arosic, M., Spada, A,, Brina, M., Bazzoni, N., Faglia, G., and Giannattasio, G. (1988) Growth hormone and prolactin secretion in acromegaly: correlations between hormonal dynamics and immunocytochemical findings. J . Clin. Endocrinol. Metab., 67: 1195-1204. Bassetti, M., Spada, A,, Pezzo, G., and Giannattasio, G. (1984) Bromocriptine treatment reduces the cell size in human macroprolactinomas: a morphometric study. J . Clin. Endocrinol. Metab., 58: 268-273.

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